WO1996031526A1 - Anti-obesity agents - Google Patents

Abstract

Proteins, including various dimeric and monomeric forms of the ob gene product, or modified ob protein-encoding DNA sequences, having anti-feeding activity and methods of preparing, purifying, formulating and using such proteins, for example, in the treatment of various feeding and metabolic disorders, including obesity and diabetes. Antibodies and immunoassays useful in the detection and quantitation of such proteins are also provided.

Description

31526 PC17US96/04909

ANTI-OBESΠΎ AGENTS

Related Applications

This application is a continuation-in-part of United States Patent Application

Serial No. 08/419,598, filed April 6, 1995, the contents of which are hereby

incorporated in their entirety by reference.

Field of the Invention

The present invention relates to specific proteins as well as recombinant

versions of these proteins which have potent anti-feeding activity, to the preparation and purification and formulation of such proteins, to antibodies and immunoassays

useful in their detection and quantitation, and to their use in methods for treating

various feeding and metabolic disorders and conditions, including obesity and

diabetes.

Background of the Invention

It has been postulated that when humans overeat, the resulting extra fat

somehow signals to the brain that the body is obese and makes it eat less and burn

more fuel. The multi-decade search for such a signal is briefly outlined in Rink, In

search of a satiety factor. (1994) Nature, 372: 406-407.

Identification of an obese (ob) gene from mice, the normal product of which is postulated to function as part of a signaling pathway from adipose tissue that acts

to regulate the size of the body fat depot, was recently reported in Zhang et al.. Positional cloning of the mouse obese gene and its human homologue. (1994)

Nature ill: 425-432. Zhang et al. reports the cloning and sequencing of the mouse

ob gene and its human homologue. According to the authors, ob encodes a 4.5-

kilobase (kb) adipose tissue messenger RNA with a highly conserved 167-amino-

acid open reading frame, the predicted amino-acid sequence of which is 84%

identical between human and mouse and has features of a secreted protein.

Briefly, Zhang et al. relates the isolation and sequencing of 22 cDNA clones

from a mouse white fat cDNA library. Zhang et al. at 428. According to this

Nature report, the authors found a putative 97-base pair 5' leader followed by a

predicted 167-amino-acid open reading frame and about a 3,700-kb 3 '-untranslated

sequence. Zhang et al. at 428. The ob cDNA sequence (reading from 5' -> 3') and

the predicted 167 amino acid sequence of the mouse ob gene product described in

Zhang etal. are set forth below:

ATG TGC TGG AGA CCC CTG TGT CGG TTC CTG TGG CTT TGG TCC TAT M C W R P L C R F L W L W S Y

15

CTG TCT TAT GTT CAA GCA GTG CCT ATC CAG AAA GTC CAG GAT GAC L S Y V O A V P I Q K V Q D D

30 ACC AAA ACC CTC ATC AAG ACC ATT GTC ACC AGG ATC AAT GAC ATT

T K T L I K T I V T R I N D I

45

TCA CAC ACG CAG TCG GTA TCC GCC AAG CAG AGG GTC ACT GGC TTG S H T Q S V S A K Q R V T G L 60

ACT TTC ATT CCT GGG CTT CAC CCC ATT CTG AGT TTG TCC AAG ATG D F I P G L H P I L S L S K M

75

GAC CAG ACT CTG GCA GTC TAT CAA CAG GTC CTC ACC AGC CTG CCT D Q T L A V Y Q Q V L T S L P 90

TCC CAA AAT GTG CTG CAG ATA GCC AAT GAC CTG GAG AAT CTC CGA S Q N V L Q I A N D L E N L R

105

GAC CTC CTC CAT CTG CTG GCC TTC TCC AAG ACD TGC TCC CTG CCT D L L H L L A F S K S C S L P

120

CAG ACC AGT GGC CTG CAG AAG CCA GAG AGC CTG GAT GGC GTC CTG Q T S G L Q K P E S L D G V L

135 GAA GCC TCA CTC TAC TCC ACA GAG GTG GTG GCT TTG AGC AGG CTG

E A S L Y S T E V V A L S R L

150

CAG GGC TCT CTG CAG GAC ATT CTT CAA CAG TTG GAT GTT AGC CCT Q G S L Q D I L Q Q L D V S P 165

GAA TGC E C

167

According to Zhang et al.. computer analysis of the predicted protein sequence suggests the presence of an N-terminal signal sequence (underlined). Zhang etal. at

428. The predicted signal sequence cleavage site is reported in Zhang etal. to be C-

terminal to an alanine at position 21.

Zhang et al. also reports use of the coding sequence of the ob gene in

hybridization experiments with genomic Southern blots of various vertebrate DNAs. The article reports that such experiments yielded detectable signals in all vertebrate

DNAs tested, from which it is concluded that ob is highly conserved among

vertebrates. Zhang et al. at 429. To determine the extent of ob sequence

conservation, Zhang et al. also reports on experiments to isolate and sequence cDNA

clones that hybridize to ob from a human adipose tissue cDNA library. According

to Zhang et al.. the results of such experiments suggested that the nucleotide

sequences from human and mouse were highly homologous in the predicted coding

sequence, but had only 30% homology in the available 5' and 3' untranslated

regions. Zhang et al. at 429. Alignment of the predicted human and mouse ob amino-acid sequences reported in Zhang et al. is set forth below:

Mouse MCWRPLCRFL WLWSYLSYVQ AVPIQKVQDD TKTLIKTTVT RINDISHTQS

50

Human MHWGTLCGFL WLWPYLFYVQ AVPIQKVQDD TKTLIKTIVT RINDISHTQS

Mouse VSAKQRVTGL DFIPGLHPIL SLSKMDQTLA VYQQVLTSLP SQNVLQIAND 100

Human VSSKQKVTGL DFIPGLHPIL TLSKMDQTLA VYQQILTSMP SRNVIQISND

Mouse LENLRDLLHL LAFSKSCSLP QTSGLQKPES LDGVLEASLY

STEVVALSRL 150

Human LENLRDLLHV LAFSKSCHLP WASGLETLDS LGGVLEASGY STEVVALSRL

Mouse QGSLQDILQQ LDVSPEC

167

Human QGSLQDMLWQ LDLSPGC

According to the authors, this alignment shows an 84% overall identity, and more

extensive identity in the N-terminus of the mature protein, with only four conservative and three non-conservative substitutions among the residues between

the presumed signal sequence cleavage site and the cysteine at position 117. Zhang et al. at 430-431. As in mouse, states Zhang et al.. 30% of the clones were missing

the codon for glutamine at position 49.

In vitro translation of human ob RNA is also reported in Zhang et al. at 429.

A human ob cDNA was said to have been subcloned by the authors into a pGEM

cloning vector, and plus-strand RNA then synthesized using SP6 polymerase.

Zhang et al. at 429. The in vitro synthesized RNA was translated in both the

presence and absence of canine pancreatic microsomal membranes, the former

revealing a single protein having an approximate molecular weight of 18 kD. Zhang

et al. at 429. Zhang et al. reported that the addition of the microsomal membranes

led to the appearance of a second translation product, also a single protein, having a

molecular weight about 2 kD less than the primary translation product.

Although no evidence of functional activity was reported, based on their genetic investigations, Zhang et al. suggested that the ob gene product is a secreted,

circulating factor that may represent at least one component of a satiety signaling

system in the body, and that the level of expression of this gene may signal the size

of the adipose depot. Thus, states Zhang et al., an increase in the level of the ob

signal (for example, after a period of overeating) may act directly or indirectly on the

central nervous system to inhibit food intake and/or regulate energy expenditure as

part of a homeostatic mechanism to maintain constancy of the adipose mass. Zhang

et al. at 431.

Obesity, excess adipose tissue, is becoming increasingly prevalent in

developed societies. For example, approximately 30% of adults in the U.S. were

estimated to be 20 percent above desirable body weight — an accepted measure of

obesity sufficient to impact a health risk (Harrison 's Principles of Internal Medicine 6 31526 PC17US96/04909

12th Edition, McGraw Hill, Inc. (1991) p. 411). The pathogenesis of obesity is

believed to be multifactorial but the basic problem is that in obese subjects food

intake and energy expenditure do not come into balance until there is excess adipose

tissue. Attempts to reduce food intake are usually fruitless in the medium term

because the weight loss induced by dieting results in both increased appetite and decreased energy expenditure. Leibel et al.. (1995) New England Journal of

Medicine 322: 621-628. The intensity of physical exercise required to expend

enough energy to materially lose adipose mass is too great for most people to

undertake on a sufficiently frequent basis. Thus, obesity is currently a poorly treatable, chronic, essentially intractable metabolic disorder. Not only is obesity itself undesirable for social reasons, but obesity also carries serious risk of co- morbidities including, Type 2 diabetes, hypertension, atherosclerosis, degenerative

arthritis, and increased incidence of complications of surgery involving general

anesthesia. In those few subjects who do succeed in losing weight, by about 10

percent of body weight, there can be striking improvements in co-morbid conditions,

most especially Type 2 diabetes in which dieting and weight loss are the primary

therapeutic modality, albeit relatively ineffective in many patients for the reasons

stated above.

Thus, it can be appreciated that an effective means to sustain weight loss is a

major challenge and a superior method of treatment would be of great utility. Such a

method, and compounds and compositions which are useful therefor, have been

invented and are described and claimed herein. Summary of the Invention

The present invention is directed to the manufacture and use of dimeric

forms of the ob gene product. We refer to these proteins as ""ob dimers."

Surprisingly, we have discovered that ob dimer proteins exhibit potent, prolonged

inhibition of food intake in vivo. The invention is also directed to modified

monomeric ob protein DNA sequences, to processes for the manufacture and

purification of ob dimer proteins and ob monomer proteins, to formulations of ob dimers and ob monomer proteins, and to the use of these ob proteins and

compositions in the treatment of subjects with disorders or conditions that would benefit from administration of these proteins and compositions.

In one aspect, then, the invention relates to ob dimers, which include human

ob dimers, rat ob dimers, mouse ob dimers, as well as other vertebrate ob dimers. The invention also relates to ob dimer fusion proteins, including ob dimer fusion

proteins that incorporate, for example, short marker or "reporter" peptides (for

example, poly-histidine, and the eight amino acid marker peptide known in the art as

"FLAG") which are useful in the detection and purification of the expressed protein,

and are usually removable, but we have discovered in fact need not be removed in

order to retain the activity of a non-fused ob dimer. We have also discovered that ob monomer fusion proteins, including human ob monomer fusion proteins, rat ob

monomer fusion proteins, mouse ob monomer fusion proteins, as well as other

vertebrate ob monomer fusion proteins, can be prepared as described above and elsewhere herein to yield in vivo appetite suppressant activity. According to this

aspect of the present invention, there are provided substantially pure and pure ob

dimers, ob fusion monomer proteins and ob fusion dimer proteins, which include substantially pure and pure human ob dimers, human ob fusion monomer proteins and human ob fusion dimer proteins, substantially pure and pure rat ob dimers, rat

ob fusion monomer proteins and rat ob fusion dimer proteins, and substantially pure

and pure mouse ob dimers, mouse ob fusion monomer proteins and mouse ob fusion

dimer proteins, as well as other substantially pure and pure vertebrate ob dimers, ob

fusion monomers and ob fusion dimers. By "substantially pure" is meant purity in

excess of about 50%, particularly at least about 80% by weight of protein. By

"pure" is meant purity greater than or equal to about 90% and particularly greater

than or equal to about 95% by weight of protein. Also described and claimed herein

are preparations of pure, unfused ob monomer proteins.

In another aspect, the invention relates to recombinant methods of preparing

and isolating ob dimers and ob dimer fusion proteins. One such method for the

production of an ob dimer comprises the steps of (a) preparing a vertebrate cDNA

library, preferably a vertebrate adipose cDNA library, and more preferably a human

adipose cDNA library, (b) ligating said cDNA library into a cloning vector, (c)

introducing said cloning vector containing said cDNA library into a first host cell,

(d) contacting the cDNA molecules of said first host cell with a solution containing a

suitable ob gene hybridization probe, (e) detecting a cDNA molecule which

hybridizes to said probe, (f) isolating said cDNA molecule, (g) ligating the nucleic

acid sequence of said cDNA molecule which encodes an ob protein into an

expression vector, (h) transforming a second host cell with said expression vector

containing said nucleic acid sequence of said cDNA molecule which encodes said ob

protein, (i) culturing the transformed second host cell under conditions that favor the production of said ob protein as a dimer, and (j) isolating said ob protein expressed

by said second host cell.

In still another aspect, the invention relates to recombinant methods for the

production of ob dimers which do not include the steps of making and screening a

cDNA library or libraries, but use instead a method of amplification of cDNA

prepared from tissue total RNA, preferably adipose tissue total RNA. Such a

method is described herein and comprises the steps of (a) isolating a preparation of

total RNA from a vertebrate tissue, preferably adipose tissue, (b) converting said

isolated RNA to cDNA, (c) amplifying a cDNA sequence from said cDNA using oligonucleotide primers suitable for annealing to a target ob protein gene sequence,

(d) detecting a cDNA molecule using oligonucleotides suitable for hybridization to

said target ob protein gene sequence, (e) isolating said cDNA molecule, (f) ligating

the nucleic acid sequence of said cDNA molecule which encodes an ob protein into

an expression vector, (g) transforming a host cell with said expression vector

containing said nucleic acid sequence of said cDNA molecule which encodes said ob

protein, (h) culturing the transformed host cell under conditions that favor the

production of said ob protein as a dimer, and (i) isolating said ob protein expressed

by said host cell. In a variation of this method, tissue poly-A⁺ RNA, preferably adipose tissue poly-A⁺ RNA, is used in place of tissue total RNA or adipose tissue

total RNA.

Another method of producing an ob dimer comprises the steps of (a)

culturing a transformed host cell containing a DNA sequence encoding a vertebrate ob protein, preferably a human ob protein, under conditions that favor the production of said vertebrate ob protein as a dimer, and (b) isolating said ob dimer expressed by

said transformed host cell.

In any of the above methods of ob dimer production, any portions of the

transformed host cell isolate that may be found to contain ob monomer may

optionally be converted to ob dimer as described herein.

Still another method of producing an ob dimer comprises the steps of (a)

culturing a transformed host cell containing a DNA sequence encoding a vertebrate ob protein, preferably a human ob protein, under conditions that favor the production

of said vertebrate ob protein as a monomer, (b) isolating said ob protein expressed

by said transformed host cell, and (c) dimerizing said ob protein by subsequent

chemical transformation.

In another aspect, the invention relates to the production of ob dimer fusion proteins using recombinant methods. One such method for the production of an ob

dimer fusion protein comprises the steps of (a) preparing a vertebrate cDNA library,

preferably a vertebrate adipose cDNA library, and more preferably a human adipose

cDNA library, (b) ligating said cDNA library into a cloning vector, (c) introducing

said cloning vector containing said cDNA library into a first host cell, (d) contacting

the cDNA molecules of said first host cell with a solution containing a suitable ob gene hybridization probe, (e) detecting a cDNA molecule which hybridizes to said

probe, (f) isolating said cDNA molecule, (g) ligating the nucleic acid sequence of said cDNA molecule which encodes an ob protein to a second DNA sequence,

preferably a marker DNA sequence encoding the FLAG peptide, to create a fusion

DNA sequence, (h) ligating said fusion DNA sequence into an expression vector, (i)

transforming a second host cell with said expression vector containing said fusion DNA sequence, (j) culturing the transformed second host cell under conditions that

favor the production of said ob fusion protein as a dimer, and (k) isolating said ob

protein expressed by said second host cell.

Ob dimer fusion proteins may also be prepared by recombinant methods

which do not include the steps of making and screening a cDNA library or libraries, but use instead a technique involving the amplification of cDNA prepared from, for

example, tissue total RNA, preferably adipose tissue total RNA. Such a method

comprises the steps of (a) isolating a preparation of total RNA from a vertebrate

tissue, preferably adipose tissue, (b) converting said isolated RNA to cDNA, (c) amplifying a cDNA sequence from said cDNA using oligonucleotide primers suitable for annealing to a target ob protein gene sequence, (d) detecting a cDNA

molecule using oligonucleotides suitable for hybridization to said target ob protein

gene sequence, (e) isolating said cDNA molecule, (f) ligating the nucleic acid

sequence of said cDNA molecule which encodes an ob protein to a second DNA

sequence to create a fusion DNA sequence encoding an ob fusion protein, (g)

ligating said fusion DNA sequence into an expression vector, (h) transforming a host

cell with said expression vector containing said fusion DNA sequence, (i) culturing

the transformed host cell under conditions that favor the production of said ob fusion protein as a dimer, and (j) isolating said ob dimer fusion protein expressed by said host cell. In a variation of this method, tissue poly-A⁺ RNA, preferably adipose

tissue poly-A⁺ RNA, is used in place of tissue total RNA or adipose tissue total

RNA.

Another method of producing a recombinant ob dimer fusion protein

comprises the steps of (a) culturing a transformed host cell containing a DNA sequence encoding a vertebrate ob protein, preferably a human ob protein, coupled

to a marker DNA sequence, preferably a marker DNA sequence encoding the FLAG

peptide, under conditions that favor the production of said vertebrate ob fusion protein as a dimer, and (b) isolating said ob dimer fusion protein expressed by said

transformed host cell.

In any of the above methods of ob dimer fusion protein production, any portions of the transformed host cell isolate that may be found to contain ob fusion

monomer may optionally be converted to ob dimer fusion protein as described

herein. Still another method of producing a recombinant ob dimer fusion protein

comprises the steps of (a) culturing a transformed host cell containing a DNA

sequence encoding a vertebrate ob protein, preferably a human ob protein, coupled to a marker DNA sequence, preferably a marker DNA sequence encoding the FLAG

peptide, under conditions that favor the production of said vertebrate ob fusion

protein as a monomer, (b) isolating said ob fusion protein expressed by said

transformed host cell, and (c) dimerizing said ob fusion protein.

In yet another aspect, the invention relates to improved methods for the

periplasmic expression and purification of ob dimer protein, ob dimer fusion protein,

ob monomer protein, and ob fusion monomer protein, which provide increased

protein yield and quality. These methods include the use of a T7 promoter vector

construct transfected into E. coli BL21(DE3) cells which are grown at about 25° to

about 30°C in media containing a supplemental carbon source, preferably glucose,

for enhanced expression. Preferred purification methods include the use of an

osmotic shock protocol which incorporates one or more specific protease inhibitors, preferably Peflabloc SC, followed by the addition of BisTris-propane, or buffers of a

similar nature, and separation using a cellulose-based anion exchange

chromatography resin, preferably DE-52 resin. Further purification may be

undertaken using high pressure liquid chromatography, preferably reversed phase

high pressure liquid chromatography.

We have also invented improved methods for the intracellular expression

(into inclusion bodies) of recombinant ob proteins, and their subsequent

solubilization, refolding and purification, which provide greatly increased protein

yield in E. coli. In this method, certain naturally-occurring nucleotides within the codons for Val²² and Pro²³ in the coding sequences for any of the mammalian ob

proteins, including human, rat and mouse, are replaced. Solubilization, refolding and purification of the recombinant proteins are accomplished by lysing the cells and

washing inclusion bodies in an anionic buffer of approximately neutral pH,

preferably lOOmM phosphate at a pH of about 6.5, dissolving the inclusion bodies in

a buffer containing a chaotropic agent, such as urea in ammonium bicarbonate

buffer, transferring the protein by dialysis or dilution into BisTris-propane or a

similar buffer, and purifying the protein using a cellulose-based anion exchange

chromatography resin, preferably DE-52 resin. The invention also provides for the preparation of ob monomer protein or ob monomer fusion protein without unwanted dimer formation by the addition of agents such as glutathione and dithiothreitol to

any or all of the above-noted buffers. Further purification may be undertaken using

high pressure liquid chromatography, preferably reversed phase high pressure liquid

chromatography. 6 31526 PC17US96/04909

In still another aspect, the invention provides for the chemical synthesis,

using solid phase peptide synthesis or a combination of both solid phase peptide

synthesis and solution chemistries, as a further method of preparation of the ob

proteins of the invention. Following chemical synthesis and isolation of the resulting product, for example, by high pressure liquid chromatography, cation

and/or anion exchange chromatography methods, dimerization may be achieved by

incubation in an appropriate buffer at a dimer-formation-enhancing pH, preferably a

pH of from 7 to 9, or by differential protection and selective deprotection of the

cysteine residues. In a still further aspect, the invention provides ob dimer and ob

monomer compounds and pharmaceutical compositions, and methods for the

treatment of disorders and conditions which may benefit from the administration of

such compounds and compositions, including obesity and diabetes, particularly Type

2 diabetes. Such pharmaceutical compositions include therapeutically effective amounts of an ob dimer or ob dimer fusion protein or ob monomer protein or ob

monomer fusion protein in pharmaceutically acceptable carriers. Ob dimers and ob

dimer fusion proteins include human ob dimers and human ob dimer fusion proteins,

rat ob dimers and rat ob dimer fusion proteins, mouse ob dimers and mouse ob dimer fusion proteins, as well as other vertebrate ob dimers and vertebrate ob dimer fusion

proteins. Ob monomer proteins and ob monomer fusion proteins include human ob

monomer and ob monomer fusion proteins, rat ob monomer and ob monomer fusion

proteins, mouse ob monomer and ob monomer fusion proteins, as well as other

vertebrate ob monomer and ob monomer fusion proteins. Preferably, these ob dimer

and ob monomer compounds are prepared in a stable lyophilized form as trifluoroacetate, acetate, hydrochloride, or ammonium bicarbonate salts, most

preferably ammonium bicarbonate salts. Preferred stable solutions of these ob dimer and ob monomer compounds are prepared using BisTris-propane, or buffers of

similar structure, with or without a detergent, such as Tween 80, to have a pH from

about 7.5 to about 9, and most preferably a pH of about 8.5.

Treatment methods comprise the administration of a therapeutically effective amount of a pharmaceutical composition comprising an ob dimer and/or ob

monomer compound of the invention to patients in need thereof. Such patients

include obese and diabetic patients (particularly Type 2 diabetic subjects), and others

whose condition would benefit from administration of an ob dimer or ob monomer

protein in an amount useful to promote reduced food intake or increased energy

expenditure or both.

Pharmaceutical ob dimer compositions and ob monomer protein

compositions may be administered separately or together with other compounds and

compositions that exhibit a short-term satiety action including but not limited to other compounds and compositions that comprise an amylin or an amylin agonist.

Suitable amylins include, for example, human amylin and rat amylin. Suitable amylin agonists include, for example, [Pro^-^'J-human amylin and salmon

calcitonin. In still another aspect, the present invention provides novel antibodies,

including polyclonal antibodies, preferably monoclonal antibodies, and antibody fragments which can be produced in mice or by recombinant cell lines or by hybrid

cell lines, the antibodies being characterized in that they have certain predetermined

specificity to ob dimers, ob dimer fusion proteins, and ob monomer fusion proteins over their corresponding ob monomers. By virtue of their specificity, such

antibodies and antibody fragments are useful in methods for the purification of ob

dimers, ob dimer fusion proteins, and ob monomer fusion proteins, and in the

immunoassay of these target antigens.

Definitions

The term "amino acid" refers to the natural L-amino acids. Natural L-amino

acids include alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gin or Q), glutamic acid (Glu or E),

glycine (Gly or G), histidine (His or H), isoleucine (lie or I), leucine (Leu or L),

lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or

P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or

Y), and valine (Val or V).

The term "peptide" refers to a sequence of amino acids linked predominantly

though not necessarily exclusively through their alpha-amino and carboxylate

groups by peptide bonds. Such sequences as shown herein are presented in the

amino (N terminus) to carboxy (C terminus) direction, from left to right.

The term "protein" refers to a molecule comprised of one or more peptides.

The term "cDNA" refers to complementary deoxyribonucleic acid.

The term "nucleic acid" refers to polymers in which bases (e.g., purines or

pyrimidines) are attached to a sugar phosphate backbone. Nucleic acids include

DNA and RNA.

The term "mRNA" refers to messenger ribonucleic acid. 6/31526 PC17US96/04909

The term "nucleic acid sequence" refers to the sequence of nucleosides

comprising a nucleic acid. Such sequences as shown herein are presented in the 5' to

3' direction, from left to right.

The term "recombinant" refers to a DNA molecule comprising pieces of DNA that are not normally contiguous, or to a protein expressed therefrom.

Brief Description of the Drawings

FIGURE 1 shows the nucleotide and deduced amino acid sequences of the

rat ob gene. FIGURE 2 shows the change in food intake in ob/ob mice administered

various doses of rat ob protein.

FIGURE 3 shows the change in food intake in ob/ob mice administered

various doses of rat ob protein and two doses of Met-rat ob protein.

FIGURE 4 shows the change in food intake in ob/ob mice administered

various doses of rat ob protein and three doses of Met-rat ob dimer protein.

FIGURE 5 shows the change in food intake in ob/ob mice administered

various doses of rat ob protein and two doses of FLAG-rat ob protein.

FIGURE 6 shows the change in food intake in ob/ob mice administered

various doses of rat ob protein and two doses of FLAG-rat ob dimer protein. FIGURE 7 shows the change in food intake in NIH/Sw mice administered

various doses of rat ob protein.

FIGURE 8 shows the change in body weight in ob/ob mice administered

various doses of rat ob protein. FIGURE 9 shows the change in body weight in ob/ob mice administered various doses of rat ob protein and two doses of Met-rat ob protein.

FIGURE 10 shows the change in body weight in ob/ob mice administered

various doses of rat ob protein and three doses of Met-rat ob dimer protein.

FIGURE 11 shows the change in body weight in ob/ob mice administered various doses of rat ob protein and two doses of FLAG-rat ob protein.

FIGURE 12 shows the change in body weight in ob/ob mice administered

various doses of rat ob protein and two doses of FLAG-rat ob dimer protein.

FIGURE 13 shows the change in body weight in NIH/Sw mice administered

various doses of rat ob protein.

Detailed Description of the Invention

The present invention is directed to dimeric forms of the ob gene product, including ob dimers and ob dimer fusion proteins, as well as to ob monomer fusion

proteins, and to the manufacture and use of such compounds. The invention is also

directed to modified monomeric ob protein DNA sequences, to processes for the

recombinant expression and purification of ob dimer proteins and ob monomer

proteins in high yield, to formulations of these ob dimers and ob monomer proteins, and to the use of these ob proteins and compositions in the treatment of subjects with

disorders or conditions that would benefit from administration of these proteins and

compositions, including but not limited to those subjects who would benefit from

reduced food intake or increased energy expenditure or both.

Ob dimers include dimers of the ob gene product from any vertebrate source,

including but not limited to human, mouse, and rat. An example of a rat ob gene sequence useful in preparing an ob dimer or other ob product is described herein and

depicted in Figure 1. An especially preferred rat ob DNA sequence for use in the

preparation of the compounds and compositions of the invention and which is described and claimed herein includes GTT as the codon for Val²² (in place of the naturally-occurring GTG codon) and CCG as the codon for Pro²³ (in place of the

naturally-occurring CCT codon).

Examples of human and mouse ob genes useful in preparing these dimers

and other ob products of the invention are found in Zhang et al.. Positional cloning of the mouse obese gene and its human homologue. (1994) Nature 372: 425-432.

This article, and all other publications referenced herein, are hereby incorporated in

their entirety by reference. An especially preferred human ob DNA sequence for use

in the preparation of the compounds and compositions of the invention and which is described and claimed herein includes GTT as the codon for Val²² (in place of the naturally-occurring GTG codon) and CCG as the codon for Pro²³ (in place of the

naturally-occurring CCC codon). Optionally, the human ob DNA sequence also

contains CAG as the codon for Gin²⁵ (in place of the naturally-occurring codon

CAA). An especially preferred mouse ob DNA sequence includes GTT as the codon for Val²² (in place of the naturally-occurring GTG codon) and CCG as the codon for

Pro²³ (in place of the naturally-occurring CCT codon). Other vertebrate ob sequences may be isolated in accordance with the methods described herein for use

in the preparation of their corresponding dimers, dimer fusion proteins, monomers

and monomer fusion proteins.

The ob dimers and ob dimer fusion proteins of the present invention, as well as their corresponding ob monomers and ob fusion monomer proteins, include those having variations in a known or disclosed ob gene sequence or sequences, including fragments, naturally occurring mutations, allelic variants, randomly generated

artificial mutants and intentional sequence variations (including proteins having an

N-terminal methionine residue), and corresponding protein alterations, all of which

conserve relevant ob protein activity, for example, anti-feeding activity. An

example of such a variation is the microheterogeneity of the cDNA in the human and

mouse ob dimer gene sequences where about 70% of the cDNAs have a glutamine

codon at position 49 and about 30 % do not. The term "fragments" refers to any part

of the ob sequence which contains fewer amino acids than the complete protein, as

for example, partial sequences excluding portions at the amino-terminus, carboxy- terminus or between the amino-terminus and carboxy-terminus of the complete

protein. Examples of such fragments includes amino terminal fragmentation to

eliminate the first five amino acids VPIHK of the rat ob gene used to create an ob

monomer fragment or ob monomer fusion fragment and corresponding ob dimers or ob dimer fusion proteins, amino terminal fragmentation to eliminate the first five

amino acids VPIQK of the human ob gene used to create an ob monomer fragment or ob monomer fusion fragment and corresponding ob dimers or ob dimer fusion

proteins, and amino terminal fragmentation to eliminate the first five amino acids

VPIQK of the mouse ob gene used to create an ob monomer fragment or ob

monomer fusion fragment and corresponding ob dimers or ob dimer fusion proteins.

Other examples of such fragments includes amino terminal fragmentation to

eliminate the first twenty-nine amino acids of the rat ob gene used to create an ob

monomer fragment or ob monomer fusion fragment starting with the sequence

VSARQ and corresponding ob dimers or ob dimer fusion proteins, amino terminal fragmentation to eliminate the first twenty-nine amino acids of the human ob gene

used to create an ob monomer fragment or ob monomer fusion fragment starting

with the sequence VSSKQ and corresponding ob dimers or ob dimer fusion proteins,

and amino terminal fragmentation to eliminate the first twenty-nine amino acids of the mouse ob gene used to create an ob monomer fragment or ob monomer fusion

fragment starting with the sequence VSAKQ and corresponding ob dimers or ob

dimer fusion proteins. Variations in the products of the invention also include

insertion of non-peptide bonds in the peptide backbone known in the art not to

influence biological activity.

The invention also includes other modified ob dimers which conserve

relevant activity, for example the anti-feeding activity, of the ob dimer. These are

typically recombinant proteins that include hybrid proteins, such as fusion proteins, proteins resulting from the expression of multiple genes within the expression vector, proteins resulting from expression of multiple genes within the chromosome

of the host cell, and may include a protein having relevant activity of a disclosed

protein, for example anti-feeding activity, linked by peptide bonds to a second

protein or peptide. For example, the present invention provides for the manufacture and use of dimeric and monomeric forms of the ob gene product that are active in

vivo notwithstanding that they have fused to them certain marker peptides (such as

poly-histidine, or the eight amino acid FLAG peptide described below) that are

useful in the isolation and purification of both the dimeric and monomeric forms of a

recombinant ob gene product. In addition to these fusion proteins which include marker or reporter peptides of various lengths, other fusion proteins of the invention

include an ob protein having an amino acid sequence added to the N-terminus, for example, to the N-terminus of a mouse, rat or human ob protein or protein fragment.

This sequence may be linked to the ob protein by a peptide bond, a peptide bond

surrogate or an appropriate chemical linker. More specifically, a sequence of amino acids between 1 and 200 residues long, preferably between 5 and 50 residues long,

having a balance of hydrophilicity and hydrophobicity such that the overall solution

and other physico-chemical properties of the fusion protein remain roughly within the

range defined by the physicochemical properties of vertebrate ob proteins and

protein fragments, such as mouse, rat and human ob proteins and protein fragments.

Such sequences would result in a range for the pi value of the resulting fusion

protein of between 4 and 8. N-terminal and C-terminal fusion sequences include,

but are are not limited to, the following: amylins, including amylin analogues (for example, [Pro^25,28,20]human amylin); calcitonins, including salmon calcitonin;

cholecystokinins, including CCK-8; ceruletide; bombesin; enterogastrone; somatostatin; thyrotropin releasing hormone; segments of neuropeptide-Y which

possess neuropeptide-Y antagonist properties; neurotensin; α-MSH; β-endorphins;

glucagon; GLP-1; insulin; insulin like growth factor; TNFs; interleukins; apolipoprotein A-IV; and, peptide sequences which are known to target particular

cells or tissues (R. Pasqualini and E. Ruoslahti, Nature, 380:364 (1996)). Fusion ob

monomers and dimers and fragments thereof which include an N-terminal amylin,

an amylin analogue, a cholecystokinin, or a calcitonin are presently preferred.

The ob dimers and ob dimer fusion proteins of the present invention, as well

as their corresponding ob monomers and ob fusion monomer proteins, also include

variants of the ob domain amino acid sequence or sequences that differ only by

conservative amino acid substitution and conserve the anti-feeding activity of the isolated ob dimers or ob dimer fusion proteins. Conservative amino acid

substitutions are defined as "sets" in Table 1 of Taylor, W.R., (1986) J. Mol. Biol,

188: 233.

It will be appreciated by those in the art that there are various methods useful

in preparing and isolating ob dimers and ob dimer fusion proteins (as well as their corresponding ob monomer and ob monomer fusion proteins). In general,

recombinant DNA isolation techniques are now well known. An extensive

discussion embodying a number of commonly used methodologies can be found in

Sambrook et al.. Molecular Cloning, A Laboratory Manual, Second Edition, Volumes 1 to 3, Cold Spring Harbor Laboratory Press 1989). Recombinant methods allow segments of genetic information, DNA, from different organisms, to be joined

together outside of the organisms from which the DNA was obtained and this hybrid

DNA to be incorporated into a cell that will allow the production of the protein for which the original DNA encodes. Genetic information encoding a protein of the

present invention may be obtained from genomic DNA, mRNA (preferably adipose

tissue mRNA), or tissue total RNA (preferably adipose tissue total RNA) of an

organism by methods well known in the art. Preferred methods of obtaining this

genetic information include isolating mRNA from an organism, converting it to its

complementary DNA, incorporating the cDNA into an appropriate cloning vector,

and identifying the clone which contains the cDNA encoding the desired protein by

means of hybridization with appropriate oligonucleotide probes constructed from

known or postulated sequences of the protein. Especially preferred methods of

obtaining this genetic information include isolating tissue total RNA, preferably adipose tissue total RNA, from an organism, converting it to its complementary DNA, and amplifying, detecting and isolating a cDNA sequence encoding the

desired protein. The genetic information in the cDNA encoding a protein of the

present invention may be ligated into an expression vector, the vector introduced into host cells, and the genetic information expressed as the protein encoded for.

Thus, nucleic acid encoding the proteins of the invention may be cloned by

incorporating a DNA fragment coding for an ob protein or ob fusion protein in a

recombinant DNA vehicle, typically, for example, mammalian, bacterial or viral

vectors, and transforming a suitable host, for example, an E. coli cell line and

isolating clones incorporating the recombinant vectors. Such clones may be grown

and used to produce the desired ob dimer or ob dimer fusion protein or ob monomer

fusion protein.

Mixtures of mRNA can be isolated from eukaryotic cells and double-

stranded DNA copies of entire genes synthesized which are complementary to the isolated mRNA. mRNA is first reverse-transcribed to form a single-stranded cDNA

by an RNA-directed DNA polymerase, e.g., reverse transcriptase. Reverse

transcriptase synthesizes DNA in the 5' to 3¹ direction, utilizes deoxyribonucleoside

5'-triphosphates as precursors, and requires both a template and a primer strand. By

a series of additional reactions, double-stranded cDNA is produced and inserted into cloning or expression vectors by any one of many known techniques, which depend

at least in part on the vector selected. Expression vectors refer to vectors which are

capable of transcribing and translating DNA sequences contained therein, where

such sequences are linked to other regulatory sequences capable of effecting their expression. These expression vectors are replicable in the host organisms or systems

as either plasmids, bacteriophage, or as an integral part of the chromosomal DNA. Recombinant vectors and methodology are in general well known and suitable for

use in host cells over a wide range of prokaryotic and eukaryotic organisms. The

cDNA cloning and expression procedures further described below and in the

Examples are but some of a wide variety of well established methods to produce specific sequences and reagents useful in the invention.

One method of preparing the products of the invention includes the steps of

constructing a vertebrate cDNA library, preferably a vertebrate adipose cDNA

library; ligating the cDNA library into a cloning vector; introducing the cloning

vector containing the cDNA library into a first host cell; contacting the cDNA molecules of the first host cell with a solution containing a suitable ob gene hybridization probe; detecting and then isolating a cDNA molecule which hybridizes

to the ob gene hybridization probe and encodes the desired ob protein; ligating the hybridizing cDNA molecule into an expression vector; transforming a second host

cell with the expression vector containing the cDNA molecule which encodes the

desired ob protein; culturing the transformed second host cell under conditions that

favor the production of the ob protein as a dimer; and, isolating the ob protein

expressed by the second host cell.

The products of the invention may also be prepared by methods that do not

require the construction and screening of a cDNA library. Such a method which

represents one embodiment for the production of a rat ob fusion dimer is described

in Examples 1, 2 and 4-7. Examples 1 and 2 describe methods used to isolate and

clone the rat ob gene from rat adipose tissue total RNA using an RT-PCR

amplification technique. Examples 4-7, 9-19, 22, 24 and 25 describe methods useful

for the expression and isolation of the ob gene products in E. coli. The ob dimers, ob dimer fusion proteins and ob monomer fusion proteins of the invention can be

isolated by various methods or combinations of methods of protein purification as

disclosed below. These purification methods are also useful for the isolation of unfused ob monomer proteins.

Preferred natural sources of mRNA from which to construct a cDNA library

are vertebrate adipose tissue, for example, epididymal adipose tissue. Preferred

methods of isolating mRNA encoding a protein of the present invention, along with

other mRNA, from an mRNA source include poly U or poly dT chromatography.

Other methods for RNA extraction, including the acid guanidinium thiocyanate

procedure used in Example 1, which details the extraction of rat adipose tissue total RNA and the preparation of oligonucleotide primers for use in the isolation and

cloning of the rat ob gene, are known in the art.

Preferred methods of obtaining double-stranded cDNA from isolated mRNA

include synthesizing a single-stranded cDNA on the mRNA template using a reverse

transcriptase, degrading the RNA hybridized to the cDNA strand using a

ribonuclease (RNase), and synthesizing a complementary DNA strand by using a

DNA polymerase to give a double-stranded cDNA. Especially preferred methods of

preparing cDNA include those described in Example 2 wherein total RNA isolated

from vertebrate adipose tissue is converted into single-stranded cDNA using Murine

Leukemia Virus Reverse Transcriptase and RNase inhibitor, followed by a PCR

procedure to amplify the target cDNA, yielding double-stranded cDNA.

cDNA encoding a protein of the present invention, along with the other

cDNA if a library is constructed as above, are then ligated into cloning vectors. Cloning vectors include a DNA sequence which accommodates the cDNA. The vectors containing the amplified cDNA or cDNA library are introduced into host cells that can exist in a stable manner and provide an environment in which the

cloning vector is replicated. Suitable cloning vectors include plasmids,

bacteriophages, viruses and cosmids. Preferred cloning vectors include plasmids.

Cloning vectors which are especially preferred in the isolation methods described

herein for the preparation of RT-PCR products from total adipose tissue RNA

include the plasmid pAMP 1.

The construction of suitable cloning vectors containing cDNA and control

sequences employs standard ligation and restriction techniques which are well

known in the art. Isolated plasmids, DNA sequences or synthesized oligonucleotides are cleaved, tailored and religated in the form desired. With respect

to restriction techniques, site-specific cleavage of cDNA is performed by treating with suitable restriction enzyme under conditions which are generally understood in

the art, and particulars of which are specified by the manufacturers of these

commercially available restriction enzymes. See, e.g., the product catalogs of New

England Biolabs, Promega, and Stratagene Cloning Systems.

Cloning vectors containing the desired cDNA are introduced into host cells

and cultured. Cloning vectors containing a cDNA library prepared as disclosed are

introduced into host cells, the host cells are cultured, plated, and then probed with a hybridization probe to identify clones which contain the recombinant cDNA

encoding a protein of the present invention. Preferred host cells include bacteria

when plasmid cloning vectors are used. Especially preferred host cells include E.

coli strains such as E. coli DH5αMCR competent cells. 6/31526 PCI7US96/04909

Hybridization probes and primers are oligonucleotide sequences which are

complementary to all or part of the cDNA molecule that is desired. They may be

prepared using any suitable method, for example, the phosphotriester and

phosphodiester methods, described respectively in Narang et al.. Methods in Enzymology, 68: 90 (1979) and Brown et al.. Methods in Enzymology, 68: 109

(1979), or automated embodiments thereof. In one such embodiment,

diethylphosphoramidites are used as starting materials and may be synthesized as

described by Beaucage et al.. Tetrahedron Letters, 22: 1859-1862 (1981). One

method for synthesizing oligonucleotides on a modified solid support is described in

U.S. Patent No. 4,458,066. Probes differ from primers in that they are labeled with an enzyme, such as horseradish peroxidase, or with a radioactive atom, such as ³²P,

to facilitate their detection. A synthesized probe is radio-labeled by nick translation

using E. coli DNA polymerase I or by end labeling using alkaline phosphatase and

T4 bacteriophage polynucleotide kinase.

Useful hybridization probes and amplification primers include

oligonucleotide sequences which are complementary to a stretch of the cDNA

encoding a portion of the amino acid sequence of an ob protein, for example, a

portion of the amino acid sequence shown in Figure 1. Especially preferred as

hybridization probes are oligonucleotide sequences encoding substantially all of the amino acid sequence of rat, mouse, or human ob protein. Other appropriate probes

for isolation of vertebrate ob genes will be apparent to those skilled in the art.

Especially preferred as amplification primers are pairs of oligonucleotide sequences

that flank substantially all of the DNA sequence encoding a vertebrate ob protein, for example, those encoding rat, mouse, or human ob protein. A preferred cDNA molecule encoding a vertebrate protein of the present invention can be identified by screening or amplification methods through its ability to hybridize to these probes or

primers.

Upon identification of the clone containing the desired cDNA, whether by an

RT-PCR procedure or through cDNA library screening, for example, amplification

may be used to produce large quantities of a gene encoding a protein of the present

invention in the form of a recombinant cDNA molecule. Preferred methods of

amplification include the use of the polymerase chain reaction (PCR). See, e.g.,

PCR Technology, W.H. Freeman and Company, New York (Edit. Erlich, H.A.

1992). PCR is an in vitro amplification method for the synthesis of specific DNA sequences. In PCR, two oligonucleotide primers that hybridize to opposite strands

and flank the region of interest in the cDNA of the clone are used. A repetitive series of cycles involving cDNA denaturation into single strands, primer annealing

to the single-stranded cDNA, and the extension of the annealed primers by DNA

polymerase results in numbers of copies of cDNA, whose termini are defined by the

5¹ ends of the primers, approximately doubling at every cycle. Through PCR amplification, the coding domain and any additional primer encoded information

such as restriction sites or translational signals (signal sequences, start and/or stop

codons) of the recombinant cDNA molecule to be isolated is obtained. Preferred

conditions for amplification of cDNA are found in manufacturer protocols, and may

be accomplished manually or by automated thermocycling devices. An example of a cDNA prepared in this fashion is that having the nucleic acid sequence of Figure 1.

The cDNA molecules of the present invention when isolated as described are

used to obtain expression of the ob dimers and ob dimer fusion proteins described and claimed herein, as well as their corresponding ob fusion monomer proteins.

Generally, a recombinant cDNA molecule of the present invention is incorporated into an expression vector, this expression vector is introduced into an appropriate

host cell, the host cell is cultured, and the expressed protein is isolated. Various

methods for ob dimer, ob monomer fusion protein and ob dimer fusion protein

expression are described in Examples 3, 4, 9, 10, 12, 13, 15, 16, and 22.

Expression vectors are DNA sequences that are required for the transcription

of cloned copies of genes and translation of their mRNAs in an appropriate host.

These vectors can express either procaryotic or eucaryotic genes in a variety of cells such as bacteria, yeast, mammalian, plant and insect cells. Proteins may also be expressed in a number of virus systems.

Suitably constructed expression vectors contain an origin of replication for autonomous replication in host cells, or are capable of integrating into the host cell

chromosomes. Such vectors will also contain selective markers, a limited number of

useful restriction enzyme sites, a high copy number, and strong promoters.

Promoters are DNA sequences that direct RNA polymerase to bind to DNA and

initiate RNA synthesis; strong promoters cause such initiation at high frequency.

The preferred expression vectors of the present invention are operatively linked to a

cDNA or recombinant cDNA of the present invention, i.e., the vectors are capable of directing both replication of the attached cDNA or recombinant cDNA molecule and

expression of the protein encoded by the cDNA or recombinant cDNA molecule. Expression vectors may include, but are not limited to cloning vectors, modified

cloning vectors and specifically designed plasmids or viruses. With each type of host cell certain expression vectors are preferred, as described below. Procaryotes may be used and are presently preferred for expression of the ob

dimers, ob monomer fusion proteins and ob dimer fusion proteins of the present

invention. Suitable bacteria host cells include the various strains of E. coli, Bacillus

subtilis, and various species of Pseudomonas. In these systems, plasmid vectors

which contain replication sites and control sequences derived from species

compatible with the host are used. Suitable vectors for E. coli are derivatives of

pBR322, a plasmid derived from and E. coli species by Bolivar et al.. Gene, 2: 95 (1977). Common procaryotic control sequences, which are defined herein to include

promoters for transcription, initiation, optionally with an operator, along with

ribosome binding site sequences, include the beta-lactamase and lactose promoter (Chang et al.. Nature, 198: 1056 (1977)), the tryptophan promoter system (Goeddel et al.. Nucleic Acids Res., 8: 4057 (1980)) and the lambda-derived ? promoter and

N-gene ribosome binding site (Shimatake et al.. Nature, 292: 128 (1981)).

However, any available promoter system compatible with procaryotes can be used.

Preferred procaryote expression systems include E. coli and their expression vectors,

such as E. coli strains W3110 and JM105, with suitable vectors, as described in

Example 5. Especially preferred is the use of E. coli strain BL21(DE3), with

suitable vectors, as described in Examples 9, 12, 15, 22 and 24.

Eucaryotes may be used for expression of the proteins of the present

invention. Eucaryotes are usually represented by the yeast and mammalian cells. Suitable yeast host cells include Saccharomyces cerevisiae and Pichia pastoris.

Suitable mammalian host cells include COS and CHO (Chinese Hamster Ovary) cells. Expression vectors for the eucaryotes are comprised of promoters derived

from appropriate eucaryotic genes. Suitable promoters for yeast cell expression vectors include promoters for synthesis of glycolytic enzymes, including those for the 3-phosphoglycerate kinase gene in Saccharomyces cerevisiae (Hitzman et al.. J.

Biol. Chem., 255: 2073 (1980)) and those for the metabolism of methanol such as

the alcohol oxidase gene in Pichia pastoris (Stroman et al.. U.S. Patent Nos.

4,808,537 and 4,855,231). Other suitable promoters include those from the enolase

gene (Holland et_al., J. Biol. Chem., 256: 1385 (1981)) or the Leu2 gene obtained

from YEpl3 (Broach et al., Gene, 8: 121 (1978)).

Suitable promoters for mammalian cell expression vectors include the early

and late promoters from SV40 (Fiers et al.. Nature, 273: 113 (1978)) or other viral

promoters such as those derived from polyoma, adenovirus II, bovine papilloma virus or avian sarcoma viruses. Suitable viral and mammalian enhancers may also

be incorporated into these expression vectors.

Suitable promoters for plant cell expression vectors include the nopaline

synthesis promoter described by Depicker et al.. Mol. Appl. Gen., 1: 561 (1978).

Suitable promoters for insect cell expression vectors include modified versions of

the system described by Smith et al.. U.S. Patent No. 4,475,051. The expression

vector comprises a baculovirus polyhedrin promoter under whose control a cDNA

molecule encoding a protein can be placed.

Another method of producing an ob dimer comprises the steps of culturing a

transformed host cell containing a DNA sequence encoding a vertebrate ob protein,

preferably a human ob protein, under conditions that favor the production of said vertebrate ob protein as a dimer, and isolating the ob dimer expressed by the

transformed host cell. Such a method is exemplified for the rat ob protein in

Examples 5-7. Still another method of producing a recombinant ob dimer comprises the steps of culturing a transformed host cell containing a DNA sequence encoding a

vertebrate ob protein, preferably a human ob protein, under conditions that favor the

production of said vertebrate ob protein as a monomer, isolating the ob protein

expressed by the transformed host cell, and dimerizing the ob protein.

Dimerization may be achieved by initially treating ob monomer protein with

a reducing agent such as mercaptoethanol or dithiothreitol in an appropriate buffer at

pH 6-9. It is assumed that some refolding is required prior to dimerization and so

the whole is diluted by 5-10 fold, or dialyzed with an appropriate buffer at pH 6-9,

and allowed to equilibrate at 4°C. Ammonium bicarbonate buffer is preferred. Incubation and air oxidation then yields dimeric protein. Other methods of

oxidation typically used are O₂/copper, mercury salts, etc. Folding aids can also be

used, including other proteins such as albumins, chaperones, monoclonal antibodies,

and soluble receptors, along with a variety of chromatography supports and plastic

surfaces. Data also indicate, by way of example, that monomeric ΨLAG-ob protein

can be converted to dimeric FLAG-oδ protein by incubation in Tris buffer at pH 7-9.

Ob dimer fusion proteins may be produced using similar methods. One such

method for the recombinant production of an ob dimer fusion protein comprises the

steps of constructing a vertebrate cDNA library, preferably a vertebrate adipose

cDNA library, and more preferably a human adipose cDNA library; ligating the cDNA library into a cloning vector; introducing the cloning vector containing the

cDNA library into a first host cell; contacting the cDNA molecules of the first host

cell with a solution containing a suitable ob gene hybridization probe; detecting a

recombinant cDNA molecule which hybridizes to the probe; isolating the

recombinant cDNA molecule; ligating the nucleic acid sequence of the cDNA molecule which encodes an ob protein to a marker DNA sequence to create a fusion

DNA sequence; ligating the fusion DNA sequence into an expression vector;

transforming a second host cell with the expression vector containing the fusion

DNA sequence; culturing the transformed second host cell under conditions that favor the production of the ob fusion protein as a dimer; and, isolating the ob protein

expressed by the second host cell. As noted, the use of adipose tissue total RNA,

followed by conversion to cDNA and amplification of the desired ob gene sequence

may be used rather than steps involving the preparation and screening of a cDNA

library or libraries, and is presently preferred.

A preferred peptide for preparation of an ob dimer fusion peptide is the

FLAG peptide. See Hopp et al.. A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification. (1988) Biotechnology, 6: 1205-

1210. FLAG is an octapeptide with the amino acid sequence DYKDDDDK.

Antibodies are available which specifically recognize this sequence, thus allowing

identification (by Western Blotting) and purification (by affinity chromatography) of

proteins containing this sequence. See Prickett et al.. A Calcium Dependent

Antibody for Identification and Purification of Recombinant Proteins. (1989)

BioTechniques, 7: 580-589. In addition, the sequence may be specifically removed

with enterokinase if placed at the N-terminus of the desired peptide. Thus, this

particular reporter peptide allows identification, purification and liberation of a given protein to which it is attached. Additionally, cloning into the pFLAG-ATS vector

(Scientific Imaging Systems, Eastman Kodak Company) allows periplasmic expression of an N-terminally tagged FLAG fusion protein in bacteria. Another method of producing an ob dimer fusion protein comprises the steps

of culturing a transformed host cell containing a DNA sequence encoding a

vertebrate ob protein, preferably a human ob protein, coupled to a marker or other

fusion DNA sequence under conditions that favor the production of said vertebrate ob fusion protein as a dimer, and isolating the ob dimer fusion protein expressed by

the transformed host cell. Still another method of producing an ob dimer fusion protein comprises the steps of culturing a transformed host cell containing a DNA

sequence encoding a vertebrate ob protein, preferably a human ob protein, coupled to a marker DNA sequence under conditions that favor the production of said

vertebrate ob fusion protein as a monomer, isolating the ob fusion protein expressed

by the transformed host cell, and dimerizing the ob fusion protein, as described

above.

Intracellular and periplasmic expression using E coli are preferred for ob

protein expression. A number of recombinant production methods are described by

contributors to Protein Purification - Micro to Macro. R. Burgess ed., Alan R. Liss,

Inc., New York, 1987, and examples of periplasmic expression of recombinant

proteins are given by H. Lee and P. Troota in Purification and Analysis of Recombinant Proteins. R. Seetharam and S. Sharma ed. Marcel Dekker, Inc., New

York, 1991, p. 163-181. Provided herein are preferred methods for the periplasmic expression and purification of ob dimer protein, ob dimer fusion protein, ob

monomer protein, and ob fusion monomer protein, which provide increased protein

yield and quality. These methods include the use of a T7 promoter vector construct

transfected into E. coli BL21(DE3) cells which are grown at about 25° to about 30°C in media containing a supplemental carbon source, preferably glucose, for enhanced expression. Preferred purification methods include the use of an osmotic shock

protocol which incorporates one or more specific protease inhibitors, preferably

Peflabloc SC, followed by the addition of BisTris-propane, or buffers of a similar

nature, and separation using a cellulose-based anion exchange chromatography resin,

preferably DE-52 resin. Further purification may be undertaken using high pressure

liquid chromatography, preferably reversed phase high pressure liquid

chromatography. Such methods are described in the below Examples.

Intracellular expression can be used to make proteins in E. coli, but the

production process is complicated by the need to the dissolve the inclusion bodies using chaotropic agents and the difficulties inherent in refolding disulfide-bonded

proteins, as discussed in Protein Refolding. G. Georgiou and E. Bernardez-Clark

eds., (1991), American Chemical Society, Washington, DC. Also provided herein

are preferred methods for the intracellular expression (into inclusion bodies) of

recombinant ob proteins, and their subsequent solubilization, refolding and

purification, which provide greatly increased protein yield in E. coli. In this method,

certain naturally-occurring nucleotides within the codons for Val²² and Pro²³ in the

coding sequences for any of the mammalian ob proteins, including human, rat and

mouse (which are not part of the set of deleterious codons AGG/ AGA, CUA, AUA, CGA, or CCC, described by J. Kane, Curr. Opin. Biotechnol. (1995), 6:494-500),

are replaced. An improved human DNA sequence coding for an ob protein which

contains an additional nucleotide change in the codon for Gin²⁵ has also been

invented and is described herein. These changes resulted in improved protein expression compared to a construct without these codon modifications, as described

in Examples 16 and 25. Solubilization, refolding and purification of the recombinant proteins are accomplished by lysing the cells and washing inclusion

bodies in an anionic buffer of approximately neutral pH, preferably lOOmM

phosphate at a pH of about 6.5, dissolving the inclusion bodies in a buffer containing

a chaotropic agent, such as urea in ammonium bicarbonate buffer, transferring the

protein by dialysis or dilution into BisTris-propane or a similar buffer, and purifying

the protein using a cellulose-based anion exchange chromatography resin, preferably

DE-52 resin. The invention also provides for the preparation of ob monomer protein

or ob monomer fusion protein without unwanted dimer formation by the addition of agents such as glutathione and dithiothreitol to any or all of the above-noted buffers.

Further purification may be undertaken using high pressure liquid chromatography, preferably reversed phase high pressure liquid chromatography.

While recombinant DNA methods of production are preferred, chemical

synthesis, using a solid phase synthesis approach or a combination of both solid

phase peptide synthesis and solution chemistries offers a further method of

preparation of ob products of the invention, including ob dimers. Examples of solid phase peptide synthesis include that described by Merrifield, J. Amer. Chem Soc,

85: 2149 (1964), or other equivalent methods known in the chemical arts, such as the method described by Houghten, Proc. Natl. Acad. Sci., 82: 5132 (1985).

Solid phase synthesis is commenced from the C-terminus of the peptide by coupling a protected amino acid or peptide to a suitable insoluble resin. Suitable

resins include those containing chloromethyl, bromomethyl, hydroxymethyl,

aminomethyl, benzhydryl, and t-alkyloxycarbonylhydrazide groups to which the

amino acid can be directly coupled. In one embodiment of this solid phase

synthesis, for example, the carboxy terminal amino acid, having its alpha amino group and, if necessary, its reactive side chain group suitably protected, is first

coupled to the insoluble resin. After removal of the alpha amino protecting group,

such as by treatment with trifluoroacetic acid in a suitable solvent (BOC chemistry)

or piperidine (FMOC chemistry), the next amino acid or peptide, also having its

alpha amino group and, if necessary, any reactive side chain group or groups

suitably protected, is coupled to the free alpha amino group of the amino acid coupled to the resin. Additional suitably protected amino acids or peptides are

coupled in the same manner to the growing peptide chain until the desired amino

acid sequence is achieved. The synthesis may be done manually, by using

automated peptide synthesizers, or by a combination of these.

The coupling of the suitably protected amino acid or peptide to the free alpha

amino group of the resin-bound amino acid can be carried out according to

conventional coupling methods, such as the azide method, mixed anhydride method, DCC (dicyclohexylcarbodiimide) method, activated ester method (p-nitrophenyl

ester or N-hydroxysuccinimide ester), BOP (benzotriazole-1-yl-oxy-tris (diamino) phosphonium hexafluorophosphate) method or Woodward reagent K method.

It is common in peptide synthesis that the protecting groups for the alpha

amino group of the amino acids or peptides coupled to the growing peptide chain

attached to the insoluble resin will be removed under conditions which do not remove the side chain protecting groups. Upon completion of the synthesis, it is

also common that the peptide is removed from the insoluble resin, and during or

after such removal, the side chain protecting groups are removed. Suitable

protecting groups for the alpha amino groups of all amino acids, the omega amino

group of lysine, the carboxy group of aspartic acid and glutamic acid, the guanidino group of arginine, the thiol group of cysteine, the amide group of asparagine and

glutamine, the imidazole group of histidine, the hydroxy group of serine and

threonine, the indole group of tryptophan, and the phenyl hydroxy group of tyrosine

are known in the art. On use of the t-BOC method for protection/deprotection of the growing N-

terminus, the completed peptide may be cleaved from the resin by treatment with

liquid anhydrous hydrogen fluoride in ether containing one or more thio-containing

carbocation scavengers at reduced temperatures. On use of the Fmoc method for protection/deprotection of the growing N-terminus, the completed peptide may be

cleaved from the resin by treatment with trifluoroacetic acid in water containing one or more carbocation scavengers at reduced temperatures. The cleavage of the

peptide from the resin by such treatment in general will also remove all side chain

protecting groups. The cleaved peptide is dissolved in water or

acetonitrile/0.1%TF A/water and purified by conventional high pressure liquid

chromatography techniques, typically on a reverse phase column using

trifluoroacetic acid-containing solvents. The purified peptide is then allowed to refold and establish proper disulfide bond formation by dilution to an appropriate

peptide concentration, for example from about 0.025 mM to about 0.25 mM, in an

appropriate buffer of pH 7-9 and then stirred open to air for about 24 to about 72

hours. In another manner, disulfide bond formation may be performed by using a

protecting group such as Acm (acetamidomethyl) on the thiol group of a pair of cysteine residues and selectively cyclizing/deprotecting these protected amino acids

with thallium trifluoroacetate in trifluoroacetic acid at reduced temperatures. If necessary or desired, the refolded peptide is further purified by anion

exchange chromatography carried out under essentially neutral conditions (pH 7-9),

for example by Q-Sepharose anion exchange chromatography. Especially preferred

is cellulose anion exchange chromatography, preferably employing a DE-52 resin.

Upon collection of fractions containing the purified peptide, the fractions are pooled

and may be lyophilized to the solid peptide.

As indicated above, chemical synthesis may also be achieved by first

preparing and then joining together peptide fragments. Typically, an initial choice is

made as to which fragments offer the best possibility of providing a clean assembly

of final product. See, e.g., Kent, Angew. Chem. Int. Edit., 30, 113 ( 1991). Those

skilled in the art will recognize the value, for example, of choosing to couple

fragments with a glycine residue at the free C-terminus, which eliminates

complications due to C-terminal racemization. The rat ob dimer

may be prepared in this way. In the absence of signal sequence, the rat ob protein

sequence has five glycine residues, all of which may be useful for fragment

synthesis, leading to a six fragment approach. However, solid phase peptide

synthesis allows the construction of relatively large fragments of up to 60 amino

acids routinely. Thus, another choice of fragments includes but is not limited to the

following three fragment approach:

Fragment 1

VPIHKVQDDT KTLIKTIVTR INDISHTQSV SARQRVTCLD FIPC

Fragment 2

LHPILSLSKM DQTLAVYQQI LTSLPSQNVL QIAHDLENLR DLLHLLAFSK

SCSLPQTRC Fragment 3 LQKPESLDGV LEASLYSTEV VALSRLQCSL QDILQQLDLS PEC

Fully protected fragments as shown above are synthesized by standard solid phase

peptide synthesis methods as described above on a polymeric resin support. Each are cleaved from the resin by standard methods which leave the protecting groups intact.

Fragment 3 requires resin cleavage conditions which place a carboxylic acid

protecting group at the C-terminus or requires subsequent protection at the C-

terminus, whereas Fragments 1 and 2 require their N-terminal protecting groups

remain intact. Fragment 3 also requires subsequent N-terminal deprotection while leaving side chain and C-terminal protecting groups intact.

The fragments are then assembled into full length protein as follows: The C-

terminal carboxylic acid of N-terminal and side chain protected Fragment 2 is

activated, typically with DCC (dicyclohexylcarbodiimide)/N-hydroxybenzotriazole

in a suitable solvent, typically DMF (dimethylformamide). The activated Fragment 2 is then reacted with C-terminal and side chain protected, N-terminal deprotected

Fragment 3 for between 3 and 48 hours at room temperature. Alternatively, if water-

DMF mixtures are used as solvents then a water soluble carbodiimide such as EDC

(N-(3-Dime ylam opropyl)-N'-ethylcarbodiimide) can be used in place of DCC.

A standard workup gives the product which can be purified by reverse phase HPLC. Removal of the N-terminal protecting group of the assembled Fragment 2-3 is then

required and can be achieved either before or after purification under conditions

known to those skilled in the art.

The C-terminal carboxylic acid of N-terminal and side chain protected

Fragment 1 is then activated, typically with DCC/N-hydroxybenzotriazole in a suitable solvent, typically DMF. The activated Fragment 1 is then reacted with C- 6/31526 PO7US96/04909

terminal and side chain protected, N-terminal deprotected Fragment 2-3 for between

3 and 48 hours at room temperature. Alternatively, if water/DMF mixtures are used

as solvents then a water soluble carbodiimide such as EDC can be used in place of

DCC. Again, a standard workup gives the product which can be purified by reverse

phase HPLC.

Removal of all protecting groups of the assembled Fragment 1-2-3 is then

required and is typically achieved by methods described above before. This is

followed by purification, for example, by high pressure liquid chromatography or by

anion exchange chromatography or cation exchange chromatography as described in

Examples 7 and 14 below for the recombinant versions of the ob protein. Dimerization can then be achieved as described above or, if differential protection of

the cysteines has been incorporated into the synthesis, by a sequence of selective

deprotection, formation of one disulphide bond under standard conditions such as air

oxidation or iodine oxidation in solution followed by further deprotection and

disulphide bond formation. The choices made above are of course merely illustrative

of, but not limited to, a strategy for chemical synthesis of the ob protein. Thus other permutations are envisaged, including a different order of coupling, the choice of

other amino acid residues in the sequence as C-termini in fragments, and the choice

of different fragments. The use of chemical ligation (Schnolzer and Kent, (1992)

Science, 256: 221-225); Dawson et al.. (1994) Science , 266: 776-779), which involves fragment coupling under conditions such that a thio ester bond is inserted in

the peptide backbone at chosen junctures is also envisaged.

The ob proteins of the present invention may be isolated, for example from

host cell or media, by various methods known in the art, which include the use of 6/31526 PC17US96/04909

chromatographic methods, such as Q-Sepharose or Q-Sepharose HP anion

chromatography, and suitable cation-exchange methods, such as SP-Sepharose

chromatography, either alone or in sequential steps. See Examples 7 and 14.

Preferred methods of purification of an ob monomer, ob dimer, ob monomer fusion

protein and ob dimer fusion protein include chromatography of an extract through

columns containing a cellulose-based anion-exchange resin, preferably DE-52, or

containing a cellulose-based cation exchange resin, preferably CM-52. In the event

that both anion and cation exchange chromatography methods are used it is preferred

that the cation exchange chromatography be done before anion exchange

chromatography. Presently preferred is the use of anion exchange chromatography

with DE-52 resin for the initial purification of crude protein as described in

Examples 11, 18, 22, 25 and 26.

Following an anion exchange chromatography procedure (or an anion

exchange chromatography procedure preceded by cation exchange chromatography), a further purification using another chromatography method, for example, gel

filtration chromatography using Sephacryl SI 00 or the like, hydrophobic interaction

chromatography using Phenyl-Sepharose HP or the like, or reverse phase high pressure liquid chromatography, may be employed. Reverse phase high pressure liquid chromatography is preferred. See Example 19. The fractions collected after

any such chromatography procedures may be selected by methods such as gel

analysis, high pressure liquid chromatographic analysis, or by their ability to reduce

feeding in mice, as described in Example 23 below, or by a combination of such

analyses. Another method of purification of ob monomer fusion protein or ob dimer

fusion protein, such as a FLAG-ob monomer fusion protein or a FLAG-ob dimer

fusion protein includes chromatography using an anti-FLAG affinity gel, provided by the manufacturer as a suspension of agarose beads covalently linked to anti- FLAG monoclonal antibodies, optionally followed by anion and/or cation exchange

chromatography as described above, or by size fractionation using, for example, a

Superose 12 gel, a Sephacryl SI 00 gel, or the like. In carrying out this affinity

purification step, it is preferred to use CM-52 resin covalently linked to anti-FLAG monoclonal antibodies. The experiments of Example 6 indicated that capture of

FLAG-ob protein on the FLAG Ml affinity resin could be limited by competition for binding sites between intact FLAG-ob protein and an N-terminal fragment of the

FLAG-ob protein. Anion exchange fractionation may first be used to remove any

FLAG containing fragments from the intact FLAG-ob monomer and dimer to

improve the efficiency of the affinity purification step. Especially preferred for the purification of ob proteins, including ob fusion

monomer proteins, ob fusion dimer proteins, Met-ob proteins, and Met-ob fusion proteins, is cellulose resin-based anion exchange chromatography of the periplasmic

extract, or the solubilized, refolded inclusion body protein, using for example,

Whatman DE-52 resin, followed by a second chromatography step using reverse

phase high pressure liquid chromatography. Preferred methods of purification of an

ob dimer or ob dimer fusion protein, illustrated by purification of FLAG ob

monomer and dimer fusion proteins, are provided in Examples 11.

Example 6 describes the purification of a FLAG-ob protein preparation

containing a mixture of monomer and dimer, but predominantly monomer. Further purification of monomer and dimer proteins to about 95% purity was accomplished

by size exclusion chromatography using a Superose 12 gel size exclusion column.

Experiments described in Example 7 show methods useful for purification of

ob monomers, ob dimers, ob monomer fusion proteins, and ob dimer fusion proteins. These experiments detail the purification of FLAG-ob monomer and dimer proteins,

demonstrating that anion exchange chromatography could be performed under essentially neutral (pH 7.4) conditions which closely resemble physiological

conditions and were least likely to inactivate these proteins through unfolding or

degradation. We also found anion exchange purification to be effective alone at

separating, for example, the monomeric and dimeric forms of the ob-FLAG protein.

Additionally, we discovered that while elution of ob fusion protein from the FLAG affinity column with EDTA, pH 7.4, yields samples containing both ob monomer

fusion protein and ob dimer fusion protein, but predominantly monomer, isolation and purification of ob dimer fusion protein to a purity of about 90% can be

accomplished by a first anion exchange chromatography step using Q-Sepharose or

Q-Sepharose HP to obtain dimer fractions for FLAG affinity chromatography,

followed by application of these fractions to a FLAG affinity column and employing

EGTA at pH 8.2 as the elution buffer.

The Example 7 results also showed that cation exchange chromatography

may give the largest purification gain over crude material, although the low pH

needed for absorption of the FLAG-ob monomer and dimer proteins to the column

material could result in unwanted unfolding or degradation.

The present invention also contemplates antibodies and immunoassays useful

for detecting the presence or amount of an ob protein or protein fragment, for example an ob dimer and or ob dimer fusion protein, and antibodies useful therein.

The general methodology and steps of antibody assays are described by Greene, U.S.

Patent 4,376,110, entitled "Immunometric Assays Using Monoclonal Antibodies;

Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, Chapter 14 (1988); Radioimmunoassay and related methods", A. E. Bolton and W.M. Hunter,

Chapter 26 of Handbook of Experimental Immunology.. Volume I,

Immunochemistry, edited by D.M. Weir, Blackwell Scientific Publications, 1986;

"Enzyme immunoassays: heterogeneous and homogeneous systems", Nakamura, et

al., Chapter 27 of Handbook of Experimental Immunology. Volume 1, Immunochemistry, edited by D.M. Weir, Blackwell Scientific Publications, 1986;

and Current Protocols in Immunology. Chapter 2, Section I, Edited by John E.

Coligan, et al., (1991). In all such assays controls are preferably performed, which

are designed to give positive and negative results. For example, the test may include a known ob dimer and/or ob dimer fusion protein positive, control and a known non- ob dimer and/or non-ob dimer fusion protein negative control. Other control

samples may include an ob monomer or ob monomer fusion protein.

Preferably, the immunoassay is a sandwich immunoassay, and comprises the

steps of (1) reacting an immobilized anti-ob dimer and/or anti-ob dimer fusion protein antibody, preferably a monoclonal antibody, and a labeled anti-ob dimer or

anti-ob dimer fusion protein antibody, preferably a monoclonal antibody, which

recognizes a different site from that recognized by the immobilized antibody, with a

sample containing or suspected of containing an ob dimer and or ob dimer fusion protein so as to form a complex of immobilized antibody-ob dimer and or ob dimer

fusion protein-labeled antibody, and (2) detecting the presence or amount of ob dimer and/or ob dimer fusion protein by determining the presence or amount of label

in the complex. In this process the reaction of the immobilized antibody and labeled antibody with the sample may be carried out either simultaneously or separately.

Alternatively, one of the antibody pair used in such an assay, preferably the labeled

antibody, is an anti-ob monomer antibody or an anti-ob monomer fusion protein

antibody. In assaying for ob dimer, it will be understood that both the labeled and

bound antibody may be the same or different anti-ob monomer antibodies.

Suitable antibodies, for example anti-ob dimer and/or anti-ob dimer fusion

protein antibodies, preferably monoclonal antibodies, in accordance with the present

invention are specific to an ob dimer and/or ob dimer fusion protein over an ob

monomer and or ob monomer fusion protein. Such antibodies, as well as suitable anti-ob monomer antibodies or anti-ob monomer fusion protein antibodies, can be

prepared from hybridomas by the following method. Ob dimers or ob dimer fusion proteins or fragments thereof, including those fragments described in Example 9, in

an amount sufficient to promote formation of antibodies, are emulsified in an adjuvant such as Freund's complete adjuvant. The immunogen may be either crude or partially purified, and is administered to a mammal, such as mice, rats or rabbits,

by intravenous, subcutaneous, iπtradermic, intramuscular, or intraperitoneal

injection. In the preparation of polyclonal antibodies, after completion of the

immunization protocol, sera are recovered from the immunized animals. In the preparation of monoclonal antibodies, after completion of the immunization

protocol, as described for example in Example 8, animal spleens are harvested and

myeloma cells having a suitable marker such as 8-azaguanine resistance can be used

as parent cells which are then fused with the antibody-producing spleen cells to prepare hybridomas. Suitable media for the preparation of hybridomas according to

the present invention include media such as Eagle's MEM, Dulbecco's modified

medium, and RPM3-1640. Myeloma parent cells and spleen cells can be suitably fused at a ratio of approximately 1 :4. Polyethylene glycol (PEG) can be used as a

suitable fusing agent, typically at a concentration of about 35% for efficient fusion. Resulting cells may be selected by the HAT method. Littlefield, J. W., (1964)

Science 145: 709. Screening of obtained hybridomas can be performed by

conventional methods including an immunoassay using culture supernatant of the

hybridomas to identify a clone of hybridoma producing the objective immunoglobulin. The obtained antibody-producing hybridoma can then be cloned

using known methods such as the limiting dilution method. In order to produce, for example, the anti-ob dimer and or anti-ob dimer fusion protein monoclonal

antibodies of the present invention, the hybridoma obtained above may be cultured either in vitro or in vivo. If the hybridoma is cultured in vitro, the hybridoma may

be cultured in the above-mentioned media supplemented with fetal calf serum (FCS)

for 3-5 days and monoclonal antibodies recovered from the culture supernatant. If

the hybridoma is cultured in vivo, the hybridoma may be implanted in the abdominal

cavity of a mammal, and after 1-3 weeks the ascites fluid collected to recover monoclonal antibodies therefrom. Much larger quantities of the monoclonal

antibodies can efficiently be obtained using in vivo cultures rather than in vitro

cultures and, thus, in vivo cultures are preferred. The monoclonal antibody obtained

from the supernatant or ascites fluids can be purified by conventional methods such as ammonium sulfate-fractionation, Protein G-Sepharose column chromatography, or their combinations. Illustrative and detailed methods for preparing the antibodies

described and claimed herein are further provided in Example 8.

Antibodies, or the desired binding portions thereof including F(ab) and Fv

fragments, along with antibody-based constructs such as single chain Fv's can also be generated using processes which involve cloning an immunoglobulin gene library

in vivo. See, e.g., Huse et al.. Generation of a Large Combinatorial Library of the

Immunoglobulin Repertoire in Phage Lambda. (1989) Science 246: 1275-1281.

Using these methods, a vector system is constructed following a PCR amplification

of messenger RNA isolated from spleen cells with oligonucleotides that incorporate

restriction sites into the ends of the amplified product. Separate heavy chain and

light chain libraries are constructed and may be randomly combined to coexpress these molecules together and screened for antigen binding. Single chain antibodies

and fragments may also be prepared by this method.

A sandwich immunoassay for ob dimers and/or ob dimer fusion proteins can

suitably be prepared using an immobilized anti-ob dimer and/or anti-ob dimer fusion

protein monoclonal antibody and a labeled anti-ob dimer and/or anti-ob dimer fusion

protein monoclonal antibody. Anti-ob monomer antibodies and anti-ob monomer

fusion protein antibodies can also be used as the immobilized or labeled antibody in

conjunction with an anti-ob dimer and/or anti-ob dimer fusion protein monoclonal

antibody to form the antibody/antigen/antibody sandwich. If an anti-ob monomer

antibody or anti-ob monomer fusion protein antibody is used in the sandwich

immunoassay to form a part of the antibody pair, it is preferably the labeled

antibody. Antibodies according to the present invention can suitably be immobilized

on commercially available carriers for the antigen-antibody reaction including beads, balls, tubes, and plates made of glass or synthetic resin. Suitable synthetic resins

include polystyrene and polyvinyl chloride. Anti-ob dimer, anti-ob monomer, anti-

ob monomer fusion protein, and anti-ob dimer fusion protein monoclonal antibodies

are suitably absorbed onto the carrier by allowing them to stand at 2-8°C overnight

in 0.05M carbonate buffer, pH 9-10, preferably about pH 9.5. The immobilized anti-

ob dimer, anti-ob monomer, anti-ob monomer fusion protein, and/or anti-ob dimer fusion protein monoclonal antibody can be stored cold in the presence of

preservatives such as sodium azide. Both monoclonal and polyclonal antibodies can

be immobilized onto carriers using this method.

Labeled anti-ob dimer, anti-ob monomer, anti-ob monomer fusion protein, and anti-ob dimer fusion protein antibodies in accordance with the present invention can suitably be prepared by labeling anti-ob dimer, anti-ob monomer, anti-ob

monomer fusion protein, and anti-ob dimer fusion protein antibodies with any substance commonly used for an immunoassay including radioisotopes, enzymes,

and fluorescent substrates. Radioisotopes and enzymes are preferably used. When radioisotopes are used as labels, the antibody is preferably labeled with ^,25I using

conventional methods such as the Chloramine T method (Hunter et al.. Nature

(1962) 194: 495) and the Bolton-Hunter method. When enzymes are used as labels,

the antibody is labeled with an enzyme such as horseradish peroxidase, β-D-

galactosidase, or alkaline phosphatase by conventional methods including the

maleimide method and the Hingi method. Ishikawa et al. (1983) J. Immunoassay 4:

1.

The activity of the label can be detected by conventional methods. If

radioisotopes are used as labels, the activity of the label can be detected using an appropriate instrument such as a scintillation counter. If enzymes are used as labels,

the activity of the label can be detected by measuring absorbance, fluorescence

intensity, or luminescence intensity after reacting the enzyme with an appropriate

substrate. The present invention also provides a kit for assaying the amount of ob dimer

and/or ob dimer fusion protein present in a sample, including for example both

biological samples and samples of ob dimers and/or ob dimer fusion proteins. The

kit of the present invention comprises an immobilized anti-ob dimer and or anti-ob dimer fusion protein monoclonal antibody and a labeled anti-ob dimer and/or anti-ob dimer fusion protein monoclonal antibody. When ob dimers and/or ob dimer fusion

proteins are assayed using this kit, ob dimers and or ob dimer fusion proteins

become sandwiched between the immobilized monoclonal antibody and the labeled monoclonal antibody. As noted above, anti-ob monomer antibodies and anti-ob monomer fusion protein antibodies can also be used as the immobilized or labeled

antibody to form one half of the antibody pair in conjunction with an anti-ob dimer

and/or anti-ob dimer fusion protein monoclonal antibody.

Since the monoclonal antibodies useful for this kit can optionally recognize

ob dimers or ob dimer fusion proteins or both when they coexist, depending on the

monoclonal antibodies used, the total amounts of ob dimers or ob dimer fusion

proteins or both can be measured by this invention.

The ob dimers, ob dimer fusion proteins, and ob monomer fusion proteins

described and claimed herein have potent and prolonged food intake inhibition

properties in vivo. These compounds will thus have significant utility in further /31526 PC17US96/04909

investigations seeking to elucidate the actions and physiological role of the ob gene

in vivo.

Their properties also support the use of ob dimers, ob dimer fusion proteins,

and ob monomer fusion proteins in the treatment of subjects with disorders or

conditions that would benefit from reduced food intake or increased energy

expenditure. Such conditions or disorders include obesity and diabetes, particularly Type 2 diabetes. In particular, the compounds of the invention possess activity as

anti-obesity agents, as evidenced by the ability to reduce feeding in mammals. As

shown, for example, by the experiments in Example 23 below, [NEEDS TO BE

REWRITTEN/UPDATED -- the ob dimer is able to markedly depress food intake

in fasted (and presumably hungry) mice for up to 12 hours following a single injection, with no apparent "catch-up" in food intake over 24 hours, and with no

observable effects on behavior or well being, other than reduced food intake]. These

unexpected and beneficial effects can be expected to provide a safe and effective

therapeutic control of eating and energy balance suitable for the chronic (long-term) amelioration of obesity by loss of excess adipose tissue, together with expected

improvement in co-morbid conditions, including Type 2 diabetes. The ob dimers,

ob dimer fusion proteins, and ob monomer fusion proteins thus may also be used to

prepare pharmaceutical compositions, and used in methods for the treatment of

disorders and conditions which may benefit from the administration of such

compositions. Such pharmaceutical compositions include therapeutically effective

amounts of an ob dimer or ob dimer fusion protein or ob monomer fusion protein in pharmaceutically acceptable carriers. By "therapeutically effective amount" is

meant an amount useful to cause reduced food intake or increased energy expenditure or both. Ob dimers and ob dimer fusion proteins include human ob

dimers and human ob dimer fusion proteins, rat ob dimers and rat ob dimer fusion

proteins, mouse ob dimers and mouse ob dimer fusion proteins, as well as other vertebrate ob dimers and vertebrate ob dimer fusion proteins. Ob monomer fusion

proteins include human ob monomer fusion proteins, rat ob monomer fusion

proteins, mouse ob monomer fusion proteins, as well as other vertebrate ob

monomer fusion proteins. Treatment methods comprise the administration of a therapeutically effective amount of a pharmaceutical composition comprising an ob

dimer or ob dimer fusion protein or ob monomer fusion protein to patients in need

thereof.

These pharmaceutical compositions may be administered separately or

together with other compounds and compositions that may be useful in the treatment

of said disorders and conditions, including but not limited to other compounds and

compositions that comprise an amylin or an amylin agonist. See Lutz et al.. (1994)

Physiology and Behavior, 55: 891-895. Suitable amylins include, for example,

human amylin and rat amylin. Suitable amylin agonists include, for example, ²⁵' ^{28, 29}Pro-human amylin, amylin agonists as described in "Amylin Agonist Peptides and

Uses Therefor," International Patent Application Number PCT/US92/09842 (published May 27, 1993), and salmon calcitonin. The compounds referenced above, as well as ob monomer protein, form salts

with various inorganic and organic acids and bases. Such salts include but are not

limited to salts prepared with organic and inorganic acids; for example, HC1, HBr,

H₂SO₄, H₃PO₄ trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid, toluenesulfonic acid, maleic acid, fumaric acid and camphorsulfonic acid. Salts prepared with bases include ammonium salts, alkali metal salts, e.g., sodium and

potassium salts, and alkali earth salts, e.g,. calcium and magnesium salts. Acetate,

hydrochloride, and ammonium bicarbonate salts are preferred. Ammonium bicarbonate salts are especially preferred, and are also preferred for preparation of ob

monomer protein. The salts may be formed by conventional means, and by reacting

the free acid or base forms of the product with one or more equivalents of the

appropriate base or acid in a solvent or medium in which the salt is insoluble, or in a

solvent such as water which is then removed in vacuo or by freeze-drying or by

exchanging the ions of an existing salt for another ion on a suitable ion exchange

resin, or gel filtration resin. See Examples 19, 20 and 22.

Compositions or products of the invention may conveniently be provided in

the form of formulations suitable for parenteral (including intravenous,

intramuscular and subcutaneous) or nasal or oral administration. In some cases, it

will be convenient to provide an ob dimer or ob fusion protein or ob monomer

fusion protein of the invention and another shorter-acting satiety agent, such as an

amylin or an amylin agonist in a single composition or solution for administration

together. Also contemplated is ob monomer protein an amylin or an amylin agonist

in a single composition or solution for administration together. In other cases, it may be more advantageous to administer an amylin or an amylin agonist separately

from the ob dimer or ob fusion protein or ob monomer fusion protein. A suitable

administration format may best be determined by a medical practitioner for each patient individually. Suitable pharmaceutically acceptable carriers and their

formulation are described in standard formulation treatises, e.g., Remington 's

Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A. "Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers,"

Journal of Parenteral Sciences and Technology, Technical Report No. 10, Supp.

42:2S (1988). Ob dimers and ob fusion proteins should preferably be formulated in

solution at neutral pH, for example about pH 6.5 to about pH 8.5, more preferably

from about pH 7.5 to 8.5, with an excipient to bring the solution to about isotonicity,

for example 4.5% mannitol or 0.9% sodium chloride, pH buffered with art-known buffer solutions, such as sodium phosphate, that are generally regarded as safe,

together with an accepted preservative such as metacresol 0.1% to 0.75%, more

preferably from about 0.1% to about 0.37% phenol. Ob dimers and ob dimer fusion proteins may not be stable or may be partially or wholly denatured at acid pH and

thus pHs below 6 should be avoided as far as possible in manufacture, purification

and formulation of these agents. The desired isotonicity may be accomplished using

sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric

acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol), or

other inorganic or organic solutes. Sodium chloride is preferred particularly for

buffers containing sodium ions.

Especially preferred lyophilized and liquid formulations for an ob

dimer or ob fusion protein or ob monomer or ob monomer fusion protein are

described in Examples 20 and 21. In a preferred lyophilized form, the proteins of the invention are prepared as ammonium bicarbonate salts. In a preferred liquid

formulation, the proteins of the invention are prepared in BisTris-propane buffer, or buffers of similar structure, with or without a detergent, such as Tween 80, to have a

pH from about 7.5 to about 9, most preferably a pH of 8.5. Lyophilized and liquid formulations of ob monomer proteins may also be formulated as described herein and such formulations form a part of the invention.

A form of repository or "depot" slow release preparation may be used so that therapeutically effective amounts of the preparation are delivered into the

bloodstream over many hours or days following transdermal injection or delivery.

If desired, solutions of the above compositions may be prepared in

emulsified form, either water in oil or oil in water. Any of a wide variety of

pharmaceutically acceptable emulsifying agents may be employed including, for example, acacia powder, a non-ionic surfactant (such as a Tween), or an ionic

surfactant (such as alkali polyether alcohol sulfates or sulfonates, e.g., a Triton). If desired, solutions of the above compositions may also be prepared and dried in the

presence of the sugar trehalose to enhance shelf life and stability. The therapeutically useful compositions of the invention are prepared by mixing the

ingredients following generally accepted procedures. For example, the selected

components may be mixed to produce a concentrated mixture which may then be

adjusted to the final concentration and viscosity by the addition of water or

thickening agent and possibly a buffer to control pH or an additional solute to

control tonicity.

For use by the physician, the compositions will be provided in dosage unit form containing an amount of a compound of the invention which will be effective

in one or multiple doses to reduce food intake and/or increase energy expenditure at

a selected level. Therapeutically effective amounts of an ob dimer or ob dimer

fusion protein or ob monomer or ob monomer fusion protein as described herein for

the treatment of obesity, diabetes, and other such conditions in which food intake is beneficially reduced and/or energy expenditure enhanced include those that decrease

food intake, preferably to about fifty percent of pretreatment levels or such that food

intake is reduced as desired. Compounds of the invention are preferably

administered to provide peak plasma levels of ob dimer or ob dimer fusion protein or

ob monomer or ob monomer fusion protein that are about twice the plasma levels of

human ob protein observed in healthy subjects, and preferably that are about two to ten times the levels, who have been on a caloric-enriched diet, for example 50 to 80

kcal/kg for about one to four weeks. As will be recognized by those in the field, an

effective amount of therapeutic agent will vary with many factors including the age

and weight of the patient, the patient's physical condition, the ob protein level or

decrease in food intake or increase in energy expenditure to be obtained, and other

factors.

The effective dose of the ob dimer and ob dimer fusion protein compounds

[MONOMER?] of this invention will typically be in the range of 0.02 mg to about

2 mg once or twice per day, preferably about 0.05 mg to about 1 mg once per day on waking [ANY MODIFICATION TO THIS?]. As noted, the exact dose to be administered is determined by the attending clinician and is dependent upon where

the particular compound lies within the above quoted range, as well as upon the age,

weight and condition of the individual. A similar dosage range is provided for ob

monomer fusion proteins [WILL THE FUSION MONOMER BE LESS?

WHAT ABOUT THE FORMULATED UNFUSED MONOMER?].

The presently preferred mode of administration is by subcutaneous injection

of a parenteral solution via a disposal syringe and needle, for example an insulin syringe, or from a pre-fiUed cartridge fitted to a pen injector such as, by way of

example, those provided by Becton-Dickinson for insulin injection.

To assist in understanding the present invention, the following Examples are

provided which describe the results of a series of experiments. The experiments

relating to this invention should not, of course, be construed as specifically limiting

the invention and such variations of the invention, now known or later developed,

which would be within the purview of one skilled in the art are considered to fall

within the scope of the invention as described herein and hereinafter claimed.

Examples

Example 1 - Extraction of Rat RNA and Preparation of Oligonucleotide

Primers

This Example describes the extraction of rat total RNA and the preparation of oligonucleotide primers for use in the isolation and cloning of the rat ob gene.

Epididymal adipose tissue was obtained from a male Sprague Dawley rat,

immediately frozen in liquid nitrogen, and then stored at -80°C. Total RNA was

extracted by the acid guanidinium thiocyanate procedure (Chomczynski, P. and Sacchi, N. (1987) Analytical Biochemistry 162: 156-159).

Oligonucleotides were synthesized for use as primers in PCR amplifications.

Upstream primer A630 corresponds to positions 98-118 of mouse ob cDNA (Zhang,

et al.. (1994) Nature 372: 425-432) and was constructed with a non-gene-specific

triplet repeat at its 5' end to facilitate subcloning. It has the following sequence: 5'-

(CUA)₄-AAGATCCCAGGGAGGAAAATG-3\ Downstream primer A631

corresponds to positions 637-617 of the non-coding strand of mouse ob cDNA (id.) and also contains a non-gene-specific triplet repeat at its 5' end to facilitate

subcloning. Its sequence is as follows: 5' (CAU)₄-

CTGGTGGCCTTTGAAACTTCA-3^,. Oligonucleotides were synthesized on a

MilliGen/Biosearch Cyclone Plus DNA synthesizer using standard β-cyanoethyl phosphoramidite chemistry. Following synthesis they were cleaved from the

support, deprotected and eluted. After evaporation to dryness they were dissolved in

water.

Example 2 — Rat ob cDNA Isolation and Cloning

This Example describes the methods used to isolate and clone rat cDNA

coding for the rat ob protein using the primers and RNA of Example 1. The

nucleotide and deduced amino acid sequences of the rat ob gene were then

determined.

Rat ob cDNA was RT-PCR amplified from rat adipose tissue total RNA

using reagents from the Perkin Elmer RNA PCR kit (Perkin Elmer, Foster City,

CA). Conversion of RNA to cDNA was accomplished in a final volume of lOOμl

containing 10 mM Tris-HCl, pH 8.3 / 50 mM KC1 / 5 mM MgCl₂ / 1 mM each

dNTP / 5 μg rat adipose tissue total RNA / 2.5 μM random hexamers / 1 unit μl^"1

RNase inhibitor / 2.5 units μl^'1 Murine Leukemia Virus Reverse Transcriptase

(MuLVRT). The reaction was heated for 5 min at 70°C and quick chilled on ice for

1 minute before the addition of the RNase inhibitor, random hexamers, and

MuLVRT. The reaction was then covered with 75μl mineral oil and incubated as

follows: 22°C for 10 min, 42°C for 1 hour, 99°C for 5 min, and then chilled on ice.

PCR amplification was accomplished using 20 μl of the above cDNA

synthesis reaction in a final 100 μl volume containing 10 mM Tris-HCl, pH 8.3 / 50 31526 PCIYUS96/04909

mM KC1 / 2.5 mM MgCl₂ / 0.2 mM each dNTP / 0.5 μM primer A630 / 0.5 μM

primer A631 / 2.5 units AmpliTaq^® DNA Polymerase. The reaction was incubated

for 45 cycles of 1 min denaturation at 94°C, 1 min annealing at 48°C , and 1 min

extension at 72°C, followed by a final incubation for 10 min at 72°C.

Products from the RT-PCR were resolved on a 2% agarose gel and a DNA fragment having a predicted 564 bp length was excised from the gel and purified

using a silica membrane system (SpinBind^® DNA Recovery System; FMC

BioProducts, Rockland, ME) following the manufacturer's protocol. The purified

DNA fragment was subcloned into plasmid vector pAMP 1 by a uracil DNA

glycosylase methodology (CloneAmp™ System, GIBCO BRL, Gaithersburg, MD) following the manufacturer's protocol. The resulting products were used to

transform E. coli DH5αMCR competent cells (GIBCO BRL) following the

manufacturer's protocol. Plasmid DNA from transformants was purified using a

minilysate procedure (Wizard™ Minipreps DNA Purification System, Promega, Madison, WI) and sequenced by a dideoxy chain termination method (ds Cycle

Sequencing System, GIBCO BRL). The DNA sequence of rat ob clone

pAMPlROB#3 and its deduced amino acid sequence is presented in Figure 1. The

triplet nucleotides for the initiation methionine and termination codons were

introduced into the sequence by virtue of the primers employed in the PCR

amplification.

Example 3 — In vitro Transcription/Translation of the Rat Ob Gene

This Example describes the methods used in the in vitro transcription and

translation of the rat ob gene isolated as set forth above. One microgram of pAMPlROB#3 from Example 2 was used as a template

for in vitro transcription translation using the TnT Sp6 Coupled Rabbit Reticulocyte

Lysate kit (Promega Corp.) following the manufacturer's protocol. A separate reaction was prepared containing canine pancreatic microsomal membranes

(Promega Corp.) in the reaction mix. A third reaction was prepared without

pAMPlROB#3 DNA template and served as a negative control. The translation

products were labeled with L-(³⁵S)methionine (Amersham Life Sciences Inc., #

SJ.1015).

Aliquots of the reactions were run under reducing and non-reducing

conditions on a 10-20% Tricine SDS polyacrylamide gradient gel (Novex Inc.).

After electrophoresis, the gel was fixed in a solution of isopropanol/dH₂0/acetic acid and then soaked in fluorography enhancer (Amplify; Amersham Life Sciences Inc.).

The gel was subsequently dried and exposed to Kodak XAR film for 15 hours. A ~ 29,000 dalton band was observed from the reactions containing pAMPlROB#3

when run under non-reducing conditions. A second band, running slightly below the

29,000 dalton band, was observed in the reaction containing the microsomal

membranes. These bands were not visible when the same samples were reduced

prior to electrophoresis. Observation of bands running in the 16,000 to 18,000

dalton range was not possible due to non-specific background from the lysate.

In order to observe translation products in the 16,000 to 18,000 dalton range,

the rat ob gene was subcloned, utilizing Hind III and Sal I restriction sites, from

pAMPlROB#3 into vector pSP64poly(A) (Promega Corp.) creating pSP64ROBa. In

vitro transcription/translation analysis was repeated, as outlined above, using the TnT Sp6 Coupled Wheat Germ Extract kit (Promega Corp.) and using the manufacturer's protocol. A single band was observed running between the 16,525

and 18,800 dalton molecular weight markers, under both non-reducing and reducing

conditions, specific to the reaction containing pSP64ROBa as template.

Example 4 —E. coli Periplasmic Expression of the FLAG/Rat ob Gene

The coding region for mature rat ob was PCR amplified from pAMPlROB#3

using an upstream gene specific oligonucleotide (A638: 5'-

AGTCCCTATCCACAAAGT

CC-3') and a downstream oligonucleotide (A540) corresponding to the T7 promoter

primer in the pAMP 1 vector. The first nucleotide of the upstream primer

corresponds to the last nucleotide of the C-terminal Lysine codon of the FLAG epitope in vector pFLAG-ATS (IBI/Kodak Scientific Imaging Systems Inc.). The remainder of the upstream primer corresponds to the coding sequence of rat ob

beginning with Valine-22. A single silent mutation (G to C) was incorporated into primer A638 at the third position of the Valine-22 codon in order to reconstruct the

Tthlll I site in the multiple cloning site of the pFLAG-ATS vector.

PCR amplification was performed in a lOOμl volume containing 30 ng

pAMPlROB#3, 0.5 μM each primer (A638, A540), 2.5 units of AmpliTAQ DNA^®

Polymerase, 10 mM Tris-HCl pH 8.3, 50 mM KC1 , 0.2 mM each dNTP, and 2.5

mM MgCl₂ . The reaction was incubated for one cycle at 95°C/1 min, 51°C/1 min

and 72°C/1 min followed by 30 cycles at 95°C/30 sec, 51°C/30 sec and 72°C/30 sec

followed by a final incubation of 10 min at 72°C. Analytical agarose gel

electrophoresis using 10 μl of the above 100 μl reaction mixture confirmed a single

band of the expected size, 543 bp. The remaining PCR reaction material was purified

using the QIAquick PCR Purification kit (QIAGEN Inc.) following the kit protocol. The PCR fragment was digested with EcoRl and cloned into the pFLAG-

ATS vector cut with Aspϊ (an isoschizomer of 7YM 11I) and EcoRI. The resulting

subclone, pFLAGROB#5, was sequenced using the N-26 primer (IBI Kodak

Scientific Imaging Systems) and various other primers specific to the rat ob sequence, to confirm proper construction of the coding region.

To test expression of the FLAG/rat ob fusion protein, pFLAGROB#5 in E.

coli strain DH5αF' (Gibco BRL) was grown at 37°C with rotation (250 rpm) in 20

mL of LB media containing 0.4% glucose and 50μg/mL ampicillin. When the OD^

reached 0.75, IPTG was added to a final concentration of 0.5 mM and the

incubation was continued for an additional 5 hours. One mL aliquots of the culture were collected at various time points throughout the incubation. The cells were

pelleted, supernatant removed and the cells resuspended in 50 μL of 2x Tricine SDS

Loading Buffer, then heated for 5 min at 95°C. A duplicate cell pellet was

resuspended in 400 μL 0.5 M Sucrose/TE pH 8.0, pelleted in a centrifuge, and the

supematant was removed. Then the pellet was resuspended in 100 μL ice-cold H₂O followed by centrifugation at 4°C. The supernatant was collected to a new tube and

an equal volume of 2x Tricine SDS Loading Buffer was added to the sample

followed by incubation at 95°C for 5 minutes. This sample is indicative of the

protein being transported into the periplasm of the E. coli. Aliquots of the cell

samples were run under reducing and non-reducing conditions on a 10-20% Tricine

SDS gradient polyacrylamide gel. After electrophoresis, a Western blot was

performed using nitrocellulose as the transfer media. Murine anti-FLAG primary

monoclonal antibody Ml (IBI/Kodak Scientific Imaging Systems) was used at a

concentration of 2.5 μg/mL. The secondary antibody was an alkaline phosphatase conjugated, goat anti-mouse IgG F(ab')₂ (Boehringer Mannheim Corp.). The blot

was developed using an Alkaline Phosphatase Conjugate Substrate Kit (Bio-Rad, #170-6432). In the non-reduced periplasm samples, sharp immunoreactive bands

were observed at -17,000 daltons, -30,000 daltons and another at a molecular

weight > 43,000 daltons. A single band migrating at -17,000 daltons was observed

in these same samples when run under reducing conditions. Similar results were

observed with the whole cell samples but with higher background. The Ml antibody

only detects the FLAG epitope if it has a free N-terminus; therefore, the antibody

only reacts with the expressed protein transported to the periplasm where the ompA

signal sequence has been removed.

To compare expression levels in different E. coli strains, pFLAGROB#5 was used to transform competent BL21 (Novagen, Inc.), W3110 (ATCC No. 27325) and

JM105 (Pharmacia) cells. FLAG/rat ob protein expression was compared to the

original DH5αF' strain by Western Blot analysis, as outlined above. W3110 and JM105 produced approximately five to ten times more protein than either the BL21

or DH5αF' strains. Both the -17,000 dalton and -30,000 dalton immunoreactive

bands were observed under non-reducing conditions from all of the strains tested.

Example 5 — Rat ob Gene Fermentation Expression

Quantities of rat ob gene product or rat ob gene fusion product can be

produced using the below described methods for the production of FLAG/rat ob

gene product.

FLAG/rat ob gene product was produced via 10 and 60 liter fermentations. The below description refers to a 10 liter fermentation. Prior to inoculation, the

production tank was prepared with 10 liters of Luria broth (Tryptone (Difco), 10 g/L; NaCl (VWR), 10 g/L; Yeast extract (Marcor), 5 g/L); glucose (Atlas Clintose),

4 g/L; polypropyleneglycol as antifoam, 0.25 g/L. The Luria broth was steam

sterilized in place at 121 °C for 30 minutes and then allowed to cool (typically 24

hours) before fermentation commenced. Fermentation conditions were as follows:

200 mL of Luria broth was inoculated with E. coli strain W3110, which had been

previously transformed with the expression vector pFLAGROB#5, and grown

overnight in an incubated shaker (37 C, 240 rpm). Prior to inoculation, 0.05 g/L of

ampicillin was added to the fermentor for plasmid selection. The inoculum was

transferred to the production tank after approximately 17 hours in a volume ratio of 1 :100. Typically, the inoculum optical density had exceeded 1 OD at 600 nm

(ODr_øo). The fermentation was controlled at the following set points: Temperature

at 37 C; dissolved O₂ at 20% of saturation at 37°C, 5 psi; pH at 6.8; airflow rate at 5

Standard Liters per minute.

Fermentation was monitored at least every hour for the following parameters:

temperature, agitation rate (φm), pH, dissolved O₂ (%), airflow rate (SLPM),

pressure (psi), offgas O₂ (%), offgas CO₂ (%), OO_m, and glucose concentration

(g/L). When the fermentation reached an OD^ of approximately 3, the E. coli were

induced to produce FLAG/ob gene product with .238 g/L IPTG (Isopropyl-β-D-

thiogalactopyranoside; Mannheim Gmbh, Lot 13951720-98). Two hours after induction of the fermentation, the cells were harvested via centrifugation in 1 liter

Nalgene bottles. The broth was decanted away, and the cells were frozen by

immersion in liquid nitrogen. Example 6 - Affinity Purification of FLAG/rat ob Fusion Protein

A 250 mL pellet obtained from the E. coli shake flask fermentation described in Example 4 was resuspended in 50 mL of extraction buffer A (50 mM

Tris (pH = 8.0), 5 mM EDTA, 0.25 mg/mL lysozyme (Sigma, L-6876), 50 μg/mL

sodium azide, 250 μl of 100 mM AEBSF/Ethanol) and the suspension processed

with a Dounce homogenizer. The whole was incubated at room temperature for 15

minutes followed by addition of 5 mL of extraction buffer B (1.5 M NaCl, 100 mM

CaCl₂, 100 mM MgCl₂, 50 μg/mL Ovomucoid protease inhibitor (Sigma, T-9253),

0.02 mg/mL DNAse I (Sigma, D-4527)). After centrifugation (25,000 xg),

Beckman J2-21 centrifuge, J-20 rotor, 14,300 φm) for one hour at 4°C, the

supernatant was applied to an anti-FLAG Ml affinity column prepared in accordance with manufacturer's instructions and cycled three times. The column

was washed with 3 x 6 mL of 2 mM CaCl₂/TBS (50 mM Tris/150 mM NaCl) to

remove all non-FLAG proteins. The FLAG proteins were eluted with 4 mM

EDT A/TBS to give a mixture of monomer and dimer (identified by SDS-PAGE,

Western blotting) and two lower molecular weight fractions which were noted on

reverse phase HPLC analysis.

Further purification of the FLAG-ob protein was accomplished by gel

filtration using a Pharmacia Superose 12 HR 10/30 column. Approximately 4 mL

of Flag-ob protein in EDTA TBS (50mM Tris/150mM NaCl/4mM EDTA pH 7.4)

was concentrated by speedvac on medium heat in two separate 12 x 25 mm Nunc

Lo-Soφ test tubes, yielding approximately a 3 to 1 concentration in about two hours. 1.3 mL of concentrated protein was injected onto a Superose 12 HR10/12

column and the column run uphill at 0.25 mL/min using an Automated FPLC System from Pharmacia Biotech. The running buffer was 50 mM Tris/150 mM

NaCl (pH 7.4). Fractions were collected at one minute intervals. Monomer and

dimer fractions were collected and were shown to be about 95% pure by SDS-

PAGE with silver stain detection.

Example 7 — Additional Purification of FLAG/rat ob Fusion Protein by

Ion Exchange Chromatography

The initial affinity chromatography described in Example 6 was complicated

by antibody binding interference from a lower molecular weight 13 mer containing

the FLAG sequence (DYKDDDDKVPHIK). Q-Sepharose resins were determined to be efficacious in purifying crude FLAG-ob and in separating FLAG-ob monomer

from dimer protein. In particular, a Q-Sepharose HP column was used to fractionate

affinity column flow-through and to yield highly active partially purified dimer.

All ion exchange purification was done at 4°C using an Automated FPLC System from Pharmacia Biotech equipped with a Conductivity Monitor. Elution

profile data were collected and analyzed using the FPLC manager software package

running on an NEC Powermate 466 computer. FLAG-ob protein was detected by

the following methods: absorbance detection at 280 nm, SDS-PAGE with detection

using Coomassie Brilliant Blue, silver staining, or Western Blotting. Reagents, 10-

20% Tricine gels and gel apparatus from NOVEX were used for SDS-PAGE. Silver staining was done using a silver stain kit from Accurate Chemical Company and

Western Blotting was done as previously described in Example 4, but using PVDF

membranes instead of nitro-cellulose. Sequencing was done using ABI model 470A

or 476A protein sequencers using standard protocols. 200 mL of FLAG affinity column flow-through material, which contained

both monomeric and dimeric FLAG-ob proteins (as determined by Western

Blotting), was subjected to anion exchange chromatography. The material was first

clarified by filtration using a 0.65μm Durapore membrane filter (Millipore), and

concentrated using a 200 mL Amicon microfiltration unit equipped with a YM3

membrane. The concentrated material was then diluted to 50 mL with 20 mM Tris HC1, pH 7.4 and loaded onto a HiLoad 16/10 Q-Sepharose HP column (Pharmacia

Biotech) using a flow rate of 3.0 mL/min. The column was eluted at 3.0 mL/min.

using a 430 mL gradient to 20 mM Tris HC1 pH 7.4 containing 1.0M NaCl while collecting 6.0 mL fractions. Fractions from this run were analyzed by SDS-PAGE with detection using Coomassie Brilliant Blue staining and Western Blotting. The

gel results indicated that monomeric FLAG-ob protein eluted mainly in earlier

fractions and that dimeric FLAG-ob protein eluted in middle fractions. One smaller

form was observed at an apparent molecular weight of 10 kD in later fractions.

Another 100 mL of FLAG affinity column flow-through material was

subjected to anion exchange chromatography. The flow-through material was

concentrated to 23 mL using a 200 mL Amicon microfiltration unit equipped with a

YM3 membrane. The concentrated material was then diluted to 40 mL with 20 mM

Tris HC1 pH 7.4 and loaded onto a HiLoad 16/10 Q-Sepharose HP column

(Pharmacia Biotech) using a flow rate of 3.0 mL/min. The column was eluted at 3.0

mL/min. using a 430 mL gradient to 20 mM Tris HC1 pH 7.4 containing 1.0 M NaCl

while collecting 6.0 mL fractions. Fractions from this run were analyzed by SDS-

PAGE with detection using Coomassie Brilliant Blue staining and Western Blotting. Again, the gel results indicated that monomeric FLAG-ob protein eluted mainly in

earlier fractions and that dimeric FLAG-ob protein eluted in later fractions.

200 mL of FLAG affinity column flow-through material was subjected to cation exchange chromatography. The flow-through was diluted with water and

brought to pH 4.5 by the careful addition of dilute acetic acid, final volume 4L, and

batch absorbed overnight to 50 mL of SP-Sepharose Fast-Flow resin (Pharmacia

Biotech). The resin was poured into an XK 26/10 column and washed with 25 mM

sodium acetate pH 4.5. The column was eluted using a 180 mL gradient to 25 mM

sodium acetate pH 4.5 containing 0.5M NaCl using a flow rate of 6.0 mL/min. 12 mL fractions were collected into tubes containing 2 mL of 0.25M Tris HCl pH 8.0

buffer to give a final pH of about 7.5 for each fraction. Fractions from this run were analyzed using SDS-PAGE with detection using Coomassie Brilliant Blue staining

and Western Blotting. The gel results indicated that monomeric FLAG-ob protein

eluted most strongly in fractions 7-9 and that minor amount of dimeric FLAG-ob

protein eluted in fractions 6-8.

A further approach was undertaken to enhance the purity of active FLAG-ob

dimer proteins and ob monomer fusion proteins isolated as described above. 1 mL

of a Q-Sepharose dimer-containing fraction derived from Q-Sepharose purification of a cell pellet from a 60 L fermentor run described in Example 5 was brought to 10

mM CaCl₂ by the addition of IM CaCl₂. This material was then applied to an 0.5

mL bed volume Anti-FLAG Ml affinity column and washed with 20 column

volumes of TBS containing 10 mM CaCl₂. The Anti-FLAG affinity column was then eluted using 20 column volumes of 50 mM Tris containing 25 mM EGTA pH 8.2, followed by elution using 100 mM glycine HCl buffer pH 3.0. Fractions were analyzed by SDS-PAGE using silver staining and Western blotting for detection. It

was found that the majority of the FLAG-ob protein eluted from the column in the

EGTA step, and that only a trace of material remained to be eluted during the

glycine HCl step. Furthermore, it was determined that this method yielded

approximately 90% pure FLAG-ob dimer protein.

Example 8 — Preparation of Anti-ob monomer and Anti-ob dimer Antibodies

Anti-ob monomer antibodies, anti-ob monomer fusion protein antibodies,

anti-ob dimer antibodies, and/or anti-ob dimer fusion protein antibodies in

accordance with the present invention are prepared as follows. A sample of ob

monomer or dimer, such as one of those prepared by the methods of Examples 5-7, 11, 14, 20, 22 and 26, and tested as described in Example 23, or a synthetic ob

protein fragment, is conjugated to bovine thyroglobulin (THY, Sigma) as a carrier

protein using a 2:1 ratio of carrier to peptide and glutaraldehyde or

sulfosuccinimidyl 4- [N-maleimidomethyl]cyclohexane-l -carboxylate (sulfo-SMCC)

as a heterobifunctional linker to facilitate conjugation. Examples of synthetic ob

protein fragments include the following modified fragments of the mouse ob

sequence: (1) VPIQKVQDDTKTGCG-NH₂; (2) Acetyl-SNDLENLRDLLHGCG- NH₂; (3) Acetyl-CSLPQTSGLQKPESLDG-NH₂; (4) Acetyl-

GCGSLQDILQQLDVSPEA-OH; (5) Acetyl-GCGLSKMDQTLAVYQ- VLTSLPSQNVLQIANDLENLRD-NH₂; and, (6)

VPIQKVQDDTKTLIKTIVTPJNDISHTQSVG-CG-NH₂. Examples of synthetic

fragments of the human ob sequence include the following modified fragments: (1)

Ac-98-109-GCG- NH₂; (2) Ac-G-117-136- NH₂; and (3) 149-167-NH₂. Examples of

synthetic fragments of the rat ob sequence include the following: (1 ) Ac-148-167 and (2) GCG-52-71-NH₂. In addition a fragment generated by CNBr cleavage of rat

ob protein (74-167) may also be used for immunization. A 10 mg/mL solution of

carrier protein in 50 mM sodium borate buffer, pH 7.4 is made to which sulfo-

SMCC (Pierce, Rockford, IL) is added to a final concentration of 6 mg/mL. This

mixture is incubated at room temperature for 1-2 hours and then dialyzed against

water overnight at 4C to remove free SMCC. Peptide is dissolved at 1 mg/mL in

HPLC grade water and activated carrier protein added dropwise with stirring and the

pH adjusted as necessary to prevent precipitation of the peptide. The mixture is incubated with stirring at room temperature for 3 hours and then dialyzed against

water overnight at 4°C. Immunization using DNA constructs of either the human or rat ob sequence inserted into a mammalian expression vector, preferably pcDNA3

can also be prepared by injecting DNA, preferably at 100 μg/ml, in the quadriceps

muscle of the mouse, and have been successfully used to prepare anti-ob antibodies.

Glutaraldehyde conjugations were performed by mixing peptide a 2 ms/mL

in HPLC grade water with thyroglobulin made up in borate buffer as above ata a 2: 1

ratio of carrier protein to peptide. Glutaraldehyde is added to a final concentration

of 0.1%, the pH is adjusted as necessary to prevent precipitation of the peptide, and

the mixture is stirred a room temperature for 3 hours. The mixture is then dialyzed

at 4°C overnight against water. Preferably, the Balb/C (H-2D) and ND4 strains of mice (Harlan Sprague

Dawley; San Diego, CA) are used for immunizations. All animals are typically

about 8-12 weeks of age when immunization protocols are initiated. Animals are

primed intraperitoneally with 50 μg of antigen emulsified in Freund's Complete Adjuvant (Sigma). At three week intervals the animals are boosted intraperitoneally 6/31526 PCI7US96/04909

with 50 μg of antigen emulsified in monophosphoryl lipid A, trehalose dimycolate

(MPL+TDM RAS) adjuvant (Ribi Immunochem Research, INC., Hamilton, MT).

Animals are bled 7 days after the third injection and the peptide-specific antibody

measured by a solid-phase RIA, such as described below. Further boosts and bleeds

are performed as needed until a sufficient antibody titer is obtained.

Antisera are assayed by a solid-phase radioimmunoassay. Dynatech Removawell Immulon 2 plates (Dynatech, Chantilly, VA) are coated with 50 μl/well

goat anti-mouse IgG+IgM antibody (Accurate, Westbury, NY) diluted in 0.05 M

sodium carbonate buffer, pH 9.5 to a dilution of 20 μg/mL. The antibody is allowed

to passively adsorb overnight at 4°C. The plates are washed extensively with PBS+0.1% Tween 20 and water, blocked with 100 μl/well of 1% nonfat dry milk in sodium carbonate buffer for 1-2 hours, and washed again. Antisera are serially

diluted in hybridoma growth medium, RPMI 1640 (Irvine Scientific, Santa Ana,

CA) + 10% fetal bovine serum (Sigma) and 50 μl/well added to the plates and

incubated for 2 hrs. at room temperature. The plates are washed a second time, and

radiolabelled recombinant ob protein or the immunizing synthetic ob protein

fragment conjugated to BSA diluted in RIA buffer to 30,000 cpm/100 μl is added

and incubated at room temperature for 3 hours. RIA buffer consists of PBS, pH 7.4

with 0.1% Triton X-100, 0.1% teleostean gelatin (Sigma), 0.01% sodium azide, and

0.001% thimerosol. Following a final wash, the wells are separated and placed in

12 X 75 mm polystyrene tubes and counted, for example in a LKB Gammamaster

1277 gamma counter.

All cell lines are maintained in RPMI 1640 medium supplemented with 10%

FBS and 3% Origen Hybridoma Cloning Factor (HCF, Fisher Scientific, Tustin, CA) and incubated at 37°C in 95% air/5% CO2. Hybridomas are selected in growth

medium supplemented with hypoxanthine-aminopterin-thymidine (HAT)

(Boehringer Mannheim (Indianapolis, IN), as described in Littlefield, J. W., (1964)

Science 145: 709.

Three days after a final 50 μg intravenous boost, animals are sacrificed and

spleens removed. The spleens are teased apart and the splenocytes fused to P3X63- Ag8.653 myeloma cells (Kearney et al.. (1979) J. Immun. 123: 1548) at a ratio of

4:1. Polyethylene glycol 1,500 MW (Aldrich, Milwaukee, WS) at a concentration of

35% is used following a modification of the method of Gerhard (1980), in Kennet et al. (Eds.) Monoclonal Antibodies, page 370 (Plenum Press). Cells are plated at

about 1.5 X 10^ cells/well in HAT selective medium in 96- well microtiter plates and incubated for 10-14 days before the supernatants are screened for specific antibody.

Selected hybridoma cell lines are subcloned by limiting dilution, and

antibodies raised in ascites in either Balb/C or nude mice.

Example 9 - Vector Construct for FLAG-rat ob Periplasmic Expression

In order to prepare a vector constuct suitable for periplasmic expression in E.

coli BL21(DE3) cells, pFLAGROB#5 plasmid DNA (described in Example 4

above) was digested with restriction endonucleases Ndel and EcoRI (Boehringer Mannheim Coφ.) and the resulting fragments were run on a 1% agarose gel. The smaller fragment, approximately 540bp, containing the ompA signal and mature rat

ob coding region, was purified by running the DNA onto DE-81 paper and

subsequently eluting with IM NaCl. The isolated DNA fragment was ethanol

precipitated, dried and resuspended in sterile distilled water. Likewise, pET27b+ vector DNA was purchased from Novagen Inc. (Madison, WI), digested with Ndel and EcoRI and run on a 1% agarose gel. The vector DNA fragment was excised

from the gel and isolated using a silica membrane system (SpinBind^® DNA

Recovery System; FMC BioProducts, Rockland, ME) following the manufacturer's

protocol. The isolated insert DNA was ligated to the vector DNA followed by

transformation into competent E. coli BL21 cells (Novagen Inc.; Madison, WI).

Transformants were screened by isolating plasmid DNA using an alkaline lysate

method (Wizard^® Minipreps DNA Purification System, Promega, Madison, WI)

followed by restriction endonuclease digestion with Ndel and EcoRI to check for the

presence of the ompA/ratob insert. A positive clone, pET27ROB, was selected and

sequenced. pET27ROB DNA was used to transform competent E. coli BL21(DE3) (Novagen Inc., Madison WI) for expression of FLAG-rat ob protein.

Example 10 — Periplasmic expression of FLAG-rat ob protein

In order to evaluate methods for enhanced periplasmic production of ob

fusion proteins, a seed culture of pET27ROB from Example 9 was inoculated from a

previously prepared plate and grown overnight at 37°C in 150ml, of 20g/L LB

media (Gibco BRL, Gaithersburg, MD) containing 50mg/L kanamycin (Fisher

Biotech, Fair Lawn, NJ) to a final OD₆₀Q,,,,, of 3.46. Six 2L Fernbach flasks

containing 1L each of sterile filtered 25g/L LB media (Gibco BRL, Gaithersburg, MD) containing 50mg/L kanamycin were inoculated with 15mL of the overnight

seed culture. The six flasks were then grown at 27°C for 4 hours to an OD_600mn of

0.424 in the presence of 5g/L glucose and production of FLAG-rat ob protein was

induced by the addition of IPTG (isopropyl, thio β-G-galactosidase; Fisher Biotech, Fair Lawn NJ) to a final concentration of 0.5mM. After induction, the cells were

incubated for 3h at 28°C to a final ODg_oo,,,,, of 1.25 and harvested by centrifugation in a JA10 rotor for 15 min. at 5000φm using a model J2-21 centrifuge (Beckman

Instruments Inc., Fullerton, CA). The 13.4g cell pellet obtained in this manner was

then washed three times by resuspending the pellet to 25g/L with lOmM Tris buffer,

pH 8.0 and centrifuging the cells at 5000φm for 10 min. in a Beckman JA-10 rotor.

The washed cell pellet was then resuspended to 25g/L with 30mM Tris buffer, pH

8.0 containing 0.5M sucrose and ImM EDTA and incubated at room temperature for lOmin. to allow the cells to equilibrate. The cells were then removed from the

sucrose-containing buffer by centrifugation, as above, and shocked by resuspension

to 40g L with ice-cold water containing ImM Peflabloc SC (Centerchem, Stamford,

CT). The periplasmic extract obtained in this manner was clarified by centrifugation at 8500φm for 10 min. in a JA-10 rotor using a model J2-21 centrifuge (Beckman

Instruments Inc., Fullerton, CA) and stored at 4C until further purification. The

yield of FLAG-rat ob protein from this protocol was 41mg by HPLC analysis, nearly

7 mg/L of cells.

Example 11 —Purification of FLAG-Rat ob protein

FLAG-rat ob protein prepared as described in Example 10 was readied for purification by adding Tween-80 to a final concentration of 0.02%w/v and adjusting

the pH of the solution to 8.5 by the careful addition of lOOmM BisTris-propane free

base in water. The protein solution was then clarified by filtration through an

0.45μm Sartobran filter (Sartorius Coφ., Tustin, CA). The clarified solution was loaded onto DE-52 resin (Whatman Inc., Clifton, NJ) packed into an XK16 column

(Pharmacia Biotech, Piscataway, NJ) to a bed height of 6.25cm at a flow rate of

4mL/min. The column was washed with 17mM BisTris-propane containing 0.02%

Tween-80 at pH 8.5 and the rat ob protein was eluted using a linear gradient to 0.5M NaCl in 17mM BisTris-propane buffer containing 0.02% Tween-80 at pH 8.5.

Protein elution was monitored using absorbance detection at 280nm and verified by

SDS-PAGE and RP-HPLC analysis of fractions collected during the elution

gradient. Appropriate fractions were pooled, brought to pH 2.5 by the addition of

TFA to 0.3% v/v and loaded onto a VYDAC 218TP1022 HPLC column (Vydac, Hesperia, CA) using a flow rate of 15mL/min. The rat ob protein was eluted from

the column using a linear gradient from 0.1%TFA in 45% acetonitrile to 0.1% TFA

in 60% acetonitrile over 15 minutes. The elution of FLAG-rat ob protein was

detected by absorbance at 214nm and confirmed by SDS-PAGE. Monomeric

FLAG-rat ob protein and dimeric FLAG-rat ob protein were pooled separately, concentrated by evaporation using a Speedvac Plus SC110A (Savant, Farmingdale,

NY), flash frozen and lyophilized as TFA salts using a FREEZE DRYER 4.5

(LABCONCO, Kansas City, MO). The proteins obtained in this manner were >95%

pure by SDS-PAGE analysis. Monomeric and dimeric FLAG-rat ob proteins were

tested and shown to be active in vivo as described in Example 23.

Example 12 — Vector Construct for Rat ob Periplasmic Expression

In order to prepare a vector constuct suitable for periplasmic expression of

unfused rat ob proteins in BL21(DE3) E. coli cells, the coding region for mature rat

ob, Val²² to Cys¹⁴⁶, was PCR amplified from a cloning vector using reagents from

the Perkin Elmer PCR kit (Perkin Elmer, Foster City, CA). The first 18 bases of the

upstream primer, 5'-GCT ACC GTT GCG CAA GCT GTG CCT ATC CAC AAA GTC CAG G, are homologous to the coding sequence of the last 18 bases of the

ompA leader sequence. The remaining bases are homologous to the first 22 bases of the coding region for mature rat ob. The downstream primer used for the PCR was a vector specific, T7 terminator primer found in the pET series of expression vectors

(Novagen , Madison , WI). PCR amplification was performed in a lOOμl volume

containing 30ng of template DNA, 0.5μM of each primer, 2.5 units of AmpliTAQ

DNA^®Polymerase, lOmM Tris-HCl, pH 8.3, 50mM KC1, 0.2mM each dNTP, and

2.5mM MgCl₂ . The reaction was incubated for two cycles at 95°C/1 min, 65°C/1

min, and 72°C/lmin followed by 30 cycles of 95°C/30 sec, 65°C/30 sec, 72°C/30sec

followed by a final incubation of 10 min at 72°C. In a separate PCR reaction, the

ompA leader sequence was amplified from the pFLAGROB#5 vector of Example 4 using, as the upstream primer, the pFLAG vector specific, N26 primer. The first 18 bases of the downstream primer, 5'- GAC TTT GTG GAT AGG CAC AGC TTG

CGC AAC GGT AGC GAA AC, correspond to the sequence of the non-coding

strand of the first 18 bases of mature rat ob. The remaining 23 bases of the downstream primer correspond to the sequence of the non-coding strand of the last 23 bases of the ompA leader sequence. The PCR reaction conditions and cycling

times were set up exactly as for the previous reaction with the only difference being

the annealing temperature, which was 53°C for this reaction. A secondary PCR

reaction was performed in a lOOμl volume containing equimolar concentrations of the two primary PCR fragments along with the N26 and T7 terminator primers following the PCR procedure outlined above. The annealing temperature in the cycling profile was 65°C. The resulting PCR product is a fusion of the ompA leader

sequence with the mature rat ob coding sequence. The PCR fragment was purified using the QIAquick PCR Purification kit (QIAGEN Inc.) following the kit protocol,

digested with Ndel and EcoRI, and run on a 1% agarose gel. The digested fragment

was isolated from the gel and purified using a silica membrane system (SpinBind^® DNA Recovery System; FMC BioProducts, Rockland, ME) following the

manufacturer's protocol. The fragment was ligated into the pET27b+ vector also cut

with Ndel and EcoRI. The resulting subclone, pΕT27ROB(-), was sequenced to

confirm proper construction of the coding region.

Example 13 — Periplasmic expression of Rat ob protein

In order to evaluate methods for enhanced periplasmic production of unfused

ob proteins, a seed culture of pET27ROB(-) was inoculated from a frozen glycerol

stock and grown overnight at 37°C in 150mL of 20g/L LB media (Gibco BRL, Gaithersburg, MD) containing 50mg/L kanamycin (Fisher Biotech, Fair Lawn NJ) to

a final ODβoo,,,,, of 3.5. Six 2L Fernbach flasks containing 1L each of sterile filtered 25g/L LB media (Gibco BRL, Gaithersburg, MD) containing 5g L glucose and

50mg/L kanamycin were inoculated with 15mL of the overnight seed culture. The

six flasks were then grown at 30°C for 2.5 hours to an OD,-^,,, of 0.49 and

production of rat ob protein was induced by the addition of IPTG (isopropyl, thio β-

G-galactosidase; Fisher Biotech, Fair Lawn NJ) to a final concentration of 0.5mM.

After induction, the cells were incubated for 3h at 30°C to a final OD_600mn of 1.53

and harvested by centrifugation in a JA10 rotor for 15 min. at 5000φm using a

model J2-21 centrifuge (Beckman Instruments Inc., Fullerton, CA). The 20g cell

pellet obtained in this manner was then washed three times by resuspending the

pellet with 800mL of lOmM Tris buffer, pH 8.0 and centrifuging the cells at 5000φm for 10 min. in a Beckman JA-10 rotor. The washed cell pellet was then

resuspended in 800mL of 30mM Tris buffer, pH 8.0 containing 0.5M sucrose and

ImM EDTA and incubated at room temperature for lOminutes to allow the cells to

equilibrate. The cells were then removed from the sucrose-containing buffer by centrifugation, as above, and shocked by resuspension in 500mL of ice-cold water containing ImM Peflabloc. The periplasmic extract obtained in this manner was

clarified by centrifugation at 8500φm for 10 minutes in a Beckman JA-10 rotor and

stored at 4°C until further purification. The yield of rat ob protein from this protocol

was 40.6mg by HPLC analysis, nearly 7 mg/L of cells.

Example 14 — Purification of Rat ob protein

Rat ob protein prepared as described in Example 13 was readied for

purification by adding Tween-80 to a final concentration of 0.02%w/v and adjusting

the pH of the solution to 8.5 by the careful addition of lOOmM BisTris-propane free

base in water. The protein solution was then clarified by centrifugation at 13,000xg for 25min., and filtration through a 5μm Millex-SV filter (Millipore, ) followed by

filtration through an 0.45μm Sterivex-HV filter. The clarified solution was loaded

onto a 14mL Q-Sepharose HP column (Pharmacia Biotech, ) at a flow rate of

6mL/min. The column was then washed with 17mM BisTris-propane containing

0.02% Tween-80 at pH 8.5 and the rat ob protein was eluted using a linear gradient

to 0.17M NaCl in 17mM BisTris-propane buffer containing 0.02% Tween-80 at pH

8.5. Protein elution was monitored using absorbance detection at 280nm and

verified by SDS-PAGE and RP-HPLC analysis of fractions collected during the elution gradient. Appropriate fractions were pooled, brought to pH 2 by the addition

of TFA to 0.2% v/v and loaded onto a VYDAC 218TP 1022 HPLC column (Vydac,

Hesperia, CA) using a flow rate of 15mL/min. The rat ob protein was eluted from

the column using a linear gradient from 0.1%TFA in 45% acetonitrile to 0.1% TFA

in 60% acetonitrile over 15 minutes. The elution of rat ob protein was detected by absorbance at 214nm and confirmed by SDS-PAGE. Monomeric rat ob protein and dimeric rat ob protein were pooled separately, concentrated by evaporation using a

Speedvac Plus SCI 10A (Savant, Farmingdale, NY), flash frozen and lyophilized as a TFA salt using a FREEZE DRYER 4.5 (LABCONCO, Kansas City, MO). The

proteins obtained in this manner was >95% pure by SDS-PAGE analysis.

Monomeric rat ob protein prepared as described herein was tested and shown to be

active in vivo as described in Example 23.

Example 15 - Vector Construct for Met-rat ob Intracellular Expression

A vector construct containing a modified rat ob coding sequence was prepared to evaluate its utility for intracellular protein expression. The coding

region for mature rat ob, Val²² to Cys¹⁴⁶, was PCR amplified from a cloning vector

using reagents from the Perkin Elmer PCR kit (Perkin Elmer, Foster City, CA). The

upstream primer, 5'-TATACAT ATG GTT CCG ATC CAC AAA GTC CAG GAT

GAC, incoφorated an Ndel site (underlined) onto the 5' end of the PCR fragment to facilitate cloning. The Ndel site also has within it the initiating methionine codon,

ATG. The codons for Val²² (GTT) and Pro²³ (CCG) of the rat ob protein were

changed from the original DNA sequence to preferred codons for E. coli expression.

The downstream primer used for PCR was a vector (pET15b, Novagen Inc,

Madison, WI) specific, T7 terminator primer. PCR amplification was performed in

a lOOμl volume containing 50ng of pET15ROB (a construct containing a T7

promoter and the native codons for the Met-rat ob sequence), 0.5μM each primer,

2.5 units of AmpliTAQ DNA ^® Polymerase, lOmM Tris-HCl, pH 8.3, 50mM KC1,

0.2mM each dNTP, and 2.5mM MgCl₂. The reaction was incubated for 2 cycles at

95°C/1 min, 53°C/1 min, and 72°C/1 min followed by 30 cycles at 95°C/30 sec,

53°C/30sec, and 72°C/30 sec followed by a final incubation of 10 min at 72°C. Analytical agarose gel electrophoresis confirmed a single band of the expected size,

approximately 540bp. The remaining PCR reaction material was purified using the

QIAquick PCR Purification kit (QIAGEN Inc.) following the kit protocol. The PCR

fragment was digested with Ndel and ΛTioI and cloned into the pET27b+ vector

(Novagen Inc, Madison WI) previously digested with the same enzymes and treated

with Calf Intestinal Alkaline Phosphatase (Boehringer Mannheim Coφ.). The

resulting subclone, pET27OPTII #4, was sequenced using rat ob specific primers to

confirm proper construction of the coding region. pET27OPTII DNA was used to

transform competent E. coli BL21(DE3) for expression of the Met-rat ob protein. Example 16 — Intracellular Expression of Met-rat ob in Inclusion Bodies

In order to evaluate methods for enhanced intracellular production of unfused

ob proteins, including ob proteins having an N-terminal methionine, a seed culture

of pET27OPTII was inoculated from a frozen glycerol stock and grown overnight at

37°C in 150mL of 20g/L LB media (Gibco BRL, Gaithersburg, MD) containing

50mg/L kanamycin (Fisher Biotech, Fair Lawn NJ) to a final OD^_^, of 3.86. Six

2L Fernbach flasks containing 1L each of 25g/L LB media containing 50mg/L

kanamycin were inoculated with 20mL of the of the overnight seed culture, grown at

35°C for 2.5 hours (OD^ = 0.723) and production of Met-rat ob protein was

induced by the addition of IPTG (isopropyl, thio β-G-galactosidase; Fisher Biotech,

Fair Lawn NJ) to a final concentration of ImM. After induction, the cells were

incubated for 5.5h at 36°C and harvested by centrifugation in a JA10 rotor for 15

min. at 5000φm using a model J2-21 centrifuge (Beckman Instruments Inc.,

Fullerton, CA). The 18.1g cell pellet was flash frozen and stored at -20°C. The cell pellet was thawed and resuspended in lysis buffer (500mL of lOOmM potassium phosphate, pH 6.5) and lysed by 2 passages through a model HOY microfluidizer

(Microfluidics Coφ., Newton, MA). The inclusion bodies from the lysed cells were

collected by centrifugation at 5000xg for 25 minutes at 4°C using an RC-3B

centrifuge (Dupont Co., Willmington, DE). Met-rat ob protein-containing inclusion

bodies were washed by resuspending with 500mL of lOOmM potassium phosphate,

pH 6.5 and collected by centrifugation as above. The washed inclusion bodies were

frozen and stored at -80°C. The typical yield was 1.8g of Met-rat ob protein per 6L

of cultured cells.

Example 17 — Solubilization and Refolding of Met-rat ob Protein from Inclusion Bodies to Generate Both Monomeric and Dimeric Protein Forms

In order to generate quantities of properly folded dimeric as well as

monomeric ob protein suitable for further purification, the phosphate-washed inclusion bodies prepared as described in Example 16 were thawed and dissolved in

50mM ammonium bicarbonate containing 8M urea at 6mg of wet inclusion body

material per mL of solubilization buffer. The solubilized protein (360mL) was

dialyzed at 4°C overnight against 10L of 17mM Bis-Tris propane containing 0.02% Tween-80 at pH 8.5 using SpectraPor regenerated cellulose membrane with a stated

molecular weight cutoff of 3500 daltons. Reversed-phase HPLC analysis showed that the protein in the initial urea containing solubilization buffer contained 1-4%

dimer as a percent of the total ob protein, while the dialyzed material contained 23%

dimer, thus confirming the desired conversion of ob monomer to ob dimer form.

Proper folding was demonstrated by in vivo activity of the isolated purified proteins,

as described in Example 23.

Example 18 - Cellulose-based Anion Exchange Purification of Refolded Met- rat ob Protein This experiment shows that purification of ob proteins, including ob

monomer, ob fusion monomer, ob dimer and ob fusion dimer, using cellulose-based

resin results in suφrisingly high yield recovery of protein. DE-52 resin (Whatman

Inc., Clifton, NJ) was swollen in 17mM BisTris-propane containing IM sodium

chloride at pH 8.4 and packed into a Pharmacia Biotech XK16 column to a packed bed height of 5.6cm. The Met-rat ob protein solution (271mg monomer and 76mg

dimer in 260mL) was loaded onto the column using a flow rate of 4mL/min. The

Met-rat ob protein was eluted using a linear gradient from 17mM BisTris-propane

containing 0.02% Tween-80 at pH 8.5 to 17.5mM Bis-Tris propane containing 0.5M

NaCl and 0.02% Tween-80 at pH 8.5. Protein elution was monitored using absorbance detection at 280nm and verified by SDS-PAGE and RP-HPLC analysis

of fractions collected during the elution gradient. Appropriate fractions were pooled

to give an average 70% yield of purified monomeric and dimeric Met-rat ob protein.

Example 19 — Separation and Further Purification of Met-rat ob Protein

Monomer and Dimer with Reversed Phase-HPLC

Pooled anion exchange fractions prepared as described in Example 18 were

carefully acidified to a final concentration of 0.3% TFA using a 10% solution of

TFA in water. The acidified protein fractions were loaded onto a Vydac 218TP 1022

HPLC column (Vydac, Hesperia, CA) at a flow rate of 15mL/minute and eluted using a linear gradient from 0.1% TFA in 45% acetonitrile to 0.1% TFA in 65%

acetonitrile over 20 minutes. Protein elution was monitored by absorbance at 214nm

and appropriate fractions were pooled and concentrated to remove acetonitrile by evaporation using a Speedvac Plus SCI 10A (Savant, Farmingdale, NY). Using this technique, monomer was separated from dimer and trace remaining endotoxin was removed from the preparation (to a level less than 1 EU/mg). When the preparation

of the TFA salt of the protein was desired, the concentrated HPLC pool was flash-

frozen and lyophilized on a FREEZE DRYER 4.5 (LABCONCO, Kansas City,

MO).

Example 20 — Ob Protein as a Lyophilized Ammonium Bicarbonate Salt

Monomeric or dimeric ob proteins may be prepared as lyophilized

ammonium bicarbonate salts. In this experiment, solutions of monomeric and dimeric ob protein prepared as described in Example 19, or the TFA salt form of

these proteins dissolved in 0.1% TFA in water, were brought to pH 7 by the addition of concentrated ammonium bicarbonate, transferred to SpectraPor 3500MW cutoff

dialysis tubing and fully dialyzed vs. 20mM ammonium bicarbonate (pH 7.8 when

freshly dissolved) at 4°C. The dialyzed protein solutions were flash frozen and

lyophilized to dryness using a FREEZE DRYER 4.5 (LABCONCO, Kansas City,

MO). The resulting ammonium bicarbonate salts of Met-rat ob protein were shown

to be soluble to at least lOmg/mL in water. Both preparations of the ammonium

bicarbonate salt form of ob protein were tesed and shown to be active in vivo as

described in Example 23.

Example 21 - Ob Proteins Formulated as a Clear, Stable Buffered Solution

Monomeric or dimeric ob proteins may be prepared in a stable liquid

formulation using BisTris propane, with or without a detergent (such as Tween 80),

and optionally containing a preservative (such as phenol). In this experiment,

lyophilized monomeric ob protein prepared as described in Example 19 was

dissolved in 0.1% TFA in water to a final concentration of 2.5mg/rnL. The pH of

the resulting solution was adjusted to 8.4 by the careful addition of lOOmM Bis-Tris propane. The resulting protein solution was transferred into SpectraPor 3500MW

cutoff dialysis tubing and fully dialyzed against 20mM Bis-Tris propane pH 8.4 to

yield a clear solution. 2 mg/mL solutions of monomeric ob protein in 20 mM

BisTris propane (pH 8.4), with and without 0.02% Tween 80 with and without 0.1

M sodium chloride, have been shown to be stable for at least 30 days at room

temperature.

Example 22 — High Yield Production of Met-rat ob Protein Monomer and

Dimer

This fiirther set of experiments exemplifies the efficient production of ob proteins using the above-described techniques. A seed culture of pET27OPΗI from Example 15 was inoculated from a frozen glycerol stock and grown overnight in

150mL of 20g/L LB media (Gibco BRL, Gaithersburg, MD) containing 50mg/L

kanamycin (Fisher Biotech, Fair Lawn, NJ) at 37°C to a final OD^^ of 3.87. Six

2L Fernbach flasks containing 1L each of sterile filtered 25g/L LB media containing

50mg/L kanamycin were inoculated with 20mL of the of the overnight seed culture,

grown at 36°C for 2 hours to an OD_600nm of 0.875) and production of Met-rat ob

protein was induced by the addition of IPTG (isopropyl, thio β-G-galactosidase;

Fisher Biotech, Fair Lawn, NJ) to a final concentration of ImM. After induction, the

cells were incubated for 4.5h at 37°C and harvested by centrifugation in a JA10 rotor for 15 min. at 5000φm using a model J2-21 centrifuge (Beckman Instruments Inc.

Fullerton, CA). The 19.2g cell pellet was flash frozen and stored at -20°C. The cells were resuspended in lOOmM potassium phosphate, pH 6.5 and lysed with two

passages through a model 110Y microfluidizer (Microfluidics Coφ., Newton, MA)

in 500mL of lOOmM potassium phosphate buffer, pH 6.5. The inclusion bodies containing Met-rat ob were recovered by centrifugation at 6000xg for 25minutes in

an RC-3B centrifuge (Dupont Co., Willmington, DE). The inclusion bodies were

resuspended in phosphate buffer and recovered by centrifugation to yield 4.35g of

Met-rat ob protein-containing inclusion bodies which contained 30% by weight Met-

rat ob protein.

The inclusion body pellet was dissolved in 725mL of 50mM ammonium

bicarbonate buffer containing 8M urea and dialyzed against 20L of 17mM Bis-Tris propane overnight at 4°C in 3500 MW cut-off Spectra-Por dialysis tubing. Prior to

dialysis, the inclusion body solution contained 1.2g of monomeric met-rat ob protein and 0.07g of dimeric met-rat ob protein. After dialysis, the solution contained 0.9g

of monomeric Met-rat ob protein and 0.27g of dimeric Met-rat ob protein. The

dialyzed solution was filtered through a Sartobran 300 capsule filter (Sartorius

Coφ., Tustin, CA) and loaded onto a 29mL Whatman DE-52 (Whatman Inc., Clifton, NJ) anion exchange column at a flow rate of lOmL/min. The column was

washed with 17mM BisTris-propane containing 0.02%v/v Tween-80 at pH 8.5 and

eluted using a 30min. gradient to 0.5M sodium chloride in the same buffer. Protein

elution was monitored by absorbance at 280nm and verified by SDS-PAGE and RP- HPLC analysis of fractions collected during the salt gradient. Appropriate fractions

were pooled, acidified to 0.3% TFA and further purified by RP-HPLC on a Vydac

218TP1022 column (Vydac, Hesperia, CA) using a 20 minute gradient from

0.1%TFA in 45% acetonitrile to 0.1% TFA in 65% acetonitrile. Fractions were

collected during the acetonitrile gradient and Met-rat ob protein elution was

monitored using absorbance at 214nm. The monomeric and dimeric forms of Met-

rat ob protein were pooled separately and concentrated for lh on a Speedvac Plus SC110A (Savant, Farmingdale, NY) to remove acetonitrile, and then brought to pH

7 by the careful addition of 0.5 M ammonium bicarbonate. Each of the neutralized

protein solutions (194mL of monomer and 108mL of dimer) were dialyzed against

10L of 20mM ammonium bicarbonate buffer (pH 7.8 when freshly dissolved) with 5

changes of the dialysis buffer using SpectraPor 3500MW cutoff membranes. The

dialyzed protein solutions were filtered, flash frozen and lyophilized on a FREEZE

DRYER 4.5 (LABCONCO, Kansas City, MO) to yield 428mg of monomeric Met- rat ob protein and 74mg of dimeric Met-rat ob protein. Both proteins appeared

>95% pure by analytical HPLC and SDS-PAGE. The overall yield of the process

was 40%.

Example 23 — In vivo Testing of Bioactivity of ob Proteins The ob protein to be tested in these experiments was dissolved at 2mg/mL in

water at room temperature with vortexing. If necessary the solution was allowed to

stand for 30 min. and vortexed again. The solution was then diluted to 1 mg/mL by

the addition of 34mM BisTris-propane containing 0.02% Tween-80, pH 8.5 and

stored at room temperature prior to use.

10 - 17 week old ob/ob mice (C57BL/6J-ob/ob) were utilized for the study.

Mice were obtained from Jackson Laboratories, Bar Harbor, ME and housed in

groups of 10 animals in the vivarium with 12:12 light:dark cycle with room temperature of 23 ± 1 °C. One week prior to the start of experiments, the animals were divided in to groups of either 4 or 6 animals per group and housed 2 animals

per cage. Daily food intake and body weight data were collected and utilized as

baseline. 10-14 week old NIH/Sw mice were utilized for the study. Mice were

obtained from Harlan Laboratories, Madison, WI and housed in groups of 10

animals in the vivarium with 12:12 ligh dark cycle with room temperature of 23 ± 1

°C. One week prior to the start of experiments, the animals were divided in to

groups of either 4 or 6 animals per group and housed 2 animals per cage. Daily food intake and body weight data were collected and utilized as baseline.

All animals received subcutaneous injection of either vehicle or test material

twice daily for five days. Morning injections were given between 6 and 7 am and

evening injections were given between 5 and 6 PM. Daily food intake and body weight data was collected for all animals. Deviations from baseline food intake and body weight were calculated on a daily basis. A dose response curve of % decrease

in body weight and % decrease in food intake, as observed after five days of

treatment was plotted using Graphpad Prizm, San Diego, CA.

The compounds of the invention had the following effects on food intake.

Rat ob protein, prepared as in Example 14, dose dependently reduced food intake in

the ob/ob mice. See Figure 2. The ED₅₀ for rat ob protein, for reduction in food

intake after 5 days of treatment was calculated to be 0.04 mg/kg ± 0.06 log units

given twice a day.

Met-rat ob monomer, prepared as in Example 20, was tested at two doses

(0.05 and 0.5 mg/kg, twice a day) and showed effect on reduction in food intake,

after five days of treatment, similar to rat ob protein. See Figure 3.

Met-rat ob dimer, prepared as in Example 22, was tested at three doses (0.03,

0.3 and 3.0 mg/kg, twice a day) and showed a dose dependent reduction in food

intake. See Figure 4. The ED₅₀ for Met-rat ob dimer, for reduction in food intake, after 5 days of treatment, was calculated to be 0.41 mg/kg ± 0.20 log units given

twice a day.

FLAG-rat ob monomer, prepared as in Example 11, was tested at two doses

(0.10 and 1.0 mg/kg, twice a day) and showed effect on reduction in food intake

after five days of treatment, similar to rat ob protein. See Figure 5.

FLAG-rat ob dimer, prepared as in Example 11, was tested at two doses (0.1

and 1.0 mg/kg, twice a day) and showed effect on reduction in food intake after five days of treatment, similar to Met-rat ob dimer. See Figure 6.

Rat ob protein, prepared as in Example 14, also dose dependently reduced

food intake in the NIH/Sw mice. See Figure 7. The ED₅₀ for rat ob protein, for

reduction in food intake after 5 days of treatment was calculated to be 0.01 mg/kg ±

0.87 log units given twice a day in NIH/Sw mice.

The compounds of the invention had the following effects on body weight.

Rat ob protein, prepared as in Example 14, dose dependently reduced body weight in

the ob/ob mice. See Figure 8. The ED₅₀ for rat ob protein, for reduction in body

weight after 5 days of treatment was calculated to be 0.09 mg/kg ± 0.14 log units given twice a day.

Met-rat ob monomer, prepared as in Example 20, was tested at two doses

(0.05 and 0.5 mg/kg, twice a day) and showed effect on reduction in body weight, after five days of treatment, similar to rat ob protein. See Figure 9.

Met-rat ob dimer, prepared as in Example 22, was tested at three doses (0.03,

0.3 and 3.0 mg/kg, twice a day) and showed a dose dependent reduction in body

weight. See Figure 10. The ED₅₀ for Met-rat ob dimer, for reduction in body weight, after 5 days of treatment, was calculated to be 1.01 mg/kg ± 0.21 log units

given twice a day.

FLAG-rat ob monomer, prepared as in Example 11, was tested at two doses

(0.10 and 1.0 mg/kg, twice a day) and showed effect on reduction in body weight after five days of treatment, similar to rat ob protein. See Figure 11.

FLAG-rat ob dimer, prepared as in Example 11, was tested at two doses (0.1

and 1.0 mg/kg, twice a day) and showed effect on reduction in body weight after five

days of treatment, similar to Met-rat ob dimer. See Figure 12.

Rat ob protein, prepared as in Example 14, also dose dependently reduced body weight in the NIH/Sw mice. See Figure 13. The ED₅₀ for rat ob protein, for reduction in body weight after 5 days of treatment was calculated to be 0.06 mg/kg ±

0.15 log units given twice a day in NIH/Sw mice.

Example 24 — E. coli Expression of Met-human ob Protein

In order to prepare a vector construct for high level production of Met-human ob protein, the coding region for mature human ob, Val²² to Cys¹⁴⁶, was PCR

amplified from a cloning vector using the reagents from the Perkin Elmer PCR kit

(Perkin Elmer, Foster City, CA). The upstream primer, 5'- TATACAT ATG GTT

CCG ATC CAG AAA GTC CAA GAT GAC, incoφorated an Ndel site

(underlined) onto the 5' end of the PCR fragment to facilitate cloning. The Ndel site also has within it the initiating methionine codon, ATG. The included codons for

Val²² (GTT), Pro²³ (CCG), and Gin²⁵ (CAG) of the human ob protein have been changed from the original DNA sequence to preferred codons for E. coli expression.

The downstream primer used for PCR was a vector (pET27b+, Novagen Inc.,

Madison, WI) specific, T7 terminator primer. PCR amplification was performed in a 100 μl volume containing 50ng of template DNA, 0.5μM each primer, 2.5 units of

AmpliTAQ DNA^® Polymerase, lOmM Tris-HCl, pH 8.3, 50mM KC1, 0.2mM each

dNTP, and 2.0mM MgCl₂. The reaction was incubated for 2 cycles at 95°C/1 min,

47°C/1 min, and 72°C/1 min followed by 30 cycles at 95°C/30 sec, 65°C/ 30 sec, and

72°C/ 30 sec followed by a final incubation of 10 min at 72°C. Analytical agarose

gel electrophoresis confirmed a single band of the expected size, approximately

650bp. The remaining PCR reaction material was ethanol precipitated, resuspended in water and ligated into the holding vector, pCRII (TA Cloning Kit, Invitrogen

Coφ., San Diego, CA) following kit protocol. Positive clones were selected and characterized to confirm the presence of the inserted PCR fragment. The Met-

human ob coding region was isolated from the pCR vector by digesting the vector

DNA with the restriction enzymes Ndel and Xhόl. The insert fragment,

approximately 540bp, was isolated from an agarose gel and purified using a silica

membrane system (SpinBind^® DNA Recovery System; FMC BioProducts,

Rockland, ME) following the manufacturer's protocol. The fragment was ligated

into the pET27b+ vector also cut with Ndel and Xhόl. The resulting vector,

pET27HOPT#l, was sequenced to confirm proper construction of the coding region.

Example 25 - High Yield Production of Met-human ob Protein Monomer and Dimer

This further set of experiments exemplifies the efficient production of Met-

human ob proteins using the above-described techniques. A seed culture of

pET27HOPT#l from Example 24 was inoculated from a previously prepared plate

and grown overnight in 150mL of 25g/L LB media (Gibco BRL, Gaithersburg, MD)

containing 50mg/L kanamycin (Fisher Biotech, Fair Lawn, NJ) at 37°C to a final ODeoo_nm of 3.33. Six 2L Fembach flasks containing 1L each of sterile filtered 25g/L

LB media containing 50mg/L kanamycin were inoculated with 20mL of the of the

overnight seed culture, grown at 36°C-37°C for 2 hours to an OD^,,, of 0.801) and

production of Met-human ob protein was induced by the addition of IPTG

(isopropyl, thio β-G-galactosidase; Fisher Biotech, Fair Lawn, NJ) to a final

concentration of ImM. After induction, the cells were incubated for 4h at 37°C and

harvested by centrifugation in a JA10 rotor for 15 min. at 5000φm using a model

J2-21 centrifuge (Beckman Instruments Inc. Fullerton, CA). The 15.9g cell pellet

was resuspended in lOOmM potassium phosphate, pH 6.5 and lysed with two

passages through a model HOY microfluidizer (Microfluidics Coφ., Newton, MA)

in 500mL of lOOmM potassium phosphate buffer, pH 6.5. The inclusion bodies

containing Met-human ob were recovered by centrifugation at 6000xg for 30minutes in an RC-3B centrifuge (Dupont Co., Willmington, DE). The inclusion bodies were

resuspended in phosphate buffer and recovered by centrifugation to yield 3.9g of

Met-human ob protein-containing inclusion bodies.

The inclusion body pellet was dissolved in 650mL of 50rnM ammonium bicarbonate buffer containing 8M urea and dialyzed against 20L of 17mM Bis-Tris

propane overnight at 4°C in 3500 MW cut-off Spectra-Por dialysis tubing. After dialysis, the solution contained 0.9g of monomeric Met-human ob protein and 0.27g

of dimeric Met-human ob protein. The dialyzed solution was filtered through a

Sartobran 300 capsule filter (Sartorius Coφ., Tustin, CA) and loaded onto a 16mL

Whatman DE-52 (Whatman Inc., Clifton, NJ) anion exchange column at a flow rate

of lOmL/min. The column was washed with 17mM BisTris-propane containing

0.02%v/v Tween-80 at pH 8.5 and eluted using a 30minutes gradient to 0.5M sodium chloride in the same buffer. Protein elution was monitored by absorbance at

280nm and determined by RP-HPLC analysis of fractions collected during the salt

gradient, to yield 600 mg of monomeric Met-human ob protein and 42mg of dimeric Met-human ob protein.

Example 26 — Periplasmic expression of FLAG-human ob protein

In order to prepare a vector construct for periplasmic expression of FLAG- human ob protein, the coding region for human ob was inserted into a vector

construct as described in Example 4. A seed culture of vector pFLAGATSHOB#5 in W3110 cells was inoculated from a previously prepared plate and grown

overnight at 37°C in 150mL of 20g/L LB media (Gibco BRL, Gaithersburg, MD)

containing 50mg/L ampicillin to a final ODgoo,,,,, of 3.46. Six 2L Fernbach flasks

containing 1L each of sterile filtered 25g/L LB media (Gibco BRL, Gaithersburg, MD) containing 50mg/L ampicillin were inoculated with 20mL of the overnight

seed culture. The six flasks were then grown at 27°C for 3.5 hours to an OD_600nm of

0.401 in the presence of 5g/L glucose and production of FLAG-human ob protein

was induced by the addition of IPTG (isopropyl, thio β-G-galactosidase; Fisher

Biotech, Fair Lawn NJ) to a final concentration of 0.5mM. After induction, the cells

were incubated for 3h at 28°C to a final OD^o,,,,, of 1.94 and harvested by

centrifugation in a JA10 rotor for 15 min. at 5000φm using a model J2-21 centrifuge (Beckman Instruments Inc., Fullerton, CA). The 18.0g cell pellet

obtained in this manner was then washed three times by resuspending the pellet to

25g/L with lOmM Tris buffer, pH 8.0 and centrifuging the cells at 5000φm for 10

minutes in a Beckman JA-10 rotor. The washed cell pellet was then resuspended to 25g/L with 30mM Tris buffer containing 0.5M sucrose and ImM EDTA at pH 8.0 and incubated at room temperature for 10 minutes to allow the cells to equilibrate.

The cells were then removed from the sucrose-containing buffer by centrifugation,

as above, and shocked by resuspension to 40g/L with ice-cold water containing ImM Peflabloc SC (Centerchem, Stamford, CT). The periplasmic extract obtained

in this manner was clarified by centrifugation at 8500φm for 10 minutes in a JA-10

rotor using a model J2-21 centrifuge (Beckman Instruments Inc., Fullerton, CA) and stored at 4°C until further purification. The yield of FLAG-human ob protein from

this protocol was 58.5mg by HPLC analysis

Tween-80 was added to the periplasmic extracts to a final concentration of

0.02%w/v and the pH of the resulting solution was adjusted to 8.5 by the careful addition of lOOmM BisTris-propane free base in water. The protein solution was

then clarified by filtration through an 0.45μm Sartobran filter (Sartorius Coφ.,

Tustin, CA). The clarified solution was loaded onto DE-52 resin (Whatman Inc., Clifton, NJ) packed into an XK16 column (Pharmacia Biotech, Piscataway, NJ) to a

bed height of 4.5cm at a flow rate of 4mL/minute. The column was washed with

17mM BisTris-propane containing 0.02% Tween-80 at pH 8.5 and the FLAG-human

ob protein was eluted using a linear gradient to 0.5M NaCl in 17mM BisTris- propane buffer containing 0.02% Tween-80 at pH 8.5. Protein elution was

monitored using absorbance detection at 280nm and verified by SDS-PAGE and RP-

HPLC analysis of fractions collected during the elution gradient. Appropriate

fractions were pooled, brought to pH 2.5 by the addition of TFA to 0.3% v/v and loaded onto a VYDAC 218TP1022 HPLC column (Vydac, Hesperia, CA) using a

flow rate of 15mL/min. The FLAG-human ob protein was eluted from the column

using a linear gradient from 0.1%TFA in 45% acetonitrile to 0.1% TFA in 60% acetonitrile over 15 minutes. The elution of FLAG-human ob protein was detected

by absorbance at 214nm and confirmed by SDS-PAGE. Monomeric FLAG-human ob protein was concentrated by evaporation using a Speedvac Plus SCI 10A (Savant,

Farmingdale, NY), flash frozen and lyophilized as TFA salts using a FREEZE

DRYER 4.5 (LABCONCO, Kansas City, MO). The proteins obtained in this

manner were >95% pure by SDS-PAGE analysis.

Various modifications of the invention in addition to those shown and

described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the following claims.

Claims

WE CLAIM:

1. A method for the production of an ob dimer which comprises the steps of

(a) isolating a vertebrate cDNA library,

(b) ligating said cDNA library into a cloning vector,

(c) introducing said cloning vector containing said cDNA library into a first host cell,

(d) contacting the cDNA molecules of said first host cell with a solution containing a suitable ob gene hybridization probe,

(e) detecting a cDNA molecule which hybridizes to said probe,

(f) isolating said cDN A molecule,

(g) ligating the nucleic acid sequence of said cDNA molecule which encodes an ob protein into an expression vector,

(h) transforming a second host cell with said expression vector

containing said nucleic acid sequence of said cDNA molecule which encodes said ob protein,

(i) culturing the transformed second host cell under conditions that favor the production of said ob protein as a dimer, and

(j) isolating said ob protein expressed by said second host cell.

2. The method of claim 1 wherein said vertebrate cDNA library comprises a

vertebrate adipose tissue cDNA library.

3. The method of claim 2 wherein said vertebrate adipose tissue cDNA

library comprises a human adipose tissue cDNA library.

4. A method of producing a ob dimer which comprises the steps of (a) culturing a transformed host cell containing a DNA sequence

encoding a vertebrate ob protein under conditions that favor the production of

said vertebrate ob protein as a dimer, and

(b) isolating said ob dimer expressed by said transformed host cell.

5. The method of claim 4 wherein said vertebrate ob protein is a human ob

protein.

6. A method of producing a ob dimer which comprises the steps of

(a) culturing a transformed host cell containing a DNA sequence

encoding a vertebrate ob protein under conditions that favor the production of

said vertebrate ob protein as a monomer,

(b) isolating said ob protein expressed by said transformed host cell,

and

(c) dimerizing said ob protein.

7. The method of claim 6 wherein said vertebrate ob protein is a human ob

protein.

8. A method for the production of an ob dimer which comprises the steps of

(a) isolating a preparation of total RNA from a vertebrate tissue,

(b) converting said total RNA to cDNA,

(c) amplifying a cDNA sequence from said cDNA using

oligonucleotide primers suitable for annealing to a target ob protein gene

sequence,

(d) detecting a cDNA molecule using oligonucleotides suitable for

hybridization to said target ob protein gene sequence, (e) isolating said cDNA molecule,

(f) ligating the nucleic acid sequence of said cDNA molecule which encodes an ob protein into an expression vector,

(g) transforming a host cell with said expression vector containing said nucleic acid sequence of said cDNA molecule which encodes said ob protein,

(h) culturing the transformed host cell under conditions that favor the

production of said ob protein as a dimer, and

(i) isolating said ob protein expressed by said host cell.

9. The method of claim 8 wherein said vertebrate tissue is a vertebrate

adipose tissue.

10. The method of claim 9 wherein said vertebrate adipose tissue is human

adipose tissue.

11. A method for the production of an ob dimer fusion protein or ob monomer fusion protein which comprises the steps of ^•

(a) isolating a vertebrate cDNA library,

(b) ligating said cDNA library into a cloning vector,

(c) introducing said cloning vector containing said cDNA library into

a first host cell,

(d) contacting the cDNA molecules of said first host cell with a

solution containing a suitable ob gene hybridization probe,

(e) detecting a cDNA molecule which hybridizes to said probe,

(f) isolating said cDNA molecule, (g) ligating the nucleic acid sequence of said cDNA molecule which

encodes an ob protein to a second DNA sequence to create a fusion DNA

sequence,

(h) ligating said fusion DNA sequence into an expression vector,

(i) transforming a second host cell with said expression vector

containing said fusion DNA sequence,

(j) culturing the transformed second host cell under conditions that

favor the production of said ob fusion protein as a dimer or monomer, and

(k) isolating said ob protein expressed by said second host cell.

12. The method of claim 11 wherein said vertebrate cDNA library comprises a vertebrate adipose tissue cDNA library.

13. The method of claim 12 wherein said vertebrate adipose tissue cDNA

library comprises a human adipose tissue cDNA library.

14. A method of producing an ob dimer fusion protein or ob monomer

fusion protein which comprises the steps of

(a) culturing a transformed host cell containing a DNA sequence

encoding a vertebrate ob protein coupled to a marker DNA sequence under

conditions that favor the production of said vertebrate ob fusion protein as a

dimer or monomer, and

(b) isolating said ob dimer fusion protein or ob monomer fusion

protein expressed by said transformed host cell.

15. The method of claim 14 wherein said vertebrate ob protein is a human

ob protein.

16. A method of producing an ob dimer fusion protein which comprises the

steps of

(a) culturing a transformed host cell containing a DNA sequence

encoding a vertebrate ob protein coupled to a marker DNA sequence under conditions that favor the production of said vertebrate ob fusion protein as a

monomer,

(b) isolating said ob fusion protein expressed by said transformed

host cell, and

(c) dimerizing said ob fusion protein.

17. The method of claim 16 wherein said vertebrate ob protein is a human

ob protein.

18. A method for the production of an ob dimer fusion protein or ob

monomer fusion protein which comprises the steps of

(a) isolating a preparation of total RNA from a vertebrate tissue,

(b) converting said total RNA to cDNA,

(c) amplifying a cDNA sequence from said cDNA using

oligonucleotide primers suitable for annealing to a target ob protein gene sequence,

(d) detecting a cDNA molecule using oligonucleotides suitable for

hybridization to said target ob protein gene sequence,

(e) isolating said cDNA molecule,

(f) ligating the nucleic acid sequence of said cDNA molecule which

encodes an ob fusion protein to a second DNA sequence to create a fusion

DNA sequence encoding an ob fusion protein (g) ligating said fusion DNA sequence into an expression vector,

(h) transforming a host cell with said expression vector containing

said fusion DNA sequence,

(i) culturing the transformed host cell under conditions that favor the

production of said ob fusion protein as a dimer or monomer, and

(j) isolating said ob fusion protein expressed by said host cell.

19. The method of claim 18 wherein said vertebrate tissue is a vertebrate

adipose tissue.

20. The method of claim 19 wherein said vertebrate adipose tissue is human

adipose tissue.

21. A composition comprising an isolated ob dimer.

22. The composition of claim 21 wherein said ob dimer is a human ob

dimer.

23. The composition of claim 21 wherein said ob dimer is a rat ob dimer.

24. The composition of claim 21 wherein said ob dimer is a mouse ob dimer.

25. A composition comprising an isolated ob dimer fusion protein.

26. The composition of claim 25 wherein said ob dimer fusion protein is a

human ob dimer fusion protein.

27. The composition of claim 25 wherein said ob dimer fusion protein is a

rat ob dimer fusion protein.

28. The composition of claim 25 wherein said ob dimer fusion protein is a

mouse ob dimer fusion protein.

29. A pharmaceutical composition for use in the treatment of conditions or disorders that would benefit from reduced food intake or increased energy expenditure, which comprises a therapeutically effective amount of an ob dimer in

association with a pharmaceutically acceptable carrier.

30. The pharmaceutical composition of claim 29 wherein said ob dimer is a

human ob dimer.

31. A pharmaceutical composition for use in the treatment of conditions or

disorders that would benefit from reduced food intake or increased energy

expenditure, which comprises a therapeutically effective amount of an ob dimer

fusion protein in association with a pharmaceutically acceptable carrier.

32. The pharmaceutical composition of claim 31 wherein said ob dimer is a

human ob dimer.

33. A method for the treatment of conditions or disorders in a subject that

would benefit from reduced food intake or increased energy expenditure, which

comprises administering to said subject an amount of an ob dimer effective to

suppress appetite in said subject.

34. The method of claim 33 wherein said condition or disorder is obesity.

35. The method of claim 33 wherein said condition or disorder is diabetes.

36. The method of claim 35 wherein said diabetes condition or disorder is

Type 2 diabetes.

37. The method of any of claims 33-35 or 36 wherein said ob dimer is a

human ob dimer.

38. A method for the treatment of conditions or disorders in a subject that

would benefit from reduced food intake or increased energy expenditure, which

comprises administering to said subject an amount of an ob dimer fusion protein

effective to reduce appetite in said subject.

39. The method of claim 38 wherein said condition or disorder is obesity.

40. The method of claim 38 wherein said condition or disorder is diabetes.

41. The method of claim 40 wherein said diabetes condition or disorder is Type 2 diabetes.

42. The method of any of claims 38-40 or 41 wherein said ob dimer fusion

protein is a human ob dimer fusion protein.

43. A monoclonal antibody which binds to an ob dimer, wherein said

monoclonal antibody recognizes said ob dimer with greater specificity than the ob monomer of which said ob dimer is comprised.

44. The monoclonal antibody of claim 43 wherein said ob dimer is a human

ob dimer.

45. The monoclonal antibody of claim 43 wherein said ob dimer is selected from the group consisting of a rat ob dimer and a mouse ob dimer.

46. A monoclonal antibody which binds to an ob dimer fusion protein,

wherein said monoclonal antibody recognizes said ob dimer fusion protein with

greater specificity than the ob monomer fusion protein of which said ob dimer fusion

protein is comprised.

47. The monoclonal antibody of claim 46 wherein said ob dimer fusion

protein is a human ob dimer fusion protein.

48. The monoclonal antibody of claim 46 wherein said ob dimer fusion

protein is selected from the group consisting of a rat ob dimer fusion protein and a

mouse ob dimer fusion protein.

49. An assay using a monoclonal antibody for detecting the presence or

amount of an ob dimer comprising the steps of: (a) contacting said ob dimer with said monoclonal antibody, wherein

said monoclonal antibody binds to said ob dimer with greater specificity than the ob monomer of which said ob dimer is comprised, and

(b) determining the presence or amount of said ob dimer.

50. The assay of claim 49 wherein a second monoclonal antibody to said ob

dimer or a polyclonal antibody to said ob dimer is used in said assay.

51. The assay of claim 49 wherein a second monoclonal antibody to the ob

monomer of which said ob dimer is comprised or a polyclonal antibody to the ob monomer of which said ob dimer is comprised is used in said assay.

52. The assay of claim 50 or 51 wherein said second monoclonal antibody is

detectably labeled.

53. The assay of claim 49 wherein said assay is a competitive assay.

54. The assay of claim 49 wherein said assay is a sandwich assay.

55. An assay using a monoclonal antibody for detecting the presence or

amount of an ob dimer fusion protein comprising the steps of:

(a) contacting said ob dimer fusion protein with said monoclonal

antibody, wherein said monoclonal antibody binds to said ob dimer fusion

protein with greater specificity than the ob monomer fusion protein of which said

ob dimer fusion protein is comprised, and (b) determining the presence or amount of said ob dimer fusion

protein.

56. The assay of claim 55 wherein a second monoclonal antibody to said ob

dimer fusion protein or a polyclonal antibody to said ob dimer fusion protein is used

in said assay.

57. The assay of claim 55 wherein a second monoclonal antibody to the ob

monomer fusion protein of which said ob dimer fusion protein is comprised or a

polyclonal antibody to the ob monomer fusion protein of which said ob dimer fusion

protein is comprised is used in said assay.

58. The assay of claim 56 or 57 wherein said first or said second monoclonal

antibody is detectably labeled.

59. The assay of claim 55 wherein said assay is a competitive assay.

60. The assay of claim 55 wherein said assay is a sandwich assay.

61. An assay for determining the presence or amount of an ob dimer or ob dimer fusion protein in a sample suspected of containing an ob dimer or ob dimer

fusion protein, comprising the steps of

(a) contacting said sample suspected of containing an ob dimer or ob

dimer fusion protein with a monoclonal antibody according to any of claims

43, 44, 46 or 47; (b) contacting positive and/or negative control samples with a

monoclonal antibody according to any of claims 43, 44, 46 or 47; and

(c) determining the presence or amount of said ob dimer or ob dimer

fusion protein.

62. The assay according to claim 61 wherein said assay is a competitive

assay.

63. The assay according to claim 61 wherein said assay is a sandwich assay.

64. The assay according to claim 61 wherein said monoclonal antibody is detectably labeled and wherein said labeled monoclonal antibody binds to said ob dimer fusion protein and is used to determine the presence or amount of said ob dimer fusion protein.

65. An assay for determining the presence or amount of an ob dimer or ob

dimer fusion protein amylin in fluid sample suspected of containing an ob dimer or

ob dimer fusion protein, comprising the steps of:

(a) contacting said sample with a measured amount of a first antibody

directed to an ob dimer or ob dimer fusion protein to form a soluble complex

of said first antibody and said ob dimer or ob dimer fusion protein present in said sample, said first antibody being labeled;

(b) contacting said soluble complex with a second antibody directed to said ob dimer or ob dimer fusion protein, said second antibody being

bound to a solid carrier, to form a ternary complex of said first antibody, said ob dimer or ob dimer fusion protein and said second antibody bound to said

solid carrier; (c) separating said solid carrier from said sample and unreacted

labeled first antibody;

(d) measuring either the amount of labeled first antibody associated

with said solid carrier or the amount of unreacted labeled first antibody; and

(e) relating the amount of labeled antibody with the amount of

labeled antibody measured for a control sample prepared in accordance with steps (a)-(d), said control sample being known to be free of said ob dimer or

ob dimer fusion protein, to determine the presence of ob dimer or ob dimer

fusion protein in said fluid sample, or relating the amount of labeled antibody measured with the amount of labeled antibody measuied for samples containing known amounts of ob dimer or ob dimer fusion protein prepared

in accordance with steps (a)-(d) to determine the amount of ob dimer or ob

dimer fusion protein in said fluid sample, said first or second antibody being

according to any of claims 43, 44, 46 or 47.

66. An assay for determining the presence or amount of ob dimer or ob

dimer fusion protein in a fluid sample suspected of containing said ob dimer or ob

dimer fusion protein comprising the steps of:

(a) contacting said sample with a first antibody directed to said ob

dimer or ob dimer fusion protein, wherein said first antibody is bound to a solid

carrier, to form a complex of said ob dimer fusion protein present in said

sample and said first antibody;

(b) separating unreacted sample from said complex;

(c) contacting said complex with a measured amount of a second

antibody directed to said ob dimer or ob dimer fusion protein, wherein said

second antibody is labeled;

(d) measuring either the amount of labeled second antibody

associated with said complex or the amount of unreacted labeled second

antibody; and

(e) relating the amount of labeled antibody with the amount of labeled antibody measured for a control sample prepared in accordance with

steps (a)-(d), said control sample being known to be free of ob dimer or ob dimer

fusion protein, to determine the presence of ob dimer or ob dimer fusion protein in

said fluid sample, or relating the amount of labeled antibody measured with the amount of labeled antibody measured for samples containing known amounts of ob dimer or ob dimer fusion protein prepared in accordance with steps (a)-(d)

to determine the amount of ob dimer or ob dimer fusion protein in said fluid

sample, said first or second antibody being according to any of claims 43, 44,

46 or 47.

67. In an immunometric assay to determine the presence or amount of ob

dimer or ob dimer fusion protein in a sample suspected of containing ob dimer or ob

dimer fusion protein, the improvement comprising employing a monoclonal

antibody according to any one of claims 43, 44, 46 or 47.

68. An assay for determining the presence or amount of ob dimer or ob dimer fusion protein in a sample suspected of containing ob dimer or ob dimer fusion protein, comprising the steps of:

(a) contacting said sample with a known quantity of added labeled ob

dimer or ob dimer fusion protein;

(b) contacting said sample with a monoclonal antibody according to

any of claims 43, 44, 46 or 47; and

(c) determining the amount of labeled ob dimer or ob dimer fusion

protein bound to said monoclonal antibody or the amount of labeled ob dimer

or ob dimer fusion protein which is not bound to said monoclonal antibody.

69. The assay of any of claims 49-51, 53 or 54 wherein said ob dimer is a

human ob dimer.

70. The assay of any of claims 55-57, 59 or 60 wherein said ob dimer fusion

protein is a human ob dimer fusion protein

71. A kit comprising a monoclonal antibody which binds to an ob dimer, wherein said monoclonal antibody recognizes said ob dimer with greater specificity than the ob monomer of which said ob dimer is comprised. .

72. A kit comprising a monoclonal antibody which binds to an ob dimer

fusion protein, wherein said monoclonal antibody recognizes said ob dimer fusion

protein with greater specificity than the ob monomer fusion protein of which said ob

dimer fusion protein is comprised.

73. A kit according to claim 71 or 72 which further comprises suitable

control samples.

74. A substantially pure ob dimer.

75. A pure ob dimer.

76. A substantially pure ob fusion dimer protein.

77. A pure ob fusion dimer protein.

78. The ob dimer of claim 74 or 75 which is a human ob dimer.

79. The ob dimer fusion protein of claim 76 or 77 which is a human ob

dimer fusion protein.

80. A method of treatment which comprises the administration of a

therapeutically effective amount of a pharmaceutical composition comprising an ob

dimer according to claim 74 or 75 to patients in need thereof.

81. The method of claim 80 wherein said ob dimer is a human ob dimer.

82. A method of treatment which comprises the administration of a

therapeutically effective amount of a pharmaceutical composition comprising an ob dimer fusion protein according to claim 76 or 77 to patients in need thereof.

83. The method of claim 82 wherein said ob dimer fusion protein is an human ob dimer fusion protein.

84. A composition comprising an ob protein in BisTris propane buffer.

85. The composition of claim 84 which further comprises a detergent.

86. The composition of claim 85 wherein said detergent is a Tween.

87. The composition of any of claims 84, 85 or 86 having a pH of from about

7.5 to about 9.

88. A composition comprising an ammonium bicarbonate salt of an ob

protein.

89. The composition of claim 88 wherein said ob protein is selected from the group consisting of an ob monomer, an ob fusion monomer, an ob dimer

and an ob fusion dimer.

90. A method of purifying an ob protein which comprises the step of separating said ob protein from a sample known to contain said ob

protein using a cellulose-based anion exchange resin.

91. The method of claim 90 wherein said cellulose-based anion exchange

resin is DE-52 resin.

92. The method of either of claims 90 or 91 which include the use of a

BisTris propane running buffer.

93. The method of claim 90 which further comprises the additional step of purifying the ob protein obtained from said cellulose-based anion

exchange resin purification step, using reversed phase high pressure

liquid chromatography.

94. A composition comprising an isolated ob monomer fusion protein.

95. A pharmaceutical composition for use in the treatment of conditions or disorders that would benefit from reduced food intake or increased energy expenditure, which comprises a therapeutically effective amount of an ob monomer fusion protein in association with a pharmaceutically acceptable carrier.

96. A method for the treatment of conditions or disorders in a subject that would benefit from reduced food intake or increased energy expenditure, which comprises administering to said subject an amount of an ob monomer fusion protein effective to suppress appetite in said subject.