JPH09504164A - Novel family of pollen proteinases, methods of use and compositions derived therefrom - Google Patents

Abstract

  (57) [Summary] A new family of proteins has been identified. This protein is derived from allergic pollen and exhibits serine proteinase-like activity. This protein has a molecular weight of about 85-95 kD as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a molecular weight of about 67 kD as measured by high performance protein liquid chromatography. This protein is resistant to inhibition by α-2-macroglobulin, α-1-proteinase inhibitor, trypsin inhibitor, and phenylmethanesulfonyl fluoride, difluorophenol, benzamidine, antipain, leupeptin, and tosyl-L. -Sensitive to inhibition by lysine chloromethyl ketone.

Description

Detailed Description of the Invention   Novel family of pollen proteinases, methods of use and compositions derived therefrom   This invention was supported in part by National Institutes of Health Grant No. HL26148. The government has certain rights in this invention.Field of the invention   The present invention includes the field of pollen allergy, pollen proteinases and proteinases. And a method of using the same.                                Background of the Invention   It has long been known that pollen is allergic. Once the immune system is involved in pollen An immune response to pollen occurs when exposed to a series of proteins. Skin or mucous membrane There is also a "pre-immune" response that is largely associated with contact with pollen . These include itching, watery eyes and other well known physiological reactions. Allergue The physiological responses to The mechanism is unknown.   A family of allergenic proteins from rye grass pollen , LolpI, LolpII and LolpIII have been characterized. Perez et al. (1990)J. Bio 1． Chem .,265: 16210-16215; and Griffith et al., (1991).FEBS,279: 210-215 . This protein was obtained from Lolium perenne (Ryegrass) and LolpI It appears to be the major allergen for ryegrass allergy. matured, Glycosylated LolpI has a molecular weight (MW) of approximately 35,000 daltons and is It has been shown to be a key IgE binding protein. These proteins are flowers Found in the cytosol of flour. Their function is unknown.   Proteins from other common allergens have also been isolated. Dust mite In the (dust mite), a protein called Der pI, and anti-tick IgE antibody, It has been identified as reacting up to 80% of allergic sera. Der pI cloned And has been sequenced. Chira et al. (1988),J. Exp. Med.,167: 175-182 . Sequence analysis showed that Der p I was homologous to cysteine proteinase, And in fact it has cysteine proteinase activity.   Aspergillus fumigatus causing allergic bronchopulmonary aspergillosis Is a proteinase that has been proposed to be a proteinase capable of inducing human epithelial segregation. Including Rugen. Robinson et al. (1990)J. Allergy Clin. Immunol.,86: 726-731 . A. fumigatus grows in the sputum plug adjacent to the airway wall for a long period of time. Since proteinases are released only by the hyphae of fungi, proteinases Can cause chronic lung damage that occurs during airway colonization. Argued. Robinson et al. (1990). Causes serine protease activity The protein or proteins have a molecular weight of 20-35 kD.   Whether proteinases are involved in the allergic response to pollen is yet to be determined Not determined. For example, the Lolp protein discussed above is highly allergic. However, it has no proteolytic activity. Protease involved in immune response Inase identification tests whether an individual is allergic to pollen In identifying drugs that can reduce the allergic response. In developing a therapy involving an agent such as, and such treatment or It is particularly useful in monitoring patient response to treatment.   The present invention meets these and other needs.                               Summary of the Invention   A new family of proteins has been identified. This protein is allergenic It is derived from pollen and exhibits serine proteinase-like activity. This protein is When measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis Approximately 85-95 kD molecular weight; and fast protein liquid chromatography (Fast Prot has a molecular weight of about 67 kD as measured by ein Liquid Chromatography) . The apparent molecular weight depends on the glycosylation state of the molecule. This protein is α-2-macroglobulin (α-2-M), α-1-proteinase inhibitor (α-1-PI) , And resistant to inhibition by trypsin inhibitors, and phenylmeta N Sulfonyl fluoride (PMSF), difluorophenol (DFP), benzamidine , Antipain, leupeptin and tosyl-L-lysine chloromethyl ketone (TLCK ) Is susceptible to inhibition.                             Brief description of the drawings   FIG. 1 is a comparison of various serine proteinases.   Figure 2 is a full-length gene encoding a mesquite pollen proteinase. The subclones combined to obtain   Figure 3 shows pBlu containing the full-length gene encoding the mesquite pollen proteinase. The escript construct is shown.   Figure 4 shows pYT containing the full-length gene encoding mesquite pollen proteinase. The construct is shown.   Figure 5: Proteinase activity of recombinant pollen proteinase expressed in yeast Is shown.                               Aspects of the invention   A new family of proteinases includes mesquite, almond, and gama (typha), Ruslancia (Rhuslancia), Ragweed (ragweed), Japanese cedar (Japanes) e cedar), ryegrass and pine (including but not limited to It has been shown to be present in a wide variety of pollens. These pollen proteins Nase (hereinafter referred to as “proteinase”) is substrate-specific for Arg-Arg and Arg-Ile. Shows serine proteinase-like activity. This proteinase activity is Correlates with the allergenicity of pollen.   When isolated from pollen, this proteinase has a molecular weight (MW) of approximately 70-90 kD. You. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) The measured molecular weight is approximately 85-95 kD, and high performance protein liquid chromatography The MW is about 67 kD as measured by chromatography (FPLC). This proteiner Is resistant to inhibition by α-2-M, α-1-PI, and trypsin inhibitors. And PMSF, DFP, benzamidine, antipain, leupeptin and TLC Sensitive to inhibition by K.   Mesquite DNA encoding a pollen proteinase has now been cloned and Have been sequenced. Therefore, the present invention provides a recombinant gene and Included proteins. This proteinase has various serine protein It has homology to inase. Figure 1 shows several serine proteinase families. Shown in   The data presented in the examples below show that allergic pollen is more prevalent than non-allergenic pollen. It is shown that it has a higher proteinase activity. Therefore, proteinase The only mechanism whose activity affects allergenic and other non-allergic responses However, the degree of proteinase activity is not allergenic. Related. Trypsin-like activity (cleavage after arginine substrate) is detected by DFP or PMSF. Inactivation causes a total loss of proteinase activity in the crude pollen extract. , Show that it is the major proteinase in pollen samples.   The purified form of proteinase is used, for example, for diagnosis of allergy victims. Individuals for allergic response in prescribing both In formulating a diagnostic agent for testing In a vaccine that can treat allergies in monitoring efficacy , And regulate the effects of proteinases on cells involved in allergic responses Are useful in a method of identifying a drug. Induced against purified proteinase The resulting antibodies are useful for many purposes, including immunodiagnostics. The present invention also provides a Inase and the newly determined amino acid sequence of the purified polypeptide Cloning of a polynucleotide encoding a possible related gene and Includes recombinant expression.   These proteinases are found in most of the known allergic Since it has been detected in pollen of seeds, the compositions and methods of the present invention can be used in other Widely applicable to rugie pollen. For example, other highly advanced sources such as Japanese cedar. Allergic pollen is now thought to produce homologous peptides, hence , Included in the embodiments discussed herein.   Although proteinases have proteolytic activity, their biological The effect may not be directly related to this activity. Early non-immunological response above The answer is, for example, cell binding, or these newly digested proteins are itchy. May be due to proteolytic activity on other proteins that give Similarly, non- The immunological response can be directly triggered by proteolytic activity, while the immune response The response may be due to a non-proteolytically active epitope. In fact, a wide range of substrates Is the role of these proteinases in generating physiological responses. , May be due to non-proteolytic events. Such events are limited But not cell surface events to regulate receptor signaling Includes.   However, the important role of proteinases in the allergic response to pollen is , Allergic individuals affected by possible mechanisms of response to proteinases Not done. Provided by the fact that the exact mechanism is not specified The widespread applicability of the diagnoses, treatments and therapies undertaken does not diminish.   All references cited above (below and below) are hereby incorporated by reference. Is used as.   Proteinases have been isolated from pollen or used for chemical synthesis or recombinant means. Included are variants or fragments of the full-length proteinases produced thereby. Normal Is such a protein at least relative to the native (wild type) proteinase. More than about 50%, preferably more than about 90%, and more preferably at least 90% homologous. Is also about 95% homologous. Also, under stringent conditions, A polynucleotide that hybridizes to a polynucleotide encoding the enzyme code The protein encoded by the protein Also included are closely related polypeptides recovered by serum.   The length of polypeptide sequences compared for homology will generally be at least about 16 amino acids. Acid residues, usually at least about 20-24 residues, typically at least about 28 residues, and And preferably more than about 35 residues.   The term “substantially homologous” or “substantially identical” when referring to a polypeptide , The polypeptide or protein in question is a naturally occurring protein or its At least about 30% homology to the portion, usually at least about 70% homology, and Ma More specifically, it means having at least about 95% homology.   Homology for polypeptides is typically determined using sequence analysis software. To be measured. For example, Sequence Analysis Software from Genetics Computer Group Package, University of Wisconsin Biotechnology Center, 1710 University A See venue, Madison, Wisconsin 53705. Protein analysis software Using a measure of homology assigned to various substitutions, deletions, and other alterations. Match similar sequences. Conservative substitutions typically include substitutions within the following groups: Mu: Glycine, alanine; valine, isoleucine, leucine; aspartic acid, Glutamic acid; Asparagine, Glutamine; Serine, Threonine; Lysine, Al Ginine; and phenylalanine, tyrosine.   Polypeptide “fragments,” “portions,” or “segments” are rare Both about 5 amino acids, usually at least about 7 amino acids, typically at least about 9 ~ 13 amino acids, and in various embodiments at least about 17 or more It is a range of amino acid residues of many amino acids.   The terms “isolated,” “substantially pure,” and “substantially homologous” refer to naturally occurring Exchange to describe a protein or polypeptide separated from its components Used as possible. A substantially pure protein is typically a protein sample. About 60-90% W / W, more usually about 95%, and preferably about 99% or more.   Protein purity or homogeneity can be measured, for example, by the polyacrylic acid of a protein sample. Mid-gel electrophoresis followed by gel staining to yield a substantially single polypeptide It can be shown by many means known in the art, such as visualization of the band. Some purpose For higher resolution, use HPLC or other means known in the art. Can be provided.   Proteinases are separated from their natural contaminants in their natural state Substantially free of naturally associated ingredients. Therefore, it is chemically synthesized Alternatively, a proteinase synthesized in a cell line different from the cell of natural origin is Substantially free of naturally associated ingredients. Techniques for synthesizing polypeptides include, for example, Merrifield (1963)J. Amer. Chem. Soc.,85: 2149-2156.   Recombinant proteinases are suitable for expressing proteinase-encoding sequences. By culturing host cells transformed with the expression system described below under various conditions Can be obtained.   Chemically synthesized proteinases are said to be expressed in cognate cell types. However, the polynucleotide encoding the isolated proteinase that is part of the expression vector Proteinases produced as cleotide expression products (ie, "recombinant protein"). Rotinase ") and is therefore considered an" isolated "polypeptide.   Example 1 below is a trypsin from a natural source, mesquite. The purification of protein-like proteinase is described. Recombinant encoding such a protein Protease from other biological materials, such as cells transformed with a polynucleotide. Various methods of isolating the proteinase can be accomplished by methods known in the art. Various methods of protein purification are known in the art, for example:Guide to Prote in Purification , Deutscher Edition,182roll,Methods in Enzymology(Academic Pre ss, Inc .: San Diego, 1990), and Scopes,Protein Purification: Principle s and Practice  (Springer-Verlag: New York, 1982). You can   The partial amino acid sequence of mesquite proteinase was obtained as described in Example 1. , But trypsin, clostripain, or Staph Enzymes such as ylococcus proteinase, or cyanogen bromide (CnBr), O-iodo Sobenzoate, hydroxylamine, or 2-nitro-5-thiocyanobenzoe It was performed using a chemical reagent such as a sheet. The peptide fragment thus obtained The components were separated by, for example, reverse phase high performance liquid chromatography (HPLC), and And analyzed by gas phase sequencing. Means for the production of peptide fragments, And of such fragments or unfragmented proteins Other means for obtaining amino acid sequences are known in the art. In this specification The method used is described in Methods of Enzymology XI and its continuations. .   Then a partial internal amino acid for the fragment of mesquite proteinase A DNA encoding a proteinase as described in Example 1 using the acid residue sequence. Got the sequence.   Abbreviations for amino acid residues used herein are as follows: Ala (A), a Lanine; Val (V), Valine; Leu (L), Leucine; Ile (I), Isoleucine; Pro ( P), proline; Phe (F), phenylalanine; Trp (W), tryptophan; Met (M ), Methionine; Gly (G), glycine; Ser (S), serine; Thr (T), threonine; Cys (C), cysteine; Tyr (Y), tyrosine; Asn (N), asparagine; Gln (Q), Glutamine; Asp (D), Aspartic acid; Glu (E), Glutamic acid; Lys (K), Li Gin; Arg (R), arginine; and His (H), histidine.   Proteinases include variants of their basic polypeptide sequence, It is substantially homologous to its primary sequence and has a characteristic biological activity of the proteinase ( For example, serine proteinase-like activity, allergenicity, or immunological activity, That is, one or more antigenic determinants recognized by antibodies specific to proteinases. Hold). Such in vivo or in vitro chemical and biochemistry Modifications include, for example, rarely used amino acids, acetylations, and carboxyls. , Phosphorylation, glycosylation, ubiquitination, eg radionuclides and other modifications Labeling can be mentioned.   Various methods for labeling polypeptides, and useful devices for such purposes. Substitutions or labels are known in the art,³²Radioactive isotope such as P, labeled anti-body Ligands that bind to ligands (eg, antibodies), fluorophores, chemiluminescent reagents, enzymes , And an anti-ligand capable of acting as a specific binding pair member to a labeled ligand Is mentioned. The choice of label depends on the sensitivity required, the binding to the proteinase (con jugation), stability requirements, and available equipment. Polype The labeling method of peptide is, for example,Molecular Cloning: A Laboratory Manual,2 Edition, Volumes 1-3, edited by Sambrook et al. (1989) Cold Spring Harbor Laboratory Press. Or Current Protocols in Molecular Biology, Ausubel et al., Greene Publishi ng and Wiley-Interscience: New York (1987 and periodic updates) ing.   In addition to the substantially full-length polypeptide, the invention also includes fragments of the polypeptide. Or segment. Such fragments or segments are usually Is at least about 5 to 7 consecutive amino acid residues, typically at least about 9 to 13 consecutive amino acids, and most preferably at least about 20 to 30 amino acids. Or more consecutive amino acid residues. This fragment is a protein It may retain a proteinase-specific or proteinase-specific epitope.   Tandemly repeated proteinase fragments can also be used, for example, in immunogens. Can be useful as or as a very effective competitor for specific binding. Prote Production of antibodies specific for inase or fragments thereof is described below . Methods for recombinantly or synthetically producing tandemly repeated segments , Within the skill of the artisan. Both polyclonal and monoclonal Methods of body production are within the skill of those in the art.   The invention also provides a fusion polypeptide comprising a proteinase or fragment thereof. Provide the code. Homologous polypeptides are two or more proteinase sequences. Can be fused between or between the sequences of proteinases and related proteins. Similarly, different A species fusion is one that exhibits a combination of properties or activities of the proteins from which it is derived. Can be built. Fusion partners include immunoglobulins and ubiquitin bacterial β- Lactosidase, trpE, protein A, β-lactamase, α-amylase, al Includes cold dehydrogenase, and yeast α-mating factor However, it is not limited to these. Godowski et al. (1988)Science,241: 812-816. Fusion The protein is typically produced recombinantly, but can be chemically synthesized.   In other embodiments, proteinase fragments, homologues or variants thereof Polynucleotides encoding (including, for example, protein fusions or deletions) And expression systems are provided. The expression system is transformed into a suitable host cell. When defined as a polynucleotide capable of expressing a proteinase. Poly A nucleotide is a polynucleotide that encodes a natural proteinase or its Having a nucleotide sequence substantially similar to the fragment of   Polynucleotides include RNA, cDNA, genomic DNA, synthetic forms, and sense strands. Contains mixed polymers of both antisense and antisense strands, and chemically or biochemical Containing nucleotide bases that can be modified biologically or that are unnatural or derivatized obtain. Recombinant polynucleotides containing non-naturally occurring sequences may also be used in wild-type Provided by the present invention as a variant of the proteinase sequence. The variant is Other polynucleotides containing deletions, insertions, substitutions of one or more nucleotides, or By, but not limited to, fusion to a sequence.   To obtain the polynucleotide encoding the proteinase, the appropriate cDNA (specific The pollen cDNA library) or the genomic library is the polynucleotide of the present invention. Can be screened as a natural source of tide or Otido involves amplification of sequences present in mRNA, cDNA, or genomic DNA, which Amplification by methods such as, for example, polymerase chain reaction (PCR). Provided by the method.   Amino acid residue sequences of various peptide fragments of mesquite proteinase. Are obtained as described in the Examples section. This proteinase In order to clone the gene encoding all the homologous proteinases, Rigonucleotides are chemically synthesized to generate such peptide fragments. Most or all possible DNA sequences that can be encoded, or such peptides Hybridizes to the native proteinase coding sequence encoding the fragment. One of the few sequences with a high likelihood of being sized was shown. Then oligo A pool of octides or oligonucleotides was used as a probe to Probing and isolation of cDNA or genomic libraries containing coding DNA sequences The sequences that were processed and purified were identified. Then probe the library Or by any of the amplification methods using such degenerate oligonucleotides The resulting polynucleotide sequence itself is a full-length cDNA or Used as a probe or a primer for obtaining the sequence of the sequence.   These oligonucleotides also display homologous proteinases in various plants. Direct cloning of all or part of the encoding gene or genes , Which is useful as a primer for Genomic DNA by a tide amplification method (eg, polymerase chain reaction, or PCR) Present in mRNA or in a cDNA or genomic library This is done by selectively amplifying the proteinase gene. PCR The library can be cloned using either rolling or such degenerate oligonucleotides. Cleaning is an oligonucleotide that corresponds to two or more peptide sequences. In the case of the present invention, when cleotide is synthesized, it is promoted when it is available. It is so. Several proteinase genes have been cloned and When these nucleotide sequences are compared, regions of homology are identified and probed. The probes or primers are designed to include these homologous regions. like this Probes and primers are derived from various plants to obtain other related genes Is useful for direct cloning or screening of libraries.   A polynucleotide typically will be at least about 12-15 nucleotides (or base pairs). ), More usually at least about 21 nucleotides, and most preferably less. Both contain a sequence corresponding to about 35 nucleotides, the length of which depends on the desired use . There may also be one or more introns.   How to construct an appropriate cDNA and genomic library, Methods for screening ibaries, suitable probes, primers (eg PC R primer), polynucleotide purification, amplification, and Recombinant DNA technology suitable for rowing, transformation of host cells, and practice of the invention Other techniques in Sambrook et al. (1989); Ausubel et al. Edition); andPCR Protocols: A Guide to Methods and ApplicationsEdited by Innis et al. , Academic Press: San Diego (1990). Apply such a technique Reagents, such as restriction enzymes, expression vectors, labels, etc. Known in the field, New England BioLabs, Boehringer Mannheim, Amersham, Prom ega Biotec, U. S. Biochemicals, New England Nuclear, and many other vendors Available from sellers, such as commercial sources.   Polynucleotides or fragments thereof are best suited for comparison with other polynucleotides. , About 60% nuclei when sequenced with appropriate nucleotide insertions or deletions. Leotide base, usually about 70%, more usually about 80%, preferably about 90%, More preferably about 95-98% nucleotide base identity with the nucleotide sequence "Substantially homologous" (or "substantially homologous" with another polynucleotide, if Is similar to ").   Alternatively, the substantial homology (or similarity) is the polynucleotide or its Fragments can bind to other polynucleotides under selective hybridization conditions. Present when it hybridizes to The selectivity of hybridization is A hybridid capable of distinguishing a target polynucleotide of interest from other polynucleotides Exists under zation conditions. Typically, selective hybridization is About 55% similarity over a range of about 14 nucleotides, preferably about 65%, and more preferably It occurs when there is a similarity of preferably about 75%, and most preferably about 90%. Ka nehisa (1984)Nucl. Acids Res.,12See: 203-213. It is listed As such, the lengths for homology comparison may be longer and in certain embodiments Usually ranges from about 17 to 20 nucleotides, and preferably about 36 or It is a longer nucleotide.   Hybridization of polynucleotides depends on salt concentration, temperature, or organic Medium, as well as base composition, complementary strand length, and hybridizing polynucleotides. Affected by conditions such as the number of nucleotide base mismatches between It will be easily understood by those skilled in the art. Stringent temperature conditions generally exceed 30 ° C Including temperatures, typically above 37 ° C, and preferably above 45 ° C . Stringent salt concentrations are usually below 1M, typically below 500 mM , And preferably less than 200 mM. But how about the combination of parameters? Is much more important than the measurement of a single parameter. Wetmur and Davidson ( 1968)J. Mol. Biol.,31: 349-370.   An "isolated" or "substantially pure" polynucleotide is a natural protein. A polynucleotide that is substantially separated from other polynucleotide sequences naturally associated with the Nucleotides. This term refers to a poline derived from a naturally-occurring environment. Recombinant or cloned DNA isolates and chemistries including cleotide sequences Include chemically synthesized analogs or biochemically synthesized analogs from heterologous systems. No.   The polynucleotide is engineered in its native state or by methods known to those of skill in the art. Transcription and / or to produce a polypeptide or fragment thereof, if Alternatively, a polypeptide is "encoded" when it can be translated. like this The antisense strand of the polynucleotide is also said to encode this sequence.   Polynucleotide sequences may be functionally related to other polynucleotide sequences. Operably coupled when disposed in. For example, a promoter Operably linked to a coding sequence if it affects transcription or expression. . Generally, operably linked means that the sequences being linked are contiguous, And two protein coding regions need to be linked in And in the open reading frame. But like an enhancer Certain genetic elements are operative even distally, i.e. not continuous It is well known that they can be linked.   The term "recombinant" polynucleotide refers to two separately separated segments. A polynucleotide produced by ligation, which is produced by genetic engineering techniques. Artificial of a segment of a polynucleotide isolated by or by chemical synthesis The sequence achieved by the operation. In doing so, the desired function By combining the polynucleotide segments with each other, a desired combination of functions Can be created.   Isolated polynucleotide probe attached to a label or reporter molecule For isolating and identifying other proteinase sequences, including polynucleotides Can be used. A probe containing synthetic oligonucleotides or other polynucleotides. The lobes may be derived from naturally occurring or recombinant single or double stranded nucleic acid. Alternatively, it may be chemically synthesized. Polynucleotide probes are well known in the art. Any known method (eg random hexamer labeling, nick translation , Or Klenow fill-in reaction).   Large quantities of polynucleotides can be produced by replication in suitable host cells. Step A natural or synthetic DNA fragment encoding a proteinase or fragment thereof. Is a recombinant polynucleotide construct, typically a prokaryotic or eukaryotic cell. Introduced into a DNA construct capable of introduction and replication therein. Usually The construct is suitable for replication in a unicellular host such as yeast or bacteria, or is multicellular. Eukaryotic hosts are also suitable with or without integration within the host cell's genome. It can be painful. Commonly used prokaryotic hosts include Escherichia coli strains However, other prokaryotic hosts such as Bacillus subtilis or Pseudomonas may also be used . Mammalian or other eukaryotic host cells include yeast, filamentous fungi, plants, insects, amphibians, or Or birds are included. Ease of operation, proper glycosylation of expressed protein You Ability, degree and control of protein expression, and whether the expressed protein is a cell contaminant. Factors such as ease of purification, or other factors, may affect the selection of host cells. obtain.   Polynucleotides are also described, for example, by Beaucage and Carruthers (1981).Tetra. Letts. ,twenty two1859-1862, or the phosphoramidite method or Matteucci et al. 81)J. Am. Chem. Soc.,103: 3185 by the triester method, by chemical synthesis And can be performed on a commercially available automated oligonucleotide synthesizer. Double-stranded fragments synthesize complementary strands and anneal the strands together under appropriate conditions By using a DNA polymerase with or without the appropriate primer sequences. Can be obtained from a chemically synthesized single-stranded product by adding complementary strands.   A DNA construct prepared for introduction into a prokaryotic or eukaryotic host will typically be recombinant for the host. Includes a more recognized replication system, which is intended to encode the desired polypeptide A polypeptide containing a DNA fragment and preferably encoding a segment Also included are transcriptional and translational initiation regulatory sequences operably linked to the sequence. Expression system (expression vector Is an origin of replication or autonomously replicating sequence (ARS), and expression control sequences. Certain promoters, enhancers, and necessary processing information sites, such as , Ribosome binding site, RNA splice site, polyadenylation site, transcription termination sequence Lanes, and mRNA stabilizing sequences. The signal peptide can also be the same or Can be included in a suitable secreted polypeptide from a related species of Are allowed to pass and / or remain there, or secreted from cells.   Selection of the appropriate promoter and other necessary vector sequences is functional in the host. Is chosen to be Of operable combinations of cell lines and expression vectors Examples are Sambrook et al. (1989); Ausubel et al. (1987); Metzger et al. (1988).Nature,334: 31-36. In bacteria, yeast, mammals, insects, plants, or other cells Many vectors useful for the expression of E. coli are well known in the art, and Stratagene, Ne Available from England Biolabs, Promega Biotech, and other vendors Can be In addition, this construct is linked to an amplifiable gene (eg, DHFR). One can then obtain multiple copies of the gene. Appropriate enhancers and other expression control elements For columns,Enhancers and Eukaryotic Gene Expression, Cold Spring Harb See also or Press, N.Y. (1983). Such expression vectors replicate autonomously But they are less preferred because they are inserted into the genome of the host cell. Cannot be duplicated.   Expression and cloning vectors are designed with a selectable marker, that is, a vector. Genes encoding proteins required for survival or growth of transformed host cells It seems to include. Such a marker gene is introduced into the host cell at the same time. Cloning vectors can be included in other polynucleotide sequences, but in most cases Included in Only the host cells into which the marker gene has been introduced survive under the selection conditions And / or grow. Typical selection genes are (a) antibiotics or other toxic substances. Resistant to substances such as ampicillin, neomycin, methotrexate Feeding; (b) supplementing auxotrophy; or (c) supplying essential nutrients not available from complex media Code for a protein that does. The selection of the appropriate selectable marker will depend on the host cell. Suitable markers for different hosts are known in the art.   The vector containing the polynucleotide of interest is prepared by any of a number of suitable means. It can be introduced (transformed, transfected) into a host cell, and these means include Electroporation; calcium chloride, rubidium chloride, calcium phosphate , DEAE-dextran, or other transfections; Mike LOS PROJECT BOMBMENTMENT; Lipofection; and Infection (Retro If the vector is an infectious agent, such as a viral genome). this The choice of such means usually depends on the host cell. Large amounts of polynucleosides of the invention Tide and polypeptides are expressed in compatible vectors or other expression vehicles. A polynucleotide encoding a light proteinase, suitable for prokaryotic or eukaryotic host Transforms primary cells and achieves expression of proteinase coding genes By culturing the host cells thus transformed under conditions suitable for Can be made. The proteinase can then be recovered from the host cell and purified.   In another embodiment, the proteinase or fragment thereof is specifically Polyclonal and / or monoclonal antibodies capable of binding are provided. for The term antibody refers to a serum product composed of homologous molecular entities or multiple different molecular entities. Used to mean any of the mixtures. Reacts specifically with proteinases The monoclonal or polyclonal antibody can be prepared by a method known in the art. Can be created. For example, Harlow and Lane (1988)Antibodies: A Laboratory M anual , CSH Laborartories; Goding (1986)Monoclonal Antibodies: Principle s and Practice Second Edition, Academic Press, New York; and Ausubel et al., (1987 ). Also, recombinant immunoglobulins can be produced by methods known in the art. . This method includes, but is not limited to, the method described in U.S. Patent No. 4,816,567. Not done. Ten⁸M^-1, Preferably 10⁹~Ten^TenOr objects with higher affinity Clonal antibodies are preferred.   Antibodies specific for proteinases are, for example, clones of cDNA expression libraries. For the detection of the presence of proteinases in cleaning or test samples Can be useful as a probe. Frequently, polypeptides and antibodies will be covalently linked. Or by non-covalent attachment to a substrate that provides a detectable signal More labeled. Suitable labels include enzymes, substrates, cofactors, inhibitors, fluorescent agents , Chemiluminescent factors, magnetic particles and the like, but are not limited thereto. like this US patents that describe the use of various labels include, but are not limited to, the following: No. 817,837; No. 3,850,752; No. 3,939,350; No. 3,996,345; No. 4,277,437; 4,275,149; and 4,366,241.   Antibodies specific for proteinases and capable of inhibiting proteinase activity are , In the treatment of animals (including humans) affected by pollen proteinases Can be for. Such antibodies can be prepared by the method described above, followed by pollen proteinase specific. A selective antibody for its ability to inhibit proteinase activity Can be obtained.   In another embodiment, a substantially purified proteinase from pollen and Compositions and vaccine preparations are provided that include the same and suitable carriers thereof. This Vaccines such as It is useful to immunize   The vaccine preparation comprises an immunogenic amount of at least one proteinase or its immunity. Contains epidemiological fragments or subunits thereof. One such vaccine is One or more proteinases, or another protein or other immunogen Can be combined with a proteinase. "Immunogenic dose" is the vaccine The amount that can induce the production of antibodies against proteinase in the individual to be administered To taste.   Immunogenic carriers can be used to enhance the immunogenicity of proteinases. You. Such carriers include proteins and polysaccharides, liposomes, and bacteria. Includes, but is not limited to, cells and membranes. Protein carriers Linked to proteinases by alternative or synthetic means or chemical coupling Can be formed into a fusion protein. A useful carrier, and a carrier like this Means for coupling A to a polypeptide antigen are known in the art. You.   The vaccine may be formulated by any means in the art. like this Vaccines are injectable, typically as either a liquid solution or suspension As prepared. Suitable for solution or suspension in liquid prior to injection Crude solid forms can also be prepared. The preparation can also be emulsified, for example, or It can be a protein encapsulated in liposomes.   The active immunogenic ingredient is an excipient that is pharmaceutically acceptable and compatible with the active ingredient or Often mixed with carrier. Suitable excipients are water, saline, dextro Glucose, glycerose, ethanol, etc., and combinations thereof. It is not limited to these. The concentration of immunogenic polypeptide in an injectable formulation is , Usually in the range of 0.2-5 mg / ml.   In addition, if desired, the vaccine should contain minor amounts of adjuvants (eg, humectants or milk). Agents, pH buffers, and / or adjuvants that enhance the effectiveness of the vaccine) May be included. Examples of adjuvants that may be effective include, but are not limited to: Not determined: aluminum hydroxide; N-acetyl-muramul-L-threonyl-D-iso Glutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isogluta Min (CGP 11637, also called nor-MDP); N-acetylmuramyl-L-alanyl-D-I Soglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxy Ciphosphoryloxy) -ethylamine (CGP 19835A, termed MTP-PE); and RIBI, which was extracted from bacteria in a 2% squalene / Tween 80 emulsion 3 Components monophosphoryl lipid A, trehalose dimycolate, and cell wall Contains the skeleton (MPL + TDM + CWS). The effectiveness of the adjuvant depends on the vaccine (this (Also composed of various adjuvants) resulting from the administration of immunogens in It can be determined by measuring the amount of antibody to the epidemics. Known in the art Additional formulations and dosage forms such as can also be used.   Proteinases and their fragments, in neutral or salt form, are It may be prescribed in Chin. Pharmaceutically acceptable salts include acid addition salts (peptide free salts). Formed with a min group). This salt is an inorganic acid, For example formed with hydrochloric acid or phosphoric acid and with organic acids such as acetic acid, oxalic acid , Formed with tartaric acid, or maleic acid. Salt formed with free carboxyl groups Are also inorganic bases such as sodium, potassium, ammonium, calcium , Or ferric hydroxide, and organic bases such as isopropylamine, trime Derived from tilamine, 2-ethylaminoethanol, histidine, and procaine Can be guided.   Vaccines are administered in a manner compatible with the dosage regimen, and prophylactically and / or therapeutically. Is administered in an amount that is therapeutically effective. The amount to be administered is generally dose-specific. Protein is in the range of about 100-1000 μg, and more commonly The range of protein is about 5 to 500 μg, which depends on the subject to be treated, the antibody. It depends on the capacity of the individual's immune system to grow and the degree of protection desired. Required for administration Precise amounts of active ingredient administered may depend on the judgment of the practitioner and are peculiar to each individual. However, such decisions are within the skill of such physicians.   Vaccines may be given in a single dose schedule or a multiple dose schedule obtain. In a multiple dose schedule, the first stage of vaccination is 1-10 or It may include more than one individual administration, and then another dose for the second dose Subsequent time intervals as required to maintain and / or enhance the answer (eg, For 1 to 4 months) and, if necessary, the next one or more doses several months later. The dose is administered.   In another embodiment, a method of monitoring animal exposure to proteinases. Is provided. Such a monitoring method can be performed, for example, by using the proteinase of the present invention. Odor to determine whether the pollen spores from which it originates causes an allergic condition Or treatment advances designed to mitigate the symptoms of such allergic conditions. Useful for monitoring lines.   In general, biological samples obtained from animals (eg blood, saliva, tissues) are Incubation with proteinases or parts thereof under conditions suitable for somatic-antigen interactions. Be baited. The detection of the formation of such interactions can be detected by pre-exposure and subsequent treatment of the animal. The following progression of the immune response to Rotainase is shown. An example of such a test is Allergen adsorption test (RAST) and enzyme-linked immunosorbent assay (ELISA) Including but not limited to.   Alternatively, the subject can be exposed to a proteinase and the next reaction can be monitored. . Such exposure may be on the skin using methods well known in the art (eg Applied to the skin by stinging or scratching), intradermally (eg, Intradermal injection) or in the form of an aerosol (typically an aqueous aerosol) intranasally or in the air Bronchial pathways (nasoprovocatrion or bronchoprovon respectively) ocation)). Typical reactions such as papules in skin tests and Erythema, or asthma or rhinitis in intranasal or bronchial induction Shows immunological response to Nase. For example, for a general description,Basic an d Clinical Immunology , 6th Edition, Stites et al., Ed. (Appleton & Lange, 1987 ), Pp. 436-438.   Proteinases also provide a way to identify factors that regulate proteinase activity. Can be used in This method works on the proteinase itself, or Or does not interfere with the interaction of proteinases with cells of allergic individuals. It depends on the gap. Such a method involves the following steps: Incubating with a constant therapeutic factor; Measuring the activity of the inase; and incubating with the obtained activity and factors Comparing the activity of the control sample that was not present.   In another embodiment, of allergic rhinitis in a patient with pollen allergy In order to reduce the symptoms, the dose is increased and the proteinase is repeated over a long period of time. Immunotherapy provided by recurrent injection. For example, stit for general description See es et al. (1987), page 442.   In another embodiment, the pollen proteinase of an animal (including human) Methods are provided to treat or mitigate the effects. Such a method is effective for animals Comprising administering a physiologically acceptable pollen proteinase inhibitor I do. Known proteinase inhibitors are generally not physiologically acceptable. However, acceptable inhibitors inhibit pollen proteinases, but It includes factors that do not or only minimally affect the activity of the proteinase. Such inhibitors include, but are not limited to, inhibitory antibodies and small molecules. Available from various sources. Inhibitors are administered by a variety of methods Can be done. This method can be applied topically, by aerosol by the nasal route or by pulmonary administration. , Gastrointestinal administration, subcutaneous administration, intravenous administration, and intraperitoneal administration, Not limited. Inhibitors can be administered as needed, especially topically or in an aerosol. Can be administered when applied by. These administration methods are known in the art. As such, it is not described in detail herein.   The above discussion and the following examples illustrate, but do not limit the invention. Not. Those skilled in the art will appreciate that the present invention may be practiced in other ways and that the claims Understand that it is only defined.                                 Example 1     Purification scheme for the isolation of trypsin-like proteinase from mesquite   To determine if proteinase is present in pollen, mesquite protein The following protein purification procedure was carried out for inase. Using the same purification technique , A proteinase from this novel family also contains ragweed, amendow and It was also purified from cattail.   50 g of mesquite pollen with 5 mM CaCl₂0.02M Bis-Tris buffer containing 200 ml, pH Mixed with 6.5. The mixture was stirred in the cold room (4 ° C) for 42-48 hours. Then extract Centrifuged at 48,200 xg for 30 minutes.   Supernatant was brought to 30% saturation with solid ammonium sulfate and allowed to stand for 10 minutes, then 48,200 xg It was centrifuged for 20 minutes. The supernatant is then brought to 60% saturation with solid ammonium sulfate for 10 minutes. while It was left to stand and centrifuged as above.   Precipitate 100 ml of 5 mM CaCl₂Dissolve in 0.02M Bis-Tris buffer (pH 6.5) containing The same buffer was exchanged twice and dialyzed for 4 hours. Dialyzed protein solution with 0.4M vinegar Adjust to pH 4.5 with sodium phosphate buffer (pH 3.8) and centrifuge for 15 minutes at 48,200 xg. Was. The supernatant was adjusted to pH 6.5 with 1.0 M Tris-HCl (pH 8.0), and 5 mM CaCl₂Including 0.02M It was dialyzed against Bis-Tris buffer, pH 6.5 for 24 hours with two buffer changes.   Dialysate is 5 mM CaCl₂Cib equilibrated with 0.02M Bis-Tris buffer, pH 6.5 containing Add to acron Blue Sepharose column (3.0 x 27.0 cm (190 ml volume)) and The rum was washed with the same buffer. Because proteinase elutes in the excluded volume, The collection started later. For each fraction, as described in Example 5, Perform assays for enzyme and amidase activity and retain peak activity Was.   Pool the active fractions and use the DEAE-sephacel column (3.0 x 18.0 cm (127 ml volume). ), A_280nmWas washed until it was less than 0.050. Then 0.02M Bis-Tris Loose Buffer solution, pH 6.5, 5 mM CaCl₂Elution was performed with a linear gradient of 0-0.5 M NaCl in the medium.   Active fractions were collected, pooled and solid NaCl was added to give a final concentration of 2.0M.  NaCl was used. This solution was then added to 0.02M Bis-Tris buffer, pH 6.5, 5 mM CaCl₂, Phenyl Sepharose column (2.0 × 17.2 cm, equilibrated with 2.0 M NaCl, (54.0 ml Volume)). A column with this buffer_280nmWash until is less than 0.05 did. Then all 0.02M Bis-Tris buffer, pH 6.5, -5 mM CaCl₂2.0 M to 0 M in An enzyme eluate was obtained with a linear gradient of NaCl.   Active fractions eluted from the phenyl sepharose column were pooled, Amicon Concentrate on the filter to approximately 7.0 ml and 0.02M Bis-Tris buffer, pH 6.5, 5 mM.  CaCl₂, Sephadex G-150 column equilibrated with 0.2 M NaCl (2.2 × 90.0 cm (342 ml Volume)). The protein was eluted with the same buffer.   The active fractions were pooled, concentrated to 5-10 ml, 0.02M Bis-Tris buffer , PH 7.0, 5 mM CaCl₂Dialyzed against, and fast protein liquid chromatograph It was applied to a Mono-Q-ion exchange column connected to a FPLC system. Start buffer The column was developed with 0.1 M NaCl in solution, followed by 0.1 M NaCl to 0.25 M NaCl in the same buffer. straight It was developed with a linear gradient. The enzyme was eluted between 0.15M NaCl and 0.165M NaCl.                                 Example 2                         Molecular weight of proteinase   The proteinase purified as described in Example 1 has various apparent MW. , Which depends on the mechanism used for the measurement.   Schaggen and von Jagow (1987)Anal. Biochem.,166: 368-379 Sodium dodecyl sulfate using the Tricine-SDS-PAGE protocol according to Polyacrylamide gel electrophoresis (SDS-PAGE) was performed. 85kD by SDS-PAGE A MW of between 95kD was obtained.   The MW was also measured by gel filtration using the FPLC system. Bovine serum albumin Since the peak of mesquite proteinase eluted at the same position as MW, MW was 67 kD It seemed to be near. Therefore, the hydrodynamics of this protein depends on the method used for this measurement. Dependently gives various MW values.                                 Example 3                       Sequence of protein amino acid residues   No amino acid residue detected after 10 cycles using protein sequencer As such, this proteinase has a blocked amino terminus. Therefore, first the proteinase was cleaved into small fragments, and the fragment The protein was sequenced by sequencing the sequence.   400 μg (4.4 nmol) mesquite pollen proteinase for trypsin digestion Incubated with 6M guanidine-HCl for 30 minutes at 37 ° C. Then 3.2 nmol di Add thiothreitol (DTT), flush with nitrogen, and mix for 2 hours. After incubation, 48 nmol iodoacetamide was added and 2 more in the dark. Incubated for hours. The mixture is then treated overnight with 2 liters of 50 mM ammonium bicarbonate. It was dialyzed against Um (pH 7.0). TPCK-treated bovine trypsin (trypsin vs. protein Inase, at a ratio of 1:50) 5 mM CaCl₂Together with dialysis proteinase, The mixture was then incubated for 6 hours and then freeze dried. Lyophilized sample To 1.0 ml with HPLC grade water and Waters Associates using a Micro Pak SP column.  Developed on HPLC. Collect peptide peaks and freeze for sequencing Dried.   200 μg (2.2 nmol) mesquite pollen proteiner for cyanogen bromide (CNBr) digestion The amount of methionine residue in the proteinase was 300 μg of CNBr (CNBr: 1 : 100) and 70% formic acid in 700 μl, flushed with nitrogen in the dark for 20 hours at 20 ° C. While incubating. Dilute sample 10x with HPLC grade water and lyophilize did. Lyophilized sample was dissolved in sample buffer and developed on SDS-PAGE. It was electroeluted from the stained PVDP membrane and the band was excised for sequencing.   Applied Biosystems, In Online with High Performance Liquid Chromatography c., Sequencing was performed using model # 4a77A or 470A. Follow manufacturer's instructions This operation was carried out, and it was all based on the Edman decomposition operation.   The following amino acid residue sequence was obtained:   (1) TLAGKEYAQYXR (trypsin digestion)   (2) KGPYXYXYYX (trypsin digestion)   (3) TLAGKEYAQYXR (trypsin digestion)   (4) FNFYLDVSKPEEGLKVLTPXR (trypsin digestion)   (5) ENGLPNIIVYHLPPIGGPL (trypsin digestion)   (6) MEGIDTTVSHR (trypsin digestion)   (7) IKEDDTTAPLR (trypsin digestion)   (8) DEILRPDXAXLXRLGTEQX (cyanogen bromide digestion)   (9) XXFYFYMXSYXP (V-8 proteinase digestion)   In all cases, X represents an unidentified amino acid residue.                                 Example 4               CDNA encoding mesquite pollen proteinase   A cDNA encoding a mesquite pollen proteinase was obtained as described herein. , Subcloned and expressed in yeast. Unless otherwise stated, the molecule The biology technique is Sambrook et al., Molecular Cloning 2nd Edition, Cold Spring Harbor L. ab The method described by oratory Press (1989) was followed. Poly A + RNA, Jerome , First-strand cDNA for PCR analysis, isolated from freshly germinated mesquite anthers harvested in AZ. Used to make. Briefly, Chomczynski and Sacchi (1987) A nal. Biochem. RNA from non-defatted mesquite pollen according to the AGPC method described in 162: 156. Was isolated and subjected to oligo d (T) chromatography. Zapf et al. (1990) J. B iol. Chem. 265: 14892 using 10 μg of prepared poly A + RNA. Complexity 2 × 10⁶ΛZAPII (Stratagene, La Jolla, CA) cDNA library did.   Using a cDNA library, trypsinized mesquite pollen proteinase The sequence was determined by PCR using degenerate primers based on the known amino acid sequence of the peptide sequence. A probe was prepared by amplifying The primer is ABI model No.39 4 Follow the manufacturer's instructions by phosphoramidite chemistry on the DNA / RNA synthesizer. Therefore it was created. The following nucleotides, MK-39-50, 256x degenerate primer Corresponding to the trading chain. The following nucleotides, MK-42-44, 32-fold degenerate primer To respond. Both primers are wild boar at the wobble position encoded by the four nucleotides. And an EcoR1 restriction endon to facilitate cloning of PCR fragments. Contains a creatase site.   Using hot-start PCR to suppress non-specific annealing as follows: Was. A final concentration of 200 μM dNTP, 2.5 mM MgCl 2 was added to 100 pmol of each primer.₂And 1x PCR buffer was added and heated to 80 ° C. with one Ampliwax bead. 25 reactants Chilled to ℃, and the remaining 5 μl first strand cDNA template, 2 units Taq1 polymer Enzyme and water were added to make 100 μl. The reaction is carried out at 94 ° C for 1 minute and then at 32 ° C for 10 minutes. Perform 10 cycles of minutes, followed by 94 ° C for 1 minute; 55 ° C for 1 minute; 72 ° C for 1 minute 2 Five cycles were carried out and finished at 72 ° C for 7 minutes.   A 300 bp PCR product was generated using the trypsinized peptide-based probe, HG-3-2 A 3,12-fold degenerate non-coding strand oligomer identified by Southern blot analysis.   This 300 bp PCR fragment was PCR-primed according to the manufacturer's instructions. The produced EcoRI site was used to clone into the EcoRI site of pCR-script (Stratagene). I learned. This clone, called A6, was probed with dot HG and Identified by Zanblot. Sequence analysis revealed that this clone And containing the 3'region used as a probe.   Mesquite pollen library with a 300 bp EcoRI fragment from clone A6 Screened to identify partial cDNA clones. A 2.1 Kb cDNA was identified. Figure 2 shows the restriction endonuclease map of this clone called MPP1-1 (1-1) Show. Since MPP1-1 is not full length, the 300 bp BglII / HindIII 5'fragment Used as a probe to further screen the cDNA library, and A 1.6 Kb clone was isolated and designated MPP8-3 (8-3). Figure 2 shows the control of MAPP8-3. 1 shows a restriction endonuclease map. This clone was cloned inside the A-rich region. It appears that the immers have started and, as shown in Figure 2, the putative initiator at the 5'end. Target methionine and with MPP1-1 at a single EcoRI site near the 3'end. Duplicate.   5'BglII / EcoRI fragment from MPP8-3 and 3'EcoR1 / Xho1 fragment from MPP1-1 The lagment was ligated into the BamHI / XhoI digested pBluescript (Stratagene) vector. This recloned fragment was used to open the entire mesquite pollen proteinase. Contains a reading frame and 80 bp of 5'untranslated sequence; immediately at the 3'end of the stop codon Includes a later XhoI site. This complex full length codon is called MPP.   Both strands of the full-length cDNA were first subcloned into M13, followed by Sanger dideoxy. It was sequenced by the method.   The following nucleotide sequence was obtained: The encoded amino acid residue immediately after the sequence Shown below.   As shown in Fig. 2, the MPP cDNA contains one open reading frame of 772 amino acid residues. Code the Ding frame. The estimated molecular weight of a full-length protein is 88 At kD, the estimated pI is 5.3. 3 possible N-linked glycosylation sites, at the amino terminus A possible signal sequence of 29 amino acid residues and 200 amino acids at the carboxy terminus There is an acid residue stretch, which extends against E. coli protease II. 63% identity, and the serine protease prolyl endopeptidase Reduced homology to Millie, as well as triad amino acid residues with identical catalysis It has the basic order Ser, Asp, His. The arrangement of this particular amino acid residue Placing in a class of enzymes distinct from the subtilisin and chymotrypsin families .   Full-length MPP to express the protein encoded by the recombinant gene The sequence was cloned into the yeast expression vector pYT in a two step process as follows: Was. pYT is a yeast 2 μm sequence for autonomous replication, and URA3 for selection in yeast. And LEU2d gene, and both the E. coli origin of replication and the ampicillin resistance gene. It is a 15.2 Kb vector containing the pBR322 sequence. pYT is also BamHI and SalI Downstream of the unique cloning site of the α-factor required for translation termination -Including a terminator. Unique clonin in such a large vector Since the target site is missing, the gene of interest and promoter And / or other signal sequences require in-frame insertion. did. Glucose-regulatable alcohol dehydrogenase II (AD H2) gene (Shuster (1989))Yeast Genetic Engineering， Butterworths ， Massa chusetts, pp. 83-107) were used. This ADH2 promoter is cloned by PCR. Ligated and ligated into the pBluescript NotI / XhoI restriction endonuclease site did. Also, the in-frame insertion of the gene to be expressed should be included in the PCR fragment. Therefore, the cloning site XbaI is included at the 3'end of the promoter. Complete cassette Is excised at the 5'BamHI site of the ADH2 promoter and the XhoI site at the 3'end of MPP. Cloned into the BamHI / SalI sites of pYT, resulting in plasmid MPPpYT . The pBluescript construct is shown in Figure 3 and the PYT construct is shown in Figure 4.   The MPPpYT plasmid was replaced with LXR1 ([cir⁰], MATa, leu2, trp1, ura3-52, prb1-1122 , Pep4-3, prc1-407) were used to transform the yeast strains. This strain Is an endogenous plasmid of BJ2168 (deposited in Berkeley Type Culture Collection), Barr et al. (1987) Biotech. 5: 486 as described in uracil selection. It is a strain that has been cured by the Roplast method. Transformants with leucine Maintained on plates without. Transformants were deprived of glucose and ADH2 48 to 72 hours when the promoter is derepressed and recombinant MPP is expressed in YEPD medium Grown in.                                 Example 5                  Proteinase inhibitor profile   Purified as described in Example 1 to characterize the proteinase The protein inhibitor profile was measured as follows.   For inhibition of trypsin-like activity, pollen extract, buffer (0 .1 M sodium phosphate, pH 7.4, 0.15 M NaCl), and Bz-L-Arg-pNA (substrate) A control consisting of an incubation mixture was used. Add this mixture to 10 Incubated at room temperature for minutes. The reaction was stopped by adding 50 μl of glacial acetic acid. That Then, read at 405 nm and observe the appearance of yellow coloration indicating proteolytic cleavage. It was measured. The release of p-nitroaniline can be quantified, indicating the progress of hydrolysis.   Different amounts were used to test the inhibition of proteinases by each inhibitor sample. Bz-L-Arg-pNA-free basal incubation mixture , Pre-incubate for 5-10 minutes, then add substrate and mix The material was processed as described above. The proteinase inhibition for each sample was Measured as the difference between the values for the did.   EDTA (Baker), Leupeptin (Calbiochem), Aprotonin (Bayer) and Ovom All inhibitors except Coid (Worthington) were purchased from Sigma Chemical Company. Was. Tomato and potato inhibitors were a gift of Dr. C. Ryan. I used The concentration of inhibitors was as follows: PMSF (4 mM), B (EDTA); (4 mM); cysteine ( 4 mM); Benzamidine (10 mM); Antipain (0.1 μg / ml); Leupeptin (0.2 μg / ml); TLCK (0.5 mM) (mesquite / 12%; rhuslanci) a) / 3%); and ragweed / 6%); TPCK (1.0 mM); E-64 (0.2 mM); P -Aminobenzamidine (10 mM); Aprotonin (0.5 mg / ml); Ovomucoid (0.5 m g / ml); SBTI (0.5 mg / ml); LBTI (0.5 mg / ml) and 2-1-PI (1.2 nmol).   To measure the inhibition of casein-degrading activity, the assay was performed with 0.4 ml of pollen extract. , 0.1 ml buffer (0.1 M sodium phosphate, pH 7.4, 0.15 M NaCl) and It consisted of 0.25 ml of an incubation mixture of 3% azocasein. This The mixture was incubated at 37 ° C for 24 or 48 hours at which point 0.75 m l of 10% trichloroacetic acid (TCA) was added. Prior to the addition of the azocasein solution, the Inhibitor (5 mM, 0.4 ml) was added to the base incubation mixture for 5-10 minutes. Just added. Azocasein alone and extract alone were also controlled . When proteolytic cleavage occurs, enzyme activity, a soluble yellow color, is added to the supernatant. The rest, and that casein and uncleaved azocasein remain in the precipitate Is shown.   The data obtained are shown in Tables 1 and 2. Table 1 shows-in selected pollen The inhibition spectrum of lipsin-like activity is shown, and Table 2 shows the casein in pollen. The inhibition spectrum of decomposition activity is shown.   Proteinases are affected by any conventional proteinase inhibitor Not done. These are α-1-PI, soybean trypsin inhibitor, limametrip Syn inhibitor, tomato trypsin inhibitor I, II and PC1, potato Lipsin inhibitor PCT1 and tobacco inhibitor TlI, tosyl-L-phenyla Lanine chloromethyl ketone (TPCK), E-64 (trans-epoxysuccinyl-leucine Luamide- (4-guanido) butein) PMSF, ethylenediaminetetraacetic acid EDTA, beta Insamidine, p-aminobenzamidine and Kunitz basic pancreatic tryps Includes syn-inhibitors. Then tomato, potato and tobacco inhibitor Was tested for purified proteinase and found to be inactive .   Inhibition includes PMSF, DFP, benzamidine, antipain, leupeptin and TLC Found about K. Therefore, proteinases are enzyme metalloproteinases. Not a member of either zecras or cysteine proteinase class It seems This is because this enzyme is a member of the serine class of proteinases. Indicates that                                 Example 6           Assay of proteinase activity of various crude pollen extracts   Do other pollens in the families described herein contain proteinases? In order to determine whether or not the pollen shown in Table 3 was subjected to rough purification, Their activity was assayed. In Table 3, the starred plants are less allergenic It was found to be yes or no.   The crude purification was carried out as follows: 1.0 g in the presence of 3.0 g 0.5 mm glass beads. Pollen was mixed with 12.0 ml of 0.1 M Tris-HCl, pH 8.0; and 0.15 M NaCl. Trial The material was vortexed for 10 minutes, followed by centrifugation at 48,000 xg for 20 minutes.   The crude fraction was subsequently tested for activity in the following system: Azocasein content Solution, hide powder, elastin esterase, cathepsin G esterer Zeze and trypsin-like activity.   The azocasein degradation assay was performed as described in Example 5, and casein degradation was performed. Inhibition of activity was measured as described. The results obtained are shown in Table 4. In Table 4 Plants with an asterisk are weak or not allergenic.   Rawhide powder assay was performed as follows: 10 mg blue rawhide powder in 0.8 ml Buffer (1.0M Tris-HCl, pH8.0, 0.15M NaCl, 1% Brij) and 0.4ml flower Mixed with flour extract. A control of rawhide or pollen extract alone was also used . Incubate sample at 37 ° C for 2.5, 5.0, and 24 hours with continuous shaking did. At the end of the given time, the sample is allowed to settle and the color development in the supernatant is reduced to 59%. Read at 5 nm. The results obtained are shown in Table 5. In Table 5, plants marked with an asterisk Things are weak or non-allergenic. Blue rawhide powder is insoluble, , If this enzyme is active, the proteolytic properties of the peptide bound to the dye Is present, i.e. the soluble peptide fragment containing the dye is It is measured quantitatively as an effective indicator of the proteinase tested.   The elastin esterase assay was performed as follows: 0.1 M sodium phosphate Um buffer, pH 7.4, 0.15 M NaCl and 0.04 ml Suc-Ala-Ala-Ala-pNA (5 mM) ( Sigma), incubate 0-0.5 ml of pollen extract or purified enzyme for 20 minutes. I did it. The absorbance at 405 nm was then read as a measure of substrate cleavage, as described above. I caught it. Table 6 shows the obtained results. In Table 6, the plants marked with an asterisk are Weak or no allergy.   The cathepsin G esterase activity assay was performed as follows: 0.1M sodium phosphate Thorium buffer, pH 7.4, 0.15M NaCl and 0.01 ml Suc-Ala-Ala-Pro-Phe-p Incubate 10-0.5 ml of pollen extract or purified enzyme in NA (50 mM) for 10 minutes. I started. The absorbance at 405 nm was then used as a measure of substrate cleavage, as described above. I read it. The results obtained are shown in Table 7. In Table 7, plants marked with an asterisk are Allergic or weak.   A trypsin-like activity and plasma kallikrein activity assay was performed as follows. : 0.1 M sodium phosphate buffer, pH 7.4, 0.15 M NaCl and 40 μl Pro-Phe- 0-0.5 ml of pollen extract or purified enzyme in Arg-pNA (5 mM) (Sigma) for 10 minutes Incubated. The absorbance at 405 nm was then measured for substrate cleavage as described above. I read it as a scale. Table 8 shows the obtained results. In Table 8, marked with a star Plants are weak or non-allergenic.                                 Example 7                     Purified protein activity assay   Mesquite pollen proteinase purified from pollen was tested to further enhance its activity. The amino acid residue specificity was measured to characterize the fraction. Characteristics of amino acid residues The protocol for measuring isomerism uses peptides of known sequence, and Met. 1.0 ml 25 mM ammonium bicarbonate buffer, pH 7.8; 5 mM calci chloride Um; and 0.125% sodium azide; insulin; melittin; 250 μg of any of the norphin fragments 1-13 was added and mixed well. At time zero, 80 μl was removed, while 80 μl was also incubated at 37 ° C overnight. Was. Subsequently, 14 μl of purified mesquite purified as described in Example 1. Proteinase (10.08 μg) (molar ratio of proteinase to peptide substrate 1: 25) was added, mixed well and incubated at 37 ° C. Each time point (0, 0.2 (5, 1.0, 5.0 and 24 hours), remove 166 μl of sample and dry with dry ice. Frozen and stored frozen until analysis. Thaw sample, peptide fragment Were separated by HPLC, the peptide peaks were collected and analyzed for amino acid composition. Was analyzed.   The protocol for activation of prothrombin was as follows: described in Example 1. 0.74, 1.48 or 2.22 μg mesquite proteiner purified as described Z. 0. in 0.1 M Tris-HCl, pH 8.0; 0.15 M NaCl; and 5 mM calcium chloride. 90 min with 10 or 20 μg prothrombin (Calbiochem) in a final volume of 2 ml Incubated for up to. This is followed by 0.8 ml of the same buffer and thrombi. Substrate, H-D-Phe-Plp-Arg-pNA (Kabi Pharmaceuticals) was added. 10 minutes After incubation, stop the reaction by adding acetic acid and absorb at 405 nm. The degree was measured. Because mesquite proteinase also cleaves this substrate, Control without prothrombin, even though Controls without inase were measured and their hydrolysis rates were Subtracted from the hydrolysis rates found in the mixture of tainase and prothrombin .   The active proteinase fraction is after the arginine residue and to a much lesser extent. Did lysine residues specifically hydrolyze both synthetic substrates and proteins after? Was. The tested proteins, including melittin and oxidized insulin B chain, It was not hydrolyzed by the enzyme. Any Lys-Ile bond that requires cleavage There was no activation of lipsinogen. However, dynorphin (1-1 The small peptide fragment in 3) is cleaved between the Arg-Arg and Arg-Ile residues. Was. Furthermore, of prothrombin, which requires cleavage between Arg-Ile peptide bonds. Precursors that also require activation and cleavage at the same type of peptide bond There was reccrein (PK) conversion. Many synthetic substrates (D-pro-phe-arg-p-nitroani Lido (Sigma); and Bz-L-Arg-pNA (Kabi Pharmaceuticals)) also It was rapidly hydrolyzed by.                                 Example 8               Prekallikrein activation of pollen proteinases   To determine whether proteinases have physiological effects on the human immune system The crude extracts were tested to determine their ability to activate PK. PK is Kalicre Which is then activated by kininogen, which is the vasodilator bradykinin. Activate to. Bradykinin is responsible for several allergic reaction symptoms. You.   As shown in Table 9, proteinase activates PK in Hageman factor-deficient plasma. I do. Therefore, this enzyme produces bradykinin, a vasoagent. Can be an indirect cause of   This assay is based on 1 of crude pollen extract prepared as described in Example 5. : 60 μl of 25 dilution, 0.1 M NaPO_FourBuffer, pH 7.4; buffer containing 0.15M NaCl 60 μl PK (Dr. Bruce Zo ran, donated by Scripps Research Institute, LaJolla, California) By doing that. The sample was then divided into two equal aliquots and Treated with 0.3 μg SBTI (Sigma) and the other half with buffer did.   Then, each sample was tested using D-Pro-Phe-Arg-pNA (S-2302, Kabi Pharmaceuticals). The reagents were assayed for kallikrein activity. The results obtained are shown in Table 9.                                 Example 9         Activation of prothrombin by mesquite pollen proteinase.   The mesquite proteinase purified as described in Example 1 was treated with protron Tested for ability to activate bottles. This activity assay is described in Example 8. I went as if. The data are shown in Table 10. Here as described in Example 1 First, the proteinase was purified and the concentration was measured.   The data shown in Table 10 shows that prothrombin is associated with mesquite proteinase. Is converted to thrombin. Therefore, the activity of the aggregation system by this enzyme The oxidization could occur in vivo.   One or more enzymes release bradykinin from kininogen directly in plasma Experiments to determine whether or not an indirect mechanism is the only pathway In progress.                               Example 10     Protocol for testing vascular permeability-enhancing response in guinea pigs   Vascular permeation to determine whether proteinases have physiological effects The ability to increase sex was measured. Anesthetize guinea pigs with 80 mg / kg ketamine Then, Evans Blue Dye (30 mg / kg) was injected into the metatarsal vein. First, Prote The inase solution was subjected to a 30 minute incubation with human plasma. Then This pre-incubated proteinase solution was applied to the guinea pig's shaved back. Intradermally. After 15 minutes, while still anesthetized with Metofane (methoxyflurane), Guinea pigs were euthanized by exsanguination using the carotid transection technique. back Exfoliate skin and obtain injection site using 15 mm punch biopsy device Was. Extract the dye from the skin with formamide and spectrophotometrically at 620 nm. It was measured. The results obtained are shown in Table 11.   A time course study showed that the peak production of vascular permeability enhancers was at 30 minutes. Was shown. Injection of proteinase directly into the skin of guinea pigs Only a few or no hyperactive reactions were given. In contrast, 100n g bradykinin caused 46 μg of dye release.                               Example 11         Analysis of recombinant mesquite proteinase produced in yeast   1.Activity assay   Yeast control and MPP expression cultures (rMPP) were centrifuged and cell pelleted. Vortex with 0.5 mM glass beads in TE + 0.1% Triton X-100. And dissolved. The clarified lysate was treated according to the method described in Example 6 with 5 mM K₂HPO_Four, For 5 mM pyro-GluGlyArgpNA (pNA) in 15 mM NaCl (pH 8) Assayed for proteolytic activity. The sample was used to measure the pNA conversion rate. At 405 nm in a kinetic assay for Incubated and obtained a single reading. Various pH levels Bell-cultured rMPP cultures showed 5-8.5 times higher activity than yeast controls. did. The results obtained are shown in FIG.   2.Inhibitor binding. Lysate irreversible with chloromethylketone inhibitor Assayed for the ability to specifically bind. Inhibitor TyrAlaLysArgC-K¹²⁵I Transformed MPPpYT, labeled with and obtained as described above, and Added to 4-10 μg lysate from both untransformed LXR1 yeast. Implementation 5 μg of crude mesquite pollen extract, obtained as described in Example 1, was also Labeled with this inhibitor as a control. Proteins on 15% SDS-PAGE Separated, Coomassie stained and dried gels were exposed to X-ray film. The results obtained show that crude mesquite pollen extract is a proteinase with the same molecular weight as rMPP. It is shown to contain ze. Yeast transformed with mesquite pollen extract and MPPpYT Both extracts from Escherichia coli have similar apparent molecular weight tampers that bind to the inhibitor. Extracts of yeast that contain protein but are not transformed with MPPpYT Does not include quality.   The present invention has been described in some detail by way of illustration and examples for the sake of clarity. Although detailed, it will be apparent to those skilled in the art that certain changes and modifications may be made. It is. Accordingly, this description and examples are presented in the book as set forth in the appended claims. It is not meant to limit the scope of the invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI C12N 1/19 9637-4B C12P 21/02 C 9/50 7823-4B C12Q 1/37 C12P 21/02 0276- 2J G01N 33/53 Q C12Q 1/37 9051-4C A61K 37/547 ABB G01N 33/53 9051-4C 37/54 ABF // (C12P 21/02 C12R 1: 645) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AM, AT, AU, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, ES, FI, GB, GE, H , JP, KE, KG, KP, KR, KZ, LK, LU, LV, MD, MG, MN, MW, NL, NO, NZ, PL, PT, RO, RU, SD, SE, SI, SK, TJ, TT, UA, US, UZ, VN (72) Inventor Travis, James United States Georgia 30602, Athens, Life Sciences Building, Department of Biochemistry, The University of Georgia (No Address) (72) Inventor Bur, Philip, Jay. United States California 94705, Berkeley, Hillcrest Road 152

Claims (1)

[Claims] 1. A composition comprising substantially pure proteinase having a molecular weight of about 85-95 kD as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a molecular weight of about 67 kD as measured by high performance protein liquid chromatography; The proteinase is resistant to inhibition by α-2-macroglobulin, α-1-proteinase inhibitor, trypsin inhibitor; and phenylmethanesulfonyl fluoride, difluorophenol, benzamidine, antipain, leupeptin, and tosyl-L. -A composition that is susceptible to inhibition by lysine chloromethyl ketone. 2. The proteinase according to claim 1, wherein the source of the proteinase is pollen. 3. The proteinase according to claim 2, wherein the pollen is derived from mesquite, Rhuslancia, ragweed, cedar, amendow, cattail or pine. 4. The proteinase according to claim 1, wherein the proteinase is a serine proteinase. 5. The proteinase according to claim 1, which has a substrate specificity for Arg-Arg and Arg-Ile. 6. The proteinase according to claim 1, having the following amino acid residue sequence: 7. A substantially pure polynucleotide encoding a proteinase according to claim 1 having a sequence capable of encoding a peptide fragment of said proteinase or hybridized to a naturally occurring polynucleotide encoding said proteinase. Probing a cDNA or genomic library with at least one oligonucleotide with a high probability; identifying the presence of a polynucleotide in the library; and isolating and purifying the polynucleotide, A polynucleotide obtained by the method comprising. 8. A substantially pure and isolated form of a polynucleotide encoding the proteinase of claim 1 having a sequence capable of encoding a peptide fragment of said proteinase, or encoding said proteinase. Synthesizing an oligonucleotide with a high probability of hybridizing to a natural polynucleotide; present in genomic DNA or mRNA or by cDNA by polymerase chain reaction (PCR) using the oligonucleotide as a primer Or selectively amplifying a proteinase-encoding polynucleotide present in a genomic library; and purifying and isolating the proteinase-encoding polynucleotide, Nucleochi . 9. A recombinant polynucleotide sequence comprising the following nucleic acid residues: 10. An expression system capable of expressing the polynucleotide of claim 9 when transformed into a host cell. 11. 11. A method comprising culturing the cells transformed by the expression system of claim 10 under conditions suitable for achieving expression of the proteinase-encoding gene, and recovering the proteinase. A method for preparing an alternative proteinase. 12. A composition comprising a substantially purified pollen proteinase and a suitable carrier thereof. 13. 13. The composition of claim 12, wherein the proteinase is a serine proteinase. 14． 13. The composition according to claim 12, wherein the pollen is derived from mesquite, ruslanchia, ragweed, cedar, amendou, cattail, or pine. 15. 13. The composition of claim 12, having substrate specificity for said proteinase, Arg-Arg and Arg-Ile. 16. 13. The composition of claim 12, wherein the proteinase has the following amino acid residue sequence: 17． A vaccine comprising a substantially purified pollen proteinase and a suitable carrier therefor. 18. 18. The vaccine of claim 17, wherein the proteinase is a serine proteinase. 19. 18. The vaccine according to claim 17, wherein the pollen is derived from mesquite, ruslanchia, ragweed, cedar, amendou, cattail, or pine. 20. 18. The vaccine of claim 17, wherein the proteinase has substrate specificity for Arg-Arg and Arg-Ile. 21. 18. The vaccine of claim 17, wherein the proteinase has the following amino acid residue sequence: 22. A method of monitoring exposure of a pollen proteinase to an animal, comprising: obtaining a sample from the animal; incubating the sample with the pollen proteinase or a portion thereof under conditions suitable for antibody-antigen interactions. And a step of detecting the presence of the antibody-antigen complex; wherein the presence of the antigen-antibody complex indicates exposure of the animal to the proteinase. 23. A method of immunizing an animal against an allergic response to pollen, comprising: obtaining at least one purified pollen proteinase or an immunogenic subunit thereof; in an amount effective to elicit an immune response to the proteinase. Administering at least one pollen proteinase or an immunogenic subunit thereof. 24. A method of identifying an agent that modulates the effects of pollen proteinase on an animal, comprising: a) incubating the pollen proteinase with the agent; b) animal cells sensitive to the proteinase incubated with the agent. Exposing the pollen proteinase; c) measuring the ability of the proteinase incubated with the agent to affect the cells; and d) the effect observed in step c) being sensitive to the proteinase. Comparing the effect of a proteinase control sample that has not been incubated with the drug in an animal cell; wherein the drug that modulates the effect of the pollen proteinase has an activity of the proteinase relative to the control sample. How to change. 25. A method of identifying an agent that modulates pollen proteinase activity, comprising: a) incubating a pollen proteinase with the agent; b) measuring the activity of the proteinase incubated with the agent; and c) the step b. A.) Comparing the activity obtained in) with the activity of a control sample of proteinase that has not been incubated with the agent. 26. A method of ameliorating the effect of a pollen proteinase on an animal affected by the proteinase comprising the step of administering to said animal an effective amount of a physiologically acceptable pollen proteinase inhibitor.