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EP0371041A1 - A process for preparing a protein or polypeptide, a dna sequence coding for the polypeptide, a microorganism containing the dna sequence as well as the polypeptide and its use as a pharmaceutical preparation - Google Patents

A process for preparing a protein or polypeptide, a dna sequence coding for the polypeptide, a microorganism containing the dna sequence as well as the polypeptide and its use as a pharmaceutical preparation

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Publication number
EP0371041A1
EP0371041A1 EP88905733A EP88905733A EP0371041A1 EP 0371041 A1 EP0371041 A1 EP 0371041A1 EP 88905733 A EP88905733 A EP 88905733A EP 88905733 A EP88905733 A EP 88905733A EP 0371041 A1 EP0371041 A1 EP 0371041A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
amino
dna sequence
glu
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP88905733A
Other languages
German (de)
French (fr)
Inventor
Henrik Dalb Ge
Jens M Lvig
Kim Ry Hejn S
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DK320987A external-priority patent/DK320987D0/en
Priority claimed from DK177488A external-priority patent/DK177488A/en
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP0371041A1 publication Critical patent/EP0371041A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/545—IL-1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/034—Fusion polypeptide containing a localisation/targetting motif containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention concerns a process for preparing a protein or polypeptide which contains an amino-terminal extension, preferably with at least two amino acids, in addition to the desired polypeptide chain.
  • proteins or polypeptides may be produced biosynthe ically by cultivating a host organism which contains a vector with the DNA sequence, such as a plasmid, which codes for the desired polypeptide.
  • the resulting polypeptide is isolated from the cultivation medium and purified, e.g. chromato- graphically.
  • polypeptides that are produced biosynthetically in this way are so atostatin , insulin and human growth hormone.
  • Another advantag . e of transport of the desired protein out into the periplasma is that the protein here folds sub ⁇ stantially correctly to provide the native protein with the correct folding for the active protein. This involves essential advantages in the biosynthetic production of proteins on an industrial scale because then it is not necessary to effect subsequent folding, which is very difficult to perform in vitro.
  • the signal sequence is cut by an enzyme contained in the bacterium during or after the transport of the protein through the inner membrane. This cut, however, is not completely specific since partially non-specific cutting of the signal sequence takes place depending upon the cultivation methods, strain of bacterium, etc., or sub- sequent enzymatic reactions occur, whereby the N-terminus of the desired protein is digested by peptidases in the periplas a to an uncontrolled degree.
  • a desired or mature protein by means of a microorganism coding for a desired protein with an amino terminal extension which comprises at least two amino acids, cf. the DK Patent Application 3448/84.
  • the desired protein with the extension is collected in the cytoplasma, from which it is to be isolated and purified.
  • the extension may be digested specifically to obtain the desired protein with a correct N-terminal in a pure state, using e.g. the enzyme DAP 1 and/or DAP 4.
  • S is a signal sequence
  • E is an amino extension with at least two amino acids, at least one of which is charged
  • P is the desired mature protein.
  • the desired polypeptide is synthetized with associated extension.
  • the signal sequence will cause the amino-terminal-extended protein to be transported through the membrane enclosing the cytoplasma
  • amino-terminal- extended polypeptide will occur in correctly folded form. If a microorganism with a periplasmatic space is used, the desired amino-extended polypeptide will normally be collected in it. It happens that under certain circum- ces, which are not fully understood, the protein also passes the outer membrane to the cultivation medium it ⁇ self.
  • the isolated amino-terminal-extended polypeptide can then be used as a substrate for e.g. the enzyme DAP 1 to produce the desired polypeptide, P.
  • the amino-terminal- extended polypeptide for production of the desired mature protein in a high yield and in a pure state by utilizing at the final purification the difference in charge between the mature protein and the amino-terminal- extended protein which might be left after the enzymatic cleavage.
  • the process of the invention can be used generally for preparation of an arbitrary protein with an amino terminal extension which can be subsequently removed enzymatically .
  • proteins which can be produced by the process of the invention are amino-terminal-extended IGF 1, amino- terminal-extended 22K hGH, amino-terminal-extended 20K hGH, amino-terminal-extended IL 1 and amino-ter inal- ex ended insulin.
  • the amino terminal extension, E. preferably comprises 2 amino acids, as mentioned, and in particular preferably 4 to 16 amino acids.
  • the amino terminal extension contains at least one charged amino acid.
  • the presence of one or more charged amino acids permits effective purification and isolation of the desired protein by chromatography .
  • the invention also concerns a DNA sequence, a plasmid containing this DNA sequence, and microorganisms in which such a plasmid has been inserted.
  • any microorganism into which a plasmid may be inserted can be used in the process of the invention.
  • E. coli lends itself particularly well for the purpose, but also other microorganisms may be used.
  • An example of this is B. subtilis.
  • This microorganism does not, as is the case with E. coli, have an outer membrane and accordingly a periplasmatic space.
  • the resulting polypeptide is transported through the cell membrane and out into the cultivation medium with simultaneous cutting of the signal sequence.
  • the polypeptide will be correctly folded and can be isolated from the cultivation medium in a manner known per se, cf. by chromatography on an ion-exchange resin.
  • the polypeptide produced by the process of the invention which comprises the amino-terminal extension, is suitable as a starting material in the preparation of the mature protein, P.
  • the amino terminal extension in a suitable manner, it is possible to obtain specific enzymatic digestion of the extension.
  • one of the enzymes DAP 1 or DAP 4 may be used for this pur ⁇ pose, which causes stepwise cleavage of the extension, preferably by removal of two amino acids at a time, followed by isolation of the resulting mature protein from possibly non-reacted or partially reacted, extended polypeptide in a known manner, such as by chromatography.
  • the invention moreover concerns an amino-terminal-extended interleukin 1 (IL-1) as well as a preparation containing the biologically active polypeptide.
  • IL-1 interleukin 1
  • Interleukin 1 is a polypeptide which is produced in various animal cells, such as monocytes.
  • the full amino acid sequence of IL-1 and the precursor of IL-1 are known.
  • IL-1 has various biological effects. Thus, it was shown recently that IL-1 has a cytotoxic effect on the Langer- hans's islets, i.e. /.-cells in the pancreas, and may thus cause insulin dependent diabetes ellitus (IDDM). The effect occurs in connection with the binding of IL-1 to specific receptors of the /3-cells.
  • IDDM insulin dependent diabetes ellitus
  • IL-1 The production of excess of IL-1 can take place by various biological influences.
  • This embodiment of the invention is based on the finding that amino-terminal-extended IL-1 counteracts the cyto ⁇ toxic effect of IL-1, such as against .-cells in the pancreas.
  • Amino-terminal-extended IL—1 is believed to bind to the same receptors as IL-1 on the - c e l l s and thus block these receptors. This would prevent the cytotoxic effect of IL-1.
  • the present invention also concerns amino-terminal- extended IL-1. Further, the invention concerns prepa ⁇ rations containing amino-terminal-extended IL-1, which may e.g. be used for therapeutic or prophylactic treat ⁇ ment of IDDM.
  • the invention moreover concerns a DNA-strueture coding for amino-terminal-extended IL-1 or amino-terminal- extended IL-1 derivatives.
  • the extension of the IL-1 and IL-1 derivatives may have different lengths and e.g. compris * e most of the pre- sequence of the naturally occurring IL-1. Preferably, however, a considerably shorter amino terminal extension is used, e.g. comprising 2 to 6 amino acids.
  • polypeptides of the present type are amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids
  • the polypeptide may be modified in a manner known per se or be amended, e.g. by replacing one or more amino acids by other amino acids or by removal of one or more amino acids in the C-terminus.
  • the polypeptide of the invention can be used therapeutic- ally for combatting or preventing i.a. diabetes and autoimmune diseases. It is also proposed to use the poly ⁇ peptide for treatment of patients who are subjected to septic shock under the action of endotoxiantibodies.
  • pharmaceutical preparations in particular injectable preparations are used, containing the poly ⁇ peptide in a pharmaceutically acceptable carrier medium.
  • the present polypeptide is produced biosynthetically by cultivation of a microorganism or a cell line which contains a gene coding for the polypeptide, which is then recovered, purified and processed to the desired preparation.
  • the restriction sites EcoRI/BamHI have inserted between them a DNA segment containing a promotor, a Shine Dalgarno sequence and the signal sequence from outer membrane protein A (2), followed by a synthetic DNA linker containing the recognition sequence for the restriction enzymes Narl, Bglll, PvuII and Ba HI.
  • the plasmid pHD167 is likewise a derivative of the plasmid pAT153.
  • This plasmid contains a promotor, a Shine Dalgarno sequence, a synthetic gene coding for IGF1, and a transcription terminator.
  • the above-mentioned sequences are inserted between the EcoRI and BamHI restriction sites in pAT153.
  • the plasmid pHD176 contains the plasmid region, the promotor region and the signal sequence from the plasmid pHD165 as well as the transcription terminator and the gene for IGF1 from the plasmid pHD167, isolated as an Ace III/BamHI DNA fragment, and a small synthetic DNA linker coding for amino terminal excusion.
  • the plasmid pHD176 contains the coding region for the outer membrane protein A signal sequence, followed by the amino terminal extension Ala-Glu-Ala-Glu, and the coding sequence for IGF1.
  • the plasmid pHD141 shown in fig. 2 is a derivative of the plasmid pHD167 in which the coding sequence for IGF1 has been replaced by the coding sequence for amino-terminal- extended 20K-hGH.
  • the plasmid pHD223 contains the sequences contained in the plasmid pHD141 between Clal/EcoRI, a Clal/Narl DNA fragment coding for the signal sequence from pHD165 as well as a synthetic DNA linker coding for an amino terminal excusion.
  • the plasmid pHD223 thus contains the coding region for the outher membrane protein A signal sequence, followed by the amino terminal extension Ala-Glu-Ala-Glu , followed by the coding sequence for 20K-hGH.
  • the plasmid pHD106-9 shown in fig. 3 is a derivative of the vector pUC18 into which the coding region for the signal sequence from E. coli fimbria protein K88 (3) has been inserted between the restriction sites BamHI/Pstl.
  • the plasmid pHD117-4 SP13 is a derivative of the plasmid pHD167, in which the coding region for IGF1 has been replaced by the coding region for amino-terminal-extended 22K-hGH.
  • the plasmid pHD148-22K-hGH contains the signal sequence from the fimbria protein K88, followed by the amino terminal extension Ala-Glu-Ala-Glu-Ala-Glu, followed by the coding region for 22K-hGH.
  • the plasmid pHD163 shown in fig. 4 is a derivative of the plasmid pHD167 in which the coding region for IGF1 has been replaced by the coding region for amino-terminal- extended IL-1.
  • the plasmid pHD185 is a derivative of the plasmid pAT153, into which a synthetic DNA fragment containing the coding region for the outer membrane protein A signal sequence as well- as the recognition sites for the restriction enzymes Narl, Bglll, PuvII and BamHI have been inserted between the restriction sites EcoRI/BamHI.
  • the plasmid pHD163/185 thus contains the coding region for the signal sequence from the outer membrane protein A, followed by the amino terminal extension Met-Glu-Ala-Glu- Phe-Asp and the coding region for interleukin 1.
  • the plasmid pHD165 which contains a promotor and the above-mentioned signal sequence was cut with the enzymes Narl/Ba HI, and then the plasmid fragment was purified.
  • the plasmid pHD167 containing e.g. the coding sequence for human IGF1 followed by transcription terminator, was cut with the enzymes AccI I I/BamHI , and then a fragment of about 600 base pairs was purified. These two fragments as well as a synthetic DNA linker, containing a restriction overhang for the enzymes Narl/AccIII (coding for amino terminal extension) were then ligated together to form the plasmid pHD176.
  • the ligation mixture was subsequently transformed into the bacterium E. coli MC1061, plated on ampicillin containing aga plates and incubated overnight at 37 °C.
  • a plurality of bacteria colonies was selected and cultivated for further characterization by restriction enzyme analysis.
  • the clones pHD176-D, -F were then selected.
  • the clone pHD176-F was cultivated on a large scale, and then the bacteria were harvested by centri- fugation.
  • the bacteria were then partly exploded by means of osmotic shock, so that the proteins present in the periplasma were released from the bacteria.
  • the amino-terminal- extended IGF1 was purified. This material was then used as a substrate for DAP 1, whereby the N-terminal extension was specifically removed.
  • the expression level in the above-mentioned cultivation of the bacterium HD176-F was about 5 mg of amino-terminal- extended IGF1 per litre of bacteria culture with an 0D600 of about 1.
  • the plasmid pHD141 containing the gene for 20K human growth hormone, was cut with the enzymes Clal/EcoRI, and then the plasmid fragment was purified.
  • the plasmid pHD165 was cut with the enzymes Clal/Narl, and then a fragment of about 120 base pairs was purified. These fragments were subsequently ligated together in the presence" of a synthetic DNA linker with an overhang for the restriction enzymes Narl/EcoRI to form the plasmid pHD223.
  • the ligation mixture was transformed into E. coli MC1.061 and plated on ampicillin containing growth plates
  • the expression level was of the same order as mentioned in example 1.
  • the plasmid pHD106-9 which contains another signal sequence described above, was cut with the restriction enzymes BamHI/Hindlll . A fragment of 190 base pairs was then purified. This fragment was subsequently cut with the enzyme BstNI, treated with SI nuclease to produce a blunt end, phenol-extract ⁇ d, ethanol-precipitated, and then cut with the enzyme Clal. A fragment of 60 base pairs was then purified.
  • the plasmid pHD117-4 SP13 was cut with the enzymes Clal/EcoRIj and then the plasmid fragment was purified. The two mentioned purified fragments were ligated together in the presence of a BstNI/EcoRI linker to form the plasmid pHD148-22K-hGH.
  • the ligation mixture was transformed into the bacterium E. coli MC1061 and plated on ampicillin containing growth plates. After incubation overnight, a plurality of colonies was selected for further character- ization by restriction enzyme analysis and DNA sequence determination. Cultivation and isolation of the resulting amino-terminal- extended 22K hGH were performed in the same manner as stated in example 1.
  • the plasmid pHD185 containing the coding sequence for a signal sequence, was cut with the enzymes Clal/Narl, and then a DNA fragment of about 90 base pairs was purified. These two fragments were then ligated together to form the plasmid pHD163/185.
  • the obtained plasmid was inserted into E. coli, which was cultivated in the usual manner.
  • the resulting amino- terminal-extended human IL-1 was purified and converted into human IL-1 by reaction with DPA 1.
  • a gene coding for the amino acids 117-269 in the IL-1 precursor was produced. It has been found by comparative studies of messenger RNA sequences of high-level- expressed and low-level-expressed genes in bacteria, respectively, that there is a strong correlation between codon use and expression efficiency. Accordingly, it was decided to use the codons which are most frequently used in high-level-expressed E. coli genes. Useful cloning sites were inserted inside the gene as well as at both ends. The gene was extended with a DNA sequence which codes for the amino terminal extension Met-Glu-Ala-Glu (MEAE). The gene was produced by stepwise cloning between restriction sites of oligonucleotides having an average length of 80 to 100 nucleotides. The sequence of the gene for MEAE-IL-1 is shown in fig. 5.
  • E. coli was selected as the host organism since glyco- sylation or other modifications of IL-1 have not been reported.
  • the expression was controlled by a synthetic promotor (SP13) and an .optimum Shine Dalgarno sequence.
  • SP13 synthetic promotor
  • the used promotor was selected because it is known that it is effective for expression of e.g. bhGH in E. coli (up to 20 ⁇ of total bacteria protein) (Bio Technology,
  • IL-1 Extraction of IL-1 was performed as described in Bio Technology, Vol. 4, Dec. 1986, p. 1078. Purification of the N-terminal-extended IL-l- derivatives was performed on FF-S sepharose, FF-Q sepharose and gel filtration colonies, respec i ely. The derivatives in question were characterized by amino acid analyses, N-terminal sequence analysis, molecular weight determination by the firm atomic bombardment method (Barber M. et al.; (1981) J. Chem. Soc., Chem. Comm. Vol. 1981, p. 325-327), SDS and native electrophoresis.
  • a DNA segment with the coding region for pheny1-alanine-aspartic acid was inserted into the clone pHD230. It was inserted in such a manner that IL-1 was extended with the sequence Met- Glu-Ala-Glu-Phe-Asp- .
  • the gene for amino-terminal- extended IL-1 was expressed in E. coli and purified as described above.
  • the clone was prepared by in vitro mutagenesis on the plasmid pHD230.
  • the amino-terminal-extended IL-1 was expressed and purified as described above.
  • the N-terminal methionine group was cleaved in vivo by an E. coli enzyme Example 10
  • the resulting clone pHD228 was then cut with the enzymes Narl/Clal, and then a fragment coding for the below amino acid sequence was inserted.
  • the gene was expressed in E. coli and purified as described above.
  • the inserted amino acid sequence had the following composition:
  • the specific bioactivity (units/ng IL-1-3) is determined by simultaneous measurement of the biological response in the comitogenic mouse thymocyte profileration assay (LAF) and by quantization by means of IL-l-/_ Elisa of the corresponding amount of IL-1-3 protein.
  • LAF assay is performed as described above (Scand. 3. Immunol. 26:611, 1987, Scand. J. Immunol. : Endotoxin stimulated human onocyte secretion of interleukin 1, tumor necrosis factor alpha and prostaglandin E show stable in erindi idual difference, in press).
  • Thymocytes are isolated from 5 to 7 weeks old male mice of the C3H/he mice strain (Charles River, Federal Republic of Germany). After washing of the cells, these are plated in shallow microtiter plates (Nunc, Roskilde) in a medium consisting of a mixture of 90? ⁇ RpMI-164 and 10? ⁇ ac ⁇ cumulated heat inactivated human serum, the medium having been added to the sample in a dilution series beginning with 20 vol? ⁇ and diluted 1:2 in up to 8 steps. The test series is repeated 3 times. PHA (Difco) 5 ,ug/ml is added to each well. After 48 hours' incubation at 37 °C 1 ,uCi tritiated thy idin is added per well. The cultures are harvested on glass fibre filters (Skatron, Norway) after another 18 hours' incubation by means of a semi-automatic cell culture harvester. Then the filters are counted in a beta counter.
  • the results are calculated as follows: The average of triplicates is calculated. Linear regression is performed for each dilution series, comprising a count above 3 x background. The highest counts in the dilution series are included only if they are more than 10? ⁇ higher than the count in the next dilution step. Linear regression is performed either semi-logarithmlcly or double-logarith- micly. This results in parallel linear curves which directly allow quantization of the biological response with respect to a standard consisting of recombl ⁇ ant I -l- 100 ng/ml (WHO standard). The results can then be stated in Units/ml, the WHO standard containing 100 U/ng.
  • IL-1-5 Elisa is performed according a standardized Elisa method (Cistro ⁇ ).
  • Results appear from the enclosed table. It is noted that the specific activity of MEAE-IL-1-/3 is found to be 50 to 100 times weaker than recombinant full length IL-1-3. The results are obtained from 3 independent experiments.
  • the pancreas is removed from 5 to 7 days old rats of the Wistar strain, and the islets are isolated by collagenase degradation.
  • the islets are cultivated in RPMI-1640 + 10? ⁇ NCS at 37 °C in a moist atmosphere. After 1 week's incubation the islets are washed in RPMI-1640 + 0.5? ⁇ HS and incubated further for 6 days with and without IL-1.
  • the insulin secretion from the islets Is determined in Radioim unoassay (RIA).
  • RIA Radioim unoassay
  • Fig. 3 shows a standard curve for rIL-1. It will be seen that the insulin secretion is stimulated at very low rIL-1 concentrations (0.001 ng/ml) (about 130? ⁇ ), while the insulin secretion is inhibited to about 50? ⁇ at a somewhat higher concentration (0.1 ng/ml). References:

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Abstract

Une protéine ou un polypeptide représentés par la formule générale: E - P, où P représente un polypeptide mature désiré et E représente une extension de terminaison amino contenant de préférence au moins deux acides aminés, sont produits par culture d'une bactérie contenant une séquence d'ADN qui code pour un polypeptide représenté par la formule: S - E - P, où S représente une séquence de signaux et E et P représentent les mêmes éléments définis ci-dessus. L'extension de terminaison amino peut être extraite du polypeptide par digestion enzymatique, de façon à former la protéine ou le polypeptide matures, P, par exemple par utilisation de l'enzyme DAP 1. Le polypeptide peut être de l'interleukine 1 à extension de terminaison amino ou un dérivé de cette interleukine qui présente une activité biologique.A protein or polypeptide represented by the general formula: E - P, where P represents a desired mature polypeptide and E represents an amino terminus extension preferably containing at least two amino acids, are produced by culturing a bacterium containing a sequence of DNA which codes for a polypeptide represented by the formula: S - E - P, where S represents a sequence of signals and E and P represent the same elements defined above. The amino-terminal extension can be extracted from the polypeptide by enzymatic digestion, so as to form the mature protein or polypeptide, P, for example by using the enzyme DAP 1. The polypeptide can be interleukin-1 with extension amino terminus or a derivative of this interleukin which exhibits biological activity.

Description

A process for preparing a protein or polypeptide, a DNA sequence coding for the polypeptide, a micro¬ organism containing the DNA sequence as well as the polypeptide and its use as a pharmaceutical preparation
The present invention concerns a process for preparing a protein or polypeptide which contains an amino-terminal extension, preferably with at least two amino acids, in addition to the desired polypeptide chain.
Many valuable proteins or polypeptides, e.g. enzymes or hormones, may be produced biosynthe ically by cultivating a host organism which contains a vector with the DNA sequence, such as a plasmid, which codes for the desired polypeptide. The resulting polypeptide is isolated from the cultivation medium and purified, e.g. chromato- graphically. Examples of polypeptides that are produced biosynthetically in this way are so atostatin , insulin and human growth hormone.
It is known to produce human growth hormone by biosynthetic methods by using a microorganism with a gene coding for the growth hormone in which the N-terminus is extended with the amino acid ethionine, the purpose of which is to translate the initiation code ATG. To obtain the desired protein in a pure state, it is necessary to remove the methionine group. This is sometimes done - depending upon the conditions of cultivation - by enzymatic cleavage in the microorganism itself, or the cleavage is effected enzymatically in a subsequent step. Both cases involve a risk of various secondary reactions so that the human growth hormone occurs in a mixture of various polypeptides which are difficult to separate. Further, purification of the desired polypeptide involves difficulties because it is present in the intercellular liquid of the micro¬ organism in a relatively low concentration in mixture with many o the r po l ypep tides
It is known, cf. EP 0001930, to use a microorganism with a gene coding for a desired protein to which a signal sequence is coupled in N-terminal position. Because of the presence of the signal sequence, the resulting polypeptide is transported through the inner cell membrane enclosing the cytoplas a out into the peri¬ plasmatic space from which it may be isolated. This takes place by lysation, whereby the outer membrane is degrated, and the liquid from the periplasmatic space is released and isolated.
This provides considerable advantages in terms of purification because the liquid in the periplasmatic space only contains a fraction, e.g. about 1% of the protein produced by "the 'microorganism, so that the desired protein occurs in concentrated form.
Another advantag.e of transport of the desired protein out into the periplasma is that the protein here folds sub¬ stantially correctly to provide the native protein with the correct folding for the active protein. This involves essential advantages in the biosynthetic production of proteins on an industrial scale because then it is not necessary to effect subsequent folding, which is very difficult to perform in vitro.
The signal sequence is cut by an enzyme contained in the bacterium during or after the transport of the protein through the inner membrane. This cut, however, is not completely specific since partially non-specific cutting of the signal sequence takes place depending upon the cultivation methods, strain of bacterium, etc., or sub- sequent enzymatic reactions occur, whereby the N-terminus of the desired protein is digested by peptidases in the periplas a to an uncontrolled degree.
For the above-men ioned reasons, this known method results in the general occurrence of a population of polypeptide molecules which are difficult to separate.
Finally, it is known to produce a desired or mature protein by means of a microorganism coding for a desired protein with an amino terminal extension which comprises at least two amino acids, cf. the DK Patent Application 3448/84. The desired protein with the extension is collected in the cytoplasma, from which it is to be isolated and purified. The extension may be digested specifically to obtain the desired protein with a correct N-terminal in a pure state, using e.g. the enzyme DAP 1 and/or DAP 4. Further, it is possible to insert into such a presequence one or more charged amino acids which permit effective purifica ion, it being possible to purify chromatographically using the difference in charge" between the amino-terminal-extended protein and the mature protein which occurs in the enzyme cleavage.
It has now been found that the advantages of both of the two last-mentioned methods may be obtained without the drawbacks associated with these, when using a bacterium containing a DNA sequence which codes for a polypeptide of the formula :
E - P
wherein S is a signal sequence, E is an amino extension with at least two amino acids, at least one of which is charged, and P is the desired mature protein. In such a cultivation, the desired polypeptide is synthetized with associated extension. The signal sequence will cause the amino-terminal-extended protein to be transported through the membrane enclosing the cytoplasma,
The signal sequence will be cut during this process so that it is possible to isolate the amino-terminal- extended polypeptide of the formula:
E - P
wherein E and P are as defined above. The amino-terminal- extended polypeptide will occur in correctly folded form. If a microorganism with a periplasmatic space is used, the desired amino-extended polypeptide will normally be collected in it. It happens that under certain circum- ces, which are not fully understood, the protein also passes the outer membrane to the cultivation medium it¬ self.
The isolated amino-terminal-extended polypeptide can then be used as a substrate for e.g. the enzyme DAP 1 to produce the desired polypeptide, P.
If the signal sequence is not cut entirely correctly during the process, or if further cleavage of individual N-terminal amino acids takes place after cutting, it will nevertheless be possible to use the amino-terminal- extended polypeptide for production of the desired mature protein in a high yield and in a pure state by utilizing at the final purification the difference in charge between the mature protein and the amino-terminal- extended protein which might be left after the enzymatic cleavage.
The process of the invention can be used generally for preparation of an arbitrary protein with an amino terminal extension which can be subsequently removed enzymatically . Examples of proteins which can be produced by the process of the invention are amino-terminal-extended IGF 1, amino- terminal-extended 22K hGH, amino-terminal-extended 20K hGH, amino-terminal-extended IL 1 and amino-ter inal- ex ended insulin.
The amino terminal extension, E. preferably comprises 2 amino acids, as mentioned, and in particular preferably 4 to 16 amino acids.
According to an expedient embodiment of the invention, the amino terminal extension contains at least one charged amino acid. The presence of one or more charged amino acids permits effective purification and isolation of the desired protein by chromatography .
The invention also concerns a DNA sequence, a plasmid containing this DNA sequence, and microorganisms in which such a plasmid has been inserted.
Any microorganism into which a plasmid may be inserted can be used in the process of the invention. E. coli lends itself particularly well for the purpose, but also other microorganisms may be used. An example of this is B. subtilis. This microorganism does not, as is the case with E. coli, have an outer membrane and accordingly a periplasmatic space. When cultivating B, subtilis with inserted plasmids comprising a DNA sequence of the present type, the resulting polypeptide is transported through the cell membrane and out into the cultivation medium with simultaneous cutting of the signal sequence. The polypeptide will be correctly folded and can be isolated from the cultivation medium in a manner known per se, cf. by chromatography on an ion-exchange resin.
The polypeptide produced by the process of the invention, which comprises the amino-terminal extension, is suitable as a starting material in the preparation of the mature protein, P. When selecting the amino terminal extension in a suitable manner, it is possible to obtain specific enzymatic digestion of the extension. For example, one of the enzymes DAP 1 or DAP 4 may be used for this pur¬ pose, which causes stepwise cleavage of the extension, preferably by removal of two amino acids at a time, followed by isolation of the resulting mature protein from possibly non-reacted or partially reacted, extended polypeptide in a known manner, such as by chromatography.
The invention moreover concerns an amino-terminal-extended interleukin 1 (IL-1) as well as a preparation containing the biologically active polypeptide.
Interleukin 1 (IL-1) is a polypeptide which is produced in various animal cells, such as monocytes. The full amino acid sequence of IL-1 and the precursor of IL-1 are known.
IL-1 has various biological effects. Thus, it was shown recently that IL-1 has a cytotoxic effect on the Langer- hans's islets, i.e. /.-cells in the pancreas, and may thus cause insulin dependent diabetes ellitus (IDDM). The effect occurs in connection with the binding of IL-1 to specific receptors of the /3-cells.
The production of excess of IL-1 can take place by various biological influences.
In order to e.g. counteract or prevent IDDM it would be desirable either to prevent the production of too large amounts of IL-1 or to prevent the toxic effect of IL-1.
This embodiment of the invention is based on the finding that amino-terminal-extended IL-1 counteracts the cyto¬ toxic effect of IL-1, such as against .-cells in the pancreas. Amino-terminal-extended IL—1 is believed to bind to the same receptors as IL-1 on the - c e l l s and thus block these receptors. This would prevent the cytotoxic effect of IL-1.
Thus, the present invention also concerns amino-terminal- extended IL-1. Further, the invention concerns prepa¬ rations containing amino-terminal-extended IL-1, which may e.g. be used for therapeutic or prophylactic treat¬ ment of IDDM.
The invention moreover concerns a DNA-strueture coding for amino-terminal-extended IL-1 or amino-terminal- extended IL-1 derivatives.
The extension of the IL-1 and IL-1 derivatives may have different lengths and e.g. compris*e most of the pre- sequence of the naturally occurring IL-1. Preferably, however, a considerably shorter amino terminal extension is used, e.g. comprising 2 to 6 amino acids.
Typical examples of polypeptides of the present type are
Met-Glu-Ala-Glu-IL-1 Ala-Glu-IL-1 and Met-Glu-Ala-Glu-Phe-Asp-IL-1
all of which have a significantly reduced cytotoxic effect on /i-cells. In certain cases, the effect is considerably lower than naturally occurring IL-1.
If desired, the polypeptide may be modified in a manner known per se or be amended, e.g. by replacing one or more amino acids by other amino acids or by removal of one or more amino acids in the C-terminus. The polypeptide of the invention can be used therapeutic- ally for combatting or preventing i.a. diabetes and autoimmune diseases. It is also proposed to use the poly¬ peptide for treatment of patients who are subjected to septic shock under the action of endotoxiantibodies. For this purpose, pharmaceutical preparations, in particular injectable preparations are used, containing the poly¬ peptide in a pharmaceutically acceptable carrier medium.
The present polypeptide is produced biosynthetically by cultivation of a microorganism or a cell line which contains a gene coding for the polypeptide, which is then recovered, purified and processed to the desired preparation.
Production of plasmids suitable for performance of the invention by insertion into a host cell and cultivation of it can take place by recombinant techniques known per se. Such a procedure is illustrated in the drawing, in which figs. 1-4.illustrate the structure and the production of various plasmids suitable in the performance of the invention.
Description of the plasmids:
The plasmid shown in fig. 1, called pHD165 of a cloning medium replicable in E. coli, based on the plasmid pAT153 (1). The restriction sites EcoRI/BamHI have inserted between them a DNA segment containing a promotor, a Shine Dalgarno sequence and the signal sequence from outer membrane protein A (2), followed by a synthetic DNA linker containing the recognition sequence for the restriction enzymes Narl, Bglll, PvuII and Ba HI.
The plasmid pHD167 is likewise a derivative of the plasmid pAT153. This plasmid contains a promotor, a Shine Dalgarno sequence, a synthetic gene coding for IGF1, and a transcription terminator. The above-mentioned sequences are inserted between the EcoRI and BamHI restriction sites in pAT153. The plasmid pHD176 contains the plasmid region, the promotor region and the signal sequence from the plasmid pHD165 as well as the transcription terminator and the gene for IGF1 from the plasmid pHD167, isolated as an Ace III/BamHI DNA fragment, and a small synthetic DNA linker coding for amino terminal excusion.
Thus, the plasmid pHD176 contains the coding region for the outer membrane protein A signal sequence, followed by the amino terminal extension Ala-Glu-Ala-Glu, and the coding sequence for IGF1.
The plasmid pHD141 shown in fig. 2 is a derivative of the plasmid pHD167 in which the coding sequence for IGF1 has been replaced by the coding sequence for amino-terminal- extended 20K-hGH.
The plasmid pHD223 contains the sequences contained in the plasmid pHD141 between Clal/EcoRI, a Clal/Narl DNA fragment coding for the signal sequence from pHD165 as well as a synthetic DNA linker coding for an amino terminal excusion. The plasmid pHD223 thus contains the coding region for the outher membrane protein A signal sequence, followed by the amino terminal extension Ala-Glu-Ala-Glu , followed by the coding sequence for 20K-hGH.
The plasmid pHD106-9 shown in fig. 3 is a derivative of the vector pUC18 into which the coding region for the signal sequence from E. coli fimbria protein K88 (3) has been inserted between the restriction sites BamHI/Pstl.
The plasmid pHD117-4 SP13 is a derivative of the plasmid pHD167, in which the coding region for IGF1 has been replaced by the coding region for amino-terminal-extended 22K-hGH.
Thus, the plasmid pHD148-22K-hGH contains the signal sequence from the fimbria protein K88, followed by the amino terminal extension Ala-Glu-Ala-Glu-Ala-Glu, followed by the coding region for 22K-hGH.
The plasmid pHD163 shown in fig. 4 is a derivative of the plasmid pHD167 in which the coding region for IGF1 has been replaced by the coding region for amino-terminal- extended IL-1.
The plasmid pHD185 is a derivative of the plasmid pAT153, into which a synthetic DNA fragment containing the coding region for the outer membrane protein A signal sequence as well- as the recognition sites for the restriction enzymes Narl, Bglll, PuvII and BamHI have been inserted between the restriction sites EcoRI/BamHI.
The plasmid pHD163/185 thus contains the coding region for the signal sequence from the outer membrane protein A, followed by the amino terminal extension Met-Glu-Ala-Glu- Phe-Asp and the coding region for interleukin 1.
The process of the invention will be illustrated more fully below by means of some working examples.
Example 1
The plasmid pHD165, which contains a promotor and the above-mentioned signal sequence was cut with the enzymes Narl/Ba HI, and then the plasmid fragment was purified. The plasmid pHD167, containing e.g. the coding sequence for human IGF1 followed by transcription terminator, was cut with the enzymes AccI I I/BamHI , and then a fragment of about 600 base pairs was purified. These two fragments as well as a synthetic DNA linker, containing a restriction overhang for the enzymes Narl/AccIII (coding for amino terminal extension) were then ligated together to form the plasmid pHD176. The ligation mixture was subsequently transformed into the bacterium E. coli MC1061, plated on ampicillin containing aga plates and incubated overnight at 37 °C.
A plurality of bacteria colonies was selected and cultivated for further characterization by restriction enzyme analysis. The clones pHD176-D, -F were then selected. The clone pHD176-F was cultivated on a large scale, and then the bacteria were harvested by centri- fugation. The bacteria were then partly exploded by means of osmotic shock, so that the proteins present in the periplasma were released from the bacteria. After digestion of the partly exploded bacteria, the amino-terminal- extended IGF1 was purified. This material was then used as a substrate for DAP 1, whereby the N-terminal extension was specifically removed.
The expression level in the above-mentioned cultivation of the bacterium HD176-F was about 5 mg of amino-terminal- extended IGF1 per litre of bacteria culture with an 0D600 of about 1.
After chromatographic purification on ion-exchanger, pure IGF1 was isolated.
Example 2
The plasmid pHD141, containing the gene for 20K human growth hormone, was cut with the enzymes Clal/EcoRI, and then the plasmid fragment was purified. The plasmid pHD165 was cut with the enzymes Clal/Narl, and then a fragment of about 120 base pairs was purified. These fragments were subsequently ligated together in the presence" of a synthetic DNA linker with an overhang for the restriction enzymes Narl/EcoRI to form the plasmid pHD223. The ligation mixture was transformed into E. coli MC1.061 and plated on ampicillin containing growth plates
Cultivation and isolation of the resulting amino-terminal- extended 20K growth hormone were performed as stated in example 1.
The expression level was of the same order as mentioned in example 1.
Example 3
The plasmid pHD106-9, which contains another signal sequence described above, was cut with the restriction enzymes BamHI/Hindlll . A fragment of 190 base pairs was then purified. This fragment was subsequently cut with the enzyme BstNI, treated with SI nuclease to produce a blunt end, phenol-extractεd, ethanol-precipitated, and then cut with the enzyme Clal. A fragment of 60 base pairs was then purified.
The plasmid pHD117-4 SP13 was cut with the enzymes Clal/EcoRIj and then the plasmid fragment was purified. The two mentioned purified fragments were ligated together in the presence of a BstNI/EcoRI linker to form the plasmid pHD148-22K-hGH. The ligation mixture was transformed into the bacterium E. coli MC1061 and plated on ampicillin containing growth plates. After incubation overnight, a plurality of colonies was selected for further character- ization by restriction enzyme analysis and DNA sequence determination. Cultivation and isolation of the resulting amino-terminal- extended 22K hGH were performed in the same manner as stated in example 1.
Example 4
The plasmid pHD163, containing the coding sequence for amino-terminal-extended human IL-1, was cut with the enzyme Clal, treated with phosphatase, and purified.
The plasmid pHD185, containing the coding sequence for a signal sequence, was cut with the enzymes Clal/Narl, and then a DNA fragment of about 90 base pairs was purified. These two fragments were then ligated together to form the plasmid pHD163/185.
The obtained plasmid was inserted into E. coli, which was cultivated in the usual manner. The resulting amino- terminal-extended human IL-1 was purified and converted into human IL-1 by reaction with DPA 1.
The following examples illustrate the production of amino terminal interleukin 1 and the use of it for a pharmaceu ical preparation.
Example 5
Preparation of -MEAE-IL-1
A gene coding for the amino acids 117-269 in the IL-1 precursor was produced. It has been found by comparative studies of messenger RNA sequences of high-level- expressed and low-level-expressed genes in bacteria, respectively, that there is a strong correlation between codon use and expression efficiency. Accordingly, it was decided to use the codons which are most frequently used in high-level-expressed E. coli genes. Useful cloning sites were inserted inside the gene as well as at both ends. The gene was extended with a DNA sequence which codes for the amino terminal extension Met-Glu-Ala-Glu (MEAE). The gene was produced by stepwise cloning between restriction sites of oligonucleotides having an average length of 80 to 100 nucleotides. The sequence of the gene for MEAE-IL-1 is shown in fig. 5.
Example 6
Expression of MEAE-IL-1
E. coli was selected as the host organism since glyco- sylation or other modifications of IL-1 have not been reported. The expression was controlled by a synthetic promotor (SP13) and an .optimum Shine Dalgarno sequence. The used promotor was selected because it is known that it is effective for expression of e.g. bhGH in E. coli (up to 20^ of total bacteria protein) (Bio Technology,
Vol. 5, Feb. 1987, p. 161). The clone was called pHD230.
Analysis of total cell extract from pHD230/MC1061 on SDS gel, coloured with Coomassie blue, was found to give a strong band of the expected size. Analysis of bacteria extracts in a IL-L-specific ELISA showed that the samples contained immunoreactive material corresponding to about 10 mg of IL-1/1 bacteria culture with a bacteria density (0D 600) of 1.
Example 7
Extraction and purification of amino-terminal extended IL-1
Extraction of IL-1 was performed as described in Bio Technology, Vol. 4, Dec. 1986, p. 1078. Purification of the N-terminal-extended IL-l- derivatives was performed on FF-S sepharose, FF-Q sepharose and gel filtration colonies, respec i ely. The derivatives in question were characterized by amino acid analyses, N-terminal sequence analysis, molecular weight determination by the firm atomic bombardment method (Barber M. et al.; (1981) J. Chem. Soc., Chem. Comm. Vol. 1981, p. 325-327), SDS and native electrophoresis.
Example 8
Preperation of a clone coding for Met-Glu-Ala-Glu-Phe- Asp-Il-1
By means of in vitro mutagenesis, a DNA segment with the coding region for pheny1-alanine-aspartic acid was inserted into the clone pHD230. It was inserted in such a manner that IL-1 was extended with the sequence Met- Glu-Ala-Glu-Phe-Asp- . The gene for amino-terminal- extended IL-1 was expressed in E. coli and purified as described above.
Example 9
Preparation of a clone coding for Met-Ala-Glu-IL-1
The clone was prepared by in vitro mutagenesis on the plasmid pHD230. The amino-terminal-extended IL-1 was expressed and purified as described above. The N-terminal methionine group was cleaved in vivo by an E. coli enzyme Example 10
Preparation of a clone coding for the amino acids 72-269 of IL-1
By means of in vitro mutagenesis, and around the sequence for amino acids Nos. 4-5 (DAN sequence AAGCACCCTGT) there was introduced a sequence which codes for the same amino acids, but introduced a unique Narl restriction site (ONA sequence AGGCGCCGGT).
The resulting clone pHD228 was then cut with the enzymes Narl/Clal, and then a fragment coding for the below amino acid sequence was inserted. The gene was expressed in E. coli and purified as described above.
The inserted amino acid sequence had the following composition:
Met-Lys-Leu-Arg-Lys-Met-Leu-Val-Pro-Cys-Pro-Gln-Thr-Phr- Gln-Glu-Asn-Asp-Leu-Ser-Thr-Phe-Phe-Pro-Phe-Ile-Phe-Glu- Glu-Glu-Pro-Ile-Phe-Phe-Asp-Thr-Trp-Asp-Asn-Glu-Ala-Tyr- Val-His-Asp.
Example 11
Preparation of a clone coding for Met-Ala-Tyr-Val-His- Asp-extended IL-1
Instead of the amino termal extension Met-Glu-Ala-Glu, a DNA fragment with the coding region for the amino acids Met-Ala-Tyr-Val-His-Asp was inserted by means of in vitro mutagenesis on the clone pHD230. The amino-terminal- extended IL-1 was expressed in E. coli and purified as described above. E x amp l e 1 2
Measurement of specific bioactivity of extended IL-1- 3 products
The specific bioactivity (units/ng IL-1-3) is determined by simultaneous measurement of the biological response in the comitogenic mouse thymocyte profileration assay (LAF) and by quantization by means of IL-l-/_ Elisa of the corresponding amount of IL-1-3 protein.
1. LAF assay: is performed as described above (Scand. 3. Immunol. 26:611, 1987, Scand. J. Immunol. : Endotoxin stimulated human onocyte secretion of interleukin 1, tumor necrosis factor alpha and prostaglandin E show stable in erindi idual difference, in press).
Thymocytes are isolated from 5 to 7 weeks old male mice of the C3H/he mice strain (Charles River, Federal Republic of Germany). After washing of the cells, these are plated in shallow microtiter plates (Nunc, Roskilde) in a medium consisting of a mixture of 90?ά RpMI-164 and 10?ό ac¬ cumulated heat inactivated human serum, the medium having been added to the sample in a dilution series beginning with 20 vol?ό and diluted 1:2 in up to 8 steps. The test series is repeated 3 times. PHA (Difco) 5 ,ug/ml is added to each well. After 48 hours' incubation at 37 °C 1 ,uCi tritiated thy idin is added per well. The cultures are harvested on glass fibre filters (Skatron, Norway) after another 18 hours' incubation by means of a semi-automatic cell culture harvester. Then the filters are counted in a beta counter.
The results are calculated as follows: The average of triplicates is calculated. Linear regression is performed for each dilution series, comprising a count above 3 x background. The highest counts in the dilution series are included only if they are more than 10?ό higher than the count in the next dilution step. Linear regression is performed either semi-logarithmlcly or double-logarith- micly. This results in parallel linear curves which directly allow quantization of the biological response with respect to a standard consisting of recomblπant I -l- 100 ng/ml (WHO standard). The results can then be stated in Units/ml, the WHO standard containing 100 U/ng.
2. IL-1-5 Elisa : is performed according a standardized Elisa method (Cistroπ).
3. General protein determination by means of Bio Rad. Protein assay kit.
RESULTS:
Results appear from the enclosed table. It is noted that the specific activity of MEAE-IL-1-/3 is found to be 50 to 100 times weaker than recombinant full length IL-1-3. The results are obtained from 3 independent experiments.
COMPARISON OF THE IMMUNE REACTIVITY AND LAF ACTIVITY OF RIL-1 PROCESS (B29, B41, B44)
BATCH FRACTION VOLUME UNSPEC. DILUTED MEASUREM. ELISA LAF TOTAL TOTAL SPECIFIC
FRACTION PROTEIN SAMPLE SAMPLE X DILUTED ACTIVITY BIOACT-. ELISA ACT. ACTIVITY SAMPLE SAMPLE DIRECT
B19stand. _ _ _ 2 .041 .082 40 — _ — _ 487
B29 2d 239 - 1600 .066 105.6 500 120 100 25.2 100 4.76
3d 66.5 - 8000 .04 320 1250 83 69 21.3 85 3.9
5d 17 - 80000 .0076 608 200000 3400 2833 10.3 41 330
7d 23.1 - 80000 .00055 44 12500 293 244 1.03 4 284
B29 2d 239 _ 1600 .063 100.8 500 120 100 24.1 100 4.98
3d 66.5 - 8000 .045 360 2500 166 138 23.9 99 6.9
5d 17 - 80000 .0096 768 200000 3400 2833 13.1 54 260
7d 23.4 _ 80000 .0025 200 100000 2340 1950 4.68 19 500
B41 2d 130 - 200 .64 128 500 65 100 16.6 100 3.91
3d 28 - 1000 .62 620 1250 35 54 17.4 105 2.01
5d 10 - 10000 .052 520 200000 2000 3076 5.2 31 385
7d 14.4 _ 100000 .00159 159 62500 900 1385 2.29 14 393
B44 2d 245 - 1000 .133 133 494 121 100 32.6 100 3.71
3d 53.7 - 1000 .44 440 1220 65.5 54 23.6 73 2.78
5d 25 - 200000 .0038 760 242000 6050 5000 19 56 318
7d 14.4 200000 .0025 500 147000 2117 1750 7.2 22 294
Examp le 13
Effect of IL-1 on islets
Method:
Is performed as described in Diabetes vol. 36, p. 641-647; 1987: Islet Cytotoxicity of Interleukin-1. Influence of Culture Conditions and Islet Donor Characteristics.
The pancreas is removed from 5 to 7 days old rats of the Wistar strain, and the islets are isolated by collagenase degradation. The islets are cultivated in RPMI-1640 + 10?ό NCS at 37 °C in a moist atmosphere. After 1 week's incubation the islets are washed in RPMI-1640 + 0.5?ό HS and incubated further for 6 days with and without IL-1. The insulin secretion from the islets Is determined in Radioim unoassay (RIA). The insulin secretion from the IL-1 incubated islets is stated as ?ά of the insulin secretion" from control Islets without IL-1.
Results:
Cultivation of islets with MEAE-IL-1, AE-IL-1 and rlL-l, respectively, in the following concentrations: 10, 1, 0.1 ng/ml showed that MEAE-IL-1 is 10 x less active than AE-IL-1, which is in turn 10 x less active than rlL-l (see fig . 6) .
Fig. 3 shows a standard curve for rIL-1. It will be seen that the insulin secretion is stimulated at very low rIL-1 concentrations (0.001 ng/ml) (about 130?ό), while the insulin secretion is inhibited to about 50?ά at a somewhat higher concentration (0.1 ng/ml). References:
(1): Mandel, M. Higa, A., 1970, J. Mol, Biol. , 5J_ , 159- 62.
(2): Chen, R. et al, 1980, Proc. Natl. Acad. Sci. , 77, 4592-96.
(3): Gaastra, W. et al., 1982, Microbiol. Rev., ^6_, 129- 61.

Claims

P a t e n t C l a i m s
1. A process for preparing a protein or a polypeptide of the general formula:
E - P
wherein P is a desired polypeptide, and E is an amino terminal extension, c h a r a c t e r i z e d by cultivating a bacterium containing a DNA sequence which codes for a polypeptide of the formula:
S - E
wherein S is a signal sequence, and __ and P are as defined above, in a substrate under conditions where the polypeptide of the formula S - E - P is transported through the membrane enclosing the cytoplasma of the organism with simultaneous complete or partial cleavage of the signal sequence.
2. A process according to claim 1, c h a r a c t e r ¬ i z e d in that the amino terminal extension contains at least 2 amino acids.
3. A process according to claim 2, c h a r a c t e r ¬ i z e d in that the amino extension comprises 4 to 16 amino acids.
4. A process according to claim 2 or 3, c h a r a c ¬ t e r i z e d in that the amino extension comprises at least one charged amino acid.
5. A DNA sequence for insertion into a plasmid, c h a r a c t e r i z e d in that it contains codons coding for a polypeptide of the formula:
S - E - P
wherein S, E and P are as defined in claim 1.
6. A plasmid, c h a r a c t e r i z e d in that it contains a DNA sequence as stated in claim 5.
7. A microorganism, c h a r a c t e r i z e d in that it contains plasmids as defined in claim 6.
8. A microorganism according to claim 7, c h a r a c t e r i z e d in that it is E. coli or B. subtilis.
9. A DNA sequence, c h a r a c t e r i z e d in that it has the formula stated in claim 5, wherein P is one of the polypeptides IGF1, 22K hGH, 20K hGH, IL1 or human insulin .
10. A polypeptide, c h a r a c t e r i z e d in that it consists of interleukin 1 (IL-1) or IL-1 derivatives having an amino terminal extension comprising 1 to 45 amino acids .
11. A polypeptide according to claim 10, c h a r a c ¬ t e r i z e d in that the extension comprises 1 to 30, preferably 1 to 10 amino acids.
12. A polypeptide according to claim 10, c h a r a c - t e r i z e d in that the extension comprises 2 to 6 amino acids.
13. A polypeptide according to claim 10, c h a r a c - t e r i z e d in that the extension comprises 1 amino acid .
14. A polypeptide according to claim 12, c h a r a c ¬ t e r i z e d in that it has one of the following formulae:
Met-Glu-Ala-Glu-Il-1,
Ala-Glu-Il-l or Met-Glu-Ala-Glu-Phe-Asp-Il-1.
15. A DNA sequence for expression of an amino-termlnal- extended polypeptide, c h a r a c t e r i z e d in that it codes for the polypeptide defined in claims 10-13.
16. A DNA sequence according to claim 15, c h a r a c ¬ t e r i z e d in that it has the structure shown in fig, 5.
17. A microorganism or cells, c h a "r a *c t e r i z e d in that the gene apparatus comprises the DNA sequence defined in claim 14 or 15.
18. A pharmaceutical preparation, c h a r a c t e r ¬ i z e d in that it contains a polypeptide according to any of claims 10 to 14 and optionally a pharmaceutically acceptable carrier.
19. A therapeutic preparation, c h a r a c t e r i z e d in that it is prepared as an injection preparation.
20. A therapeutic method of treatment, c h a r a c - t e r i z e d in that it consists of administration of the pharmaceutical preparation according to claim 18 or 19.
EP88905733A 1987-06-24 1988-06-23 A process for preparing a protein or polypeptide, a dna sequence coding for the polypeptide, a microorganism containing the dna sequence as well as the polypeptide and its use as a pharmaceutical preparation Withdrawn EP0371041A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DK320987A DK320987D0 (en) 1987-06-24 1987-06-24 PROCEDURE FOR PREPARING A MATURE POLYPEPTIDE WITH AN ADMINISTRATIVE AMINO-TERMINAL EXTENSION
DK3209/87 1987-06-24
DK177488A DK177488A (en) 1988-03-30 1988-03-30 POLYPEPTIDE AND PHARMACEUTICAL PREPARATION CONTAINING POLYPEPTIDE
DK1774/88 1988-03-30

Publications (1)

Publication Number Publication Date
EP0371041A1 true EP0371041A1 (en) 1990-06-06

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Application Number Title Priority Date Filing Date
EP88905733A Withdrawn EP0371041A1 (en) 1987-06-24 1988-06-23 A process for preparing a protein or polypeptide, a dna sequence coding for the polypeptide, a microorganism containing the dna sequence as well as the polypeptide and its use as a pharmaceutical preparation

Country Status (5)

Country Link
EP (1) EP0371041A1 (en)
JP (1) JPH03503596A (en)
AU (1) AU1987088A (en)
FI (1) FI896261A0 (en)
WO (1) WO1988010299A1 (en)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6858409B1 (en) 1988-05-27 2005-02-22 Amgen Inc. Nucleic acids encoding interleukin-1 inhibitors and processes for preparing interleukin-1 inhibitors
US5075222A (en) 1988-05-27 1991-12-24 Synergen, Inc. Interleukin-1 inhibitors
DK470689A (en) * 1988-09-30 1990-03-31 Lilly Co Eli HIS UNKNOWN DNA SEQUENCES CODING FOR PROTEIN DERIVATIVES THAT ALLOW HIGH EXPRESSION OF RECOMBINANT PROTEINS WITH LOW MOLECULE WEIGHT
DK105489D0 (en) * 1989-03-03 1989-03-03 Novo Nordisk As POLYPEPTIDE
US5158875A (en) * 1989-08-25 1992-10-27 Amgen Inc. Production of biologically active insulin-like growth factor i from high expression host cell systems
DK0527778T3 (en) * 1990-04-09 1996-10-21 Upjohn Co Improved method of purifying recombinant proteins and compounds useful in such a method
US5594115A (en) * 1990-04-09 1997-01-14 Pharmacia & Upjohn Company Process of purifying recombinant proteins and compounds useful in such process
DE69126434D1 (en) * 1990-08-20 1997-07-10 Novo Nordisk As Process for the production of biologically active IGF-1 by using amino-terminally extended IGF-1
DE4105480A1 (en) * 1991-02-21 1992-08-27 Boehringer Mannheim Gmbh IMPROVED ACTIVATION OF RECOMBINANT PROTEINS
US5378613A (en) * 1991-09-24 1995-01-03 Eli Lilly And Company Method for increased expression of low molecular weight recombinant polypeptides
US5496713A (en) * 1992-09-09 1996-03-05 Mitsui Toatsu Chemicals, Inc. Process for producing 20 kD human growth hormone
CZ76297A3 (en) * 1994-09-12 1998-01-14 Schering Aktiengesellschaft Process for preparing modified palladipine - inhibitor of blood platelet aggregation induced by collagen
GB9916630D0 (en) * 1999-07-16 1999-09-15 Univ Manchester Molecules derived from interleukins

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SE8204811L (en) * 1982-08-23 1984-02-24 Sven Lofdahl DNA FRAGMENT INCLUDING A SIGNAL SEQUENCE
JPS61264000A (en) * 1985-03-21 1986-11-21 イミユネツクス　コ−ポレイシヨン Synthesis of protein by labelled peptide
FR2593518B1 (en) * 1985-05-02 1989-09-08 Transgene Sa VECTORS FOR THE EXPRESSION AND SECRETION OF HIRUDIN BY TRANSFORMED YEASTS
JPS6236183A (en) * 1985-06-20 1987-02-17 ザ・サルク・インステイチユ−ト・バイオテクノロジ−／インダストリアル・アソシエイツ・インコ−ポレ−テツド Ugenoyobibunpi
DE3526995A1 (en) * 1985-07-27 1987-02-05 Hoechst Ag FUSION PROTEINS, METHOD FOR THEIR PRODUCTION AND THEIR USE

Non-Patent Citations (1)

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Also Published As

Publication number Publication date
JPH03503596A (en) 1991-08-15
FI896261A0 (en) 1989-12-22
WO1988010299A1 (en) 1988-12-29
AU1987088A (en) 1989-01-19

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