EP0705435A1 - A novel family of pollen proteinases, methods of use thereof and compositions derived therefrom - Google Patents

Abstract

A novel family of proteins has been identified. The proteins are derived from allergenic pollens and exhibit serine-proteinase-like activity. The proteins have a molecular weight of about 85-95 kD as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis; and a molecular weight of about 67 kD as determined by Fast Protein Liquid Chromatography. The proteins are resistant to inhibition by α-2-macroglobulin, α-1-proteinase inhibitor, trypsin inhibitors and are sensitive to inhibition by phenyl methane sulfonyl fluoride, difluorophenol, benzamidine, antipain, leupeptin and tosyl-L-lysine chloromethyl ketone.

Description

A NOVEL FAMILY OF POLLEN PROTEINASES. METHODS

OF USE THEREOF AND COMPOSITIONS DERIVED THEREFROM

This invention was funded in part by National Institutes of Health Grant No. HL26148. The Government may have certain rights in the invention.

Field of the Invention

This invention relates to the field f pollen allergies, pollen proteinases, composit ons comprising the proteinases and methods of use thereof.

Background of the Invention It has long been known that pollens are allergenic. An immunological response to pollens occurs once the immune system has been exposed to the proteins associated with pollen. There are also a "pre-immune" response largely associated with contact of the skin or mucous membranes with pollen. These include itching, tearing and other well-known physiological reactions. Although the physiological responses to allergens are well defined, the precise mechanisms by which these responses are effected are unknown.

A family of allergenic proteins, LolpI, LolpII and LolpIII obtained from rye grass pollen has been characterized. Perez et al. (1990) J. Biol. Chem. , 2_£5.16210-16215; and Griffith et al. (1991) FEBS. 279;210-215. The proteins were obtained from Lolium perenne (rye grass) and it appears that LolpI is the major allergen responsible for rye grass allergy. Mature, glycosylated LolpI has a molecular weight (MW) of about 35,000 Daltons and has been shown to be the major IgE binding protein. These proteins are found in the cytosol of pollen. Their function is unknown.

Proteins from other common allergens have also been isolated. In dust mites, a protein termed Der pi has been identified as reacting with anti-mite IgE antibodies in up to 80% of allergic sera. Der pi has been cloned and sequenced. Chira et al. (1988) J. Exp. Med.. 167:175-182. Sequence analysis showed that Der pi is homologous to cysteine proteinases and in fact has cysteine proteinase activity.

Aspergillus fumigatus, responsible for allergic bronchopulmonary aspergillosis, contains an allergen that has been proposed to be a proteinase capable of inducing human epithelial detachment. Robinson et al. (1990) J. Allergy Clin. Immunol.. _86.:726-731. A. fumigatus grows within sputum plugs adjacent to the airway wall for prolonged periods. Since the proteinase is released only by the hyphal tip of the fungus, it has been theorized that the proteinase may be responsible for the resulting chronic lung damage that occurs upon colonization of the airways. Robinson et al. (1990). The protein or proteins responsible for the serine protease activity are from 20-35 kD in molecular weight. It has not yet been determined whether proteinases are involved in allergic responses to pollen. For instance, the Lolp proteins discussed above are highly allergenic but do not have proteolytic activity. Identification of proteinases involved in immune response would be particularly useful in testing whether an individual is allergic to pollen, identifying agents that could ameliorate allergic responses, developing treatments involving such agents, and in monitoring the response of a patient to such treatments or therapies. The present invention meets these and other needs. _._,„

Summary of the Invention A novel family of proteins has been identified.

The proteins are derived from allergenic pollens and exhibit serine proteinase-like activity. The proteins have a molecular weight of about 85-95 kD as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis; and a molecular weight of about 67 kD as determined by Fast Protein Liquid Chromatography. The apparent molecular weight is dependent on the glycosylation state of the molecule. The proteins are resistant to inhibition by o.-2-macroglobulin (α-2-M) , α-1-proteinase inhibitor (α-l-PI) , and trypsin inhibitors and are sensitive to. inhibition by phenyl methane sulfonyl fluoride (PMSF) , difluorophenol (DFP) , - benzamidine, antipain, leupeptin and tosyl-L-lysine chloromethyl ketone (TLCK) .

Brief Description of the Drawings Figure 1 is a comparison of various serine proteinases.

Figure 2 depicts the subclones which were combined to obtain the full length gene encoding the mesquite pollen proteinase.

Figure 3 depicts the pBluescript construct containing the full length gene encoding the mesquite pollen proteinase. Figure 4 depicts the pYT construct containing the full length gene encoding the mesquite pollen proteinase.

Figure 5 depicts the proteinase activity of the recombinant pollen proteinase expressed in yeast.

Modes of the Invention It has now been shown that a novel family of proteinases is present in a wide variety of pollens including, but not limited to, mesquite, almond, typha Rhuslancia, ragweed, Japanese cedar, ryegrass and pine. These pollen proteinases (hereinafter "the proteinases") exhibit serine proteinase-like activity, displaying a substrate specificity for Arg-Arg and Arg-lie. The proteinase activity is correlated with the allergenicity of these pollens.

When isolated from pollen, the molecular weight (MW) of the proteinases is about 70-90 kD. As determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the MW is about 85-95 kD and as determined by Fast Protein Liquid Chromatography (FPLC) the MW is about 67 kD. The proteinases are resistant to inhibition by α-2-M, α-l-PI, and trypsin inhibitors and sensitive to inhibition by PMSF, DFP, benzamidine, antipain, leupeptin and TLCK. The mesquite DNA encoding the pollen proteinase has now been cloned and sequenced. The invention thus encompasses the recombinant gene and protein encoded thereby. The proteinase has homology to various serine proteinases. Several serine proteinase families are depicted in Figure 1.

The data presented in the Examples below indicate that allergenic pollens have significantly higher proteinase activity than non-allergenic pollens. The degree of proteinase activity is thus correlated with allergenicity, although it is possible that proteinase activity may not be the sole mechanism effecting allergenicity and other nonallergic responses. Inactivation of the trypsin-like activity (cleavage after arginine ubstrates) by DFP or PMSF causes total loss of proteinase activity in the crude pollen extracts, indicating that this is the primary proteinase in the pollen sample.

The proteinases in purified form are useful, for example, in formulating both diagnostic and therapeutic regimens for allergy sufferers, in formulating diagnostics for testing individuals for allergic responses, in monitoring the effectiveness of allergy treatments and therapies, in vaccines capable of treating an allergy, and in methods for identifying agents that modulate the effect of the proteinases on cells involved in a. rgic responses. Antibodies raised against the purified proteinases are useful for a number of purposes, including immunodiagnostics. The present invention also embraces the cloning and recombinant expression of polynucleotides encoding the proteinases and related genes made possible by the newly determined amino acid sequence of the purified polypeptide.

Because these proteinases have been detected in most species of pollen known to be allergenic, the compositions and methods of the present invention are widely applicable to other allergenic pollens as well. For example, other highly allergenic pollens such as Japanese cedar are now thought to produce homologous peptides and thus are encompassed by the embodiments discussed herein.

Although the proteinases have proteolytic activity, it is possible their biological effects may not be directly related to this activity. The initial non- immunological response described above may be due to cell binding, for instance, or proteolytic activity on other proteins which renders these newly digested proteins irritating. Likewise, non-immunologic responses may be directly caused by the proteolytic activity whereas immune responses may be due to non-proteolytically active epitopes. In fact, the absence of widespread substrates indicates that the role of these proteinases in generating a physiological response may be due to a non- proteolytic event. Such events include but are not limited to modulating cell surface events so as to modulate receptor signalling.

The important role of the proteinases in allergic responses to pollen, however, is not affected by the possible mechanisms by which an allergic individual responds to a proteinase. The fact that the exact mechanism has not been defined does not diminish the broad applicability of the diagnostics, treatments and therapies provided herein.

All references cited herein, both supra and infra, are hereby incorporated herein by reference.

The proteinases include variants or fragments of full length proteinases as isolated from pollen or produced by chemical synthesis or recombinant means. Ordinarily, such proteins will be at least about 50% homologous to the native (wild-type) proteinase, preferably in excess of about 90%, and, more preferably, at least about 95% homologous. Also included are proteins encoded by polynucleotides which hybridize under stringent conditions to proteinase-encoding polynucleotides and closely related polypeptides retrieved by antisera raised against a proteinase.

The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 to 24 residues, typically at least about 28 residues, and preferably more than about 35 residues.

When referring to polypeptides, the term "substantial homology" or "substantial identity" indicates that the polypeptide or protein in question exhibits at least about 30% homology with a naturally occurring protein or a portion thereof, usually at least about 70% homology, and preferably at least about 95% homology.

Homology, for polypeptides, is typically measured using sequence analysis software. See, e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

A polypeptide "fragment, " "portion, " or "segment" is a stretch of amino acid residues of at least about 5 amino acids, often at least about 7 amino acids, typically at least about 9 to 13 amino acids, and, in various embodiments, at least about 17 or more amino acids.

The terms "isolated," "substantially pure, " and "substantially homogeneous" are used interchangeably to describe a protein or polypeptide which has been separated from components which accompany it in nature. A substantially pure protein typically comprises about 60 to 90% W/W of a protein sample, more usually about 95%, and preferably over about 99%. Protein purity or homogeneity may be indicated by a number of means known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a substantially single polypeptide band upon staining the gel. For certain purposes higher resolution may be provided by using HPLC or other means known in the art.

The proteinases are substantially free of naturally associated components when separated from the native contaminants which accompany them in their natural state. Thus, a proteinase that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Techniques for synthesis of polypeptides are described, for example, in Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156.

Recombinant proteinases can be obtained by culturing host cells transformed with the expression systems described below under conditions suitable to attain expression of the proteinase-encoding sequence. A chemically synthesized proteinase is considered an "isolated" polypeptide, as is a proteinase produced as an expression product of an isolated proteinase-encoding polynucleotide which is part of an expression vector (i.e., a "recombinant proteinase"), even if expressed in a homologous cell type.

Example 1 below describes the purification of a trypsin-like proteinase from mesquite, its natural source. Various methods for the isolation of the proteinases from other biological material, such as from cells transformed with recombinant polynucleotides encoding such proteins, may be accomplished by methods known in the art. Various methods of protein purification are known in the art, including those described, e.g., in Guide to Protein Purification, ed. Deutscher, vol. 182 of Methods in Enzymology (Academic Press, Inc.: San Diego, 1990) and Scopes, Protein Purification: Principles and Practice (Springer-Veriag: New York, 1982) .

The partial amino acid sequence of the mesquite proteinase was obtained as described in Example 1, with enzymes such as trypsin, clostripain, or Staphylococcus proteinase or with chemical agents such as cyanogen bromide (CnBr) , O-iodosobenzoate, hydroxylamine or 2- nitro-5-thiocyanobenzoate. The peptide fragments thus obtained were separated for instance by reverse-phase high performance liquid chromatography (HPLC) and analyzed by gas-phase sequencing. Other means for generating peptide fragments and obtaining the amino acid sequence of such fragments or an unfragmented protein are known in the art. The method used herein is described by Methods of Enzymology XI and subsequent volumes. The partial internal amino acid residue sequences for fragments of the mesquite proteinase were then used to obtain the DNA sequence encoding the proteinase as described in Example 1.

The abbreviations for amino acid residues as used herein are as follows: Ala (A), alanine; Val (V), valine; Leu (L) , leucine; lie (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; Gin (Q) , glutamine; Asp (D) , aspartic acid; Glu (E) , glutamic acid; Lys (K) , lysine; Arg (R) , arginine; and His (H) , histidine.

The proteinases include modified forms of the basic polypeptide sequences thereof but which are substantially homologous to that primary sequence and which possess a biological activity characteristic of a proteinase (e.g., serine proteinase-like activity, allergenicity, or immunologic activity, i.e., possession of one or more antigenic determinants recognized by an antibody specific for a proteinase) . Such .in vivo or in vitro chemical and biochemical modifications include, e.g., unusual amino acids, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labelling, e.g., with radionuclides and other modifications. A variety of methods for labelling polypeptides and substituents or labels useful for such purposes are known in the art, including radioactive isotopes such as ³²P, ligands which bind to labeled antiligands (e.g., antibodies) , fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the proteinase, stability requirements, and available instrumentation. Methods of labeling polypeptides are described, e.g., in Molecular Cloning: A Laboratory Manual. 2nd ed. , Vols. 1-3, ed. Sambrook et al. (1989) Cold Spring Harbor Laboratory Press or Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987 and periodic updates) . Besides substantially full-length pol -eptides, the present invention provides for fragments or segments of the polypeptides. Such fragments or segments will ordinarily be at least about 5 to 7 contiguous amino acid residues, typically at least about 9 to 13 contiguous amino acids, and most preferably at least about 20 to 30 or more cont; nous amino acid residue.. The fragments may retain proteinase activity or a proteinase specific epitope. Tandemly repeated proteinase fr ments may also be useful, e.g., as immunogens or as highly efficient competitors for specific binding. Production of antibodies specific for proteinases or fragments thereof is described below. Methods of recombinantly or synthetically producing tandemly repeated segments are within the skill of one in the art. Methods of producing antibodies, both polycr~nal and monoclonal, are within the skill of one in the art.

The present invention also provide:- for fusion polypeptides comprising proteinases or fragments thereof. Homologous polypeptides may be fusions between two or more proteinase sequences or between the sequences of a proteinase and a related protein. Likewise, heterologous fusions may be constructed which would exhibit a combination of properties or activities of the proteins from which they are derived. Fusion partners include, but are not limited to, immunoglobulins, ubiquitin bacterial 3-galactosidase, trpE, protein A, 3-lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factor. Godowski et al. (1988) Science. 241:812- 816. Fusion proteins will typically be made by recombinant methods, but may be chemically synthesized. In another embodiment, polynucleotides which encode the proteinase fragments, homologs or variant thereof, including, e.g., protein fusions or deletions, as well as expression systems are provided. Expression systems are defined as polynucleotides which, when transformed into an appropriate host cell, can express a proteinase. The polynucleotides possess a nucleotide sequence which is substantially similar to a natural proteinase-encoding polynucleotide or a fragment thereof. The polynucleotides include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases. Recombinant polynucleotides comprising sequences otherwise not naturally occurring are also provided by this invention, as are alterations of a wild type proteinase sequence, including but not limited to deletion, insertion, substitution of one or more nucleotides or by fusion to other polynucleotide sequences.

In order to obtain polynucleotides encoding the proteinases, suitable cDNA (particularly pollen cDNA libraries) or genomic libraries may be screened as natural sources of the polynucleotides of the present invention, or such polynucleotides may be provided by methods including but not limited to amplification of sequences resident in mRNA, cDNA or genomic DNA, e.g., by amplification methods such as the polymerase chain reaction (PCR) .

The amino acid residue sequences of various peptide fragments of a mesquite proteinase have been obtained as described in the Examples Section. In order to clone the gene encoding this or any homologous proteinase, oligonucleotides were chemically synthesized to represent most or all possible DNA sequences capable of encoding such a peptide fragment or one of a small number of sequences having a high likelihood of hybridizing to a native proteinase-encoding sequence encoding such a peptide fragment. The oligonucleotide or pool of oligonucleotides was then used to probe a cDNA or genomic library which contains a proteinase-encoding DNA sequence in order to identify the sequence, which was then isolated and purified. The polynucleotide sequence obtained either by probing a library or an amplification method using such degenerate oligonucleotides was then itself used as a probe or primer in order to obtain full length cDNA or genomic sequences of the proteinase. These oligonucleotides are also useful as primers for directly cloning all or a portion of the gene or genes encoding homologous proteinases in a variety of plants by selectively amplifying the proteinase gene present in a genomic DNA or mRNA, or in a cDNA or genomic library by polynucleotide amplification methods known in the art (e.g., the polymerase chain reaction, or PCR). Cloning by PCR or screening libraries using such degenerate oligonucleotides is facilitated where oligonucleotides are synthesized which correspond to two or more peptide sequences are available, as is true in the present case. Once several proteinase genes are cloned and their nucleotide sequences compared, homologous regions are identified, and probes or primers are fashioned which contain these homologous regions. Such probes and primers are useful in directly cloning or screening libraries derived from a variety of plants in order to obtain other, related genes.

The polynucleotides will usually comprise sequences corresponding to at least about 12 to 15 nucleotides (or base pairs) , more usually at least about 21 nucleotides, and most preferably at least about 35 nucleotides, the length depending on the desired use. One or more introns may also be present.

Methods for constructing appropriate cDNA and genomic libraries, for screening libraries for sequences of interest, for preparing suitable probes, primers (e.g., PCR primers), polynucleotide purification, amplification and subcloning, host cell transformation and other techniques of recombinant DNA technology appropriate to the practice of the present invention are provided, inter alia, in Sambrook et al. (1989); Ausubel et al. (1987 and periodic updates); and PCR Protocols: A Guide to Methods and Applications. Innis et al., eds., Academic Press: San Diego (1990) . Reagents useful in applying such techniques, such as restriction enzymes, expression vectors, labels, etc. are known in the art and commercially available from such vendors as New England BioLabs, Boehringer Mannheim, Amersham, Promega Biotec, U. S. Biochemicals, New England Nuclear, and a number of other commercial sources.

A polynucleotide or fragment thereof is "substantially homologous" (or "substantially similar") to another polynucleotide if, when optimally aligned (with appropriate nucleotide insertions or deletions) with another polynucleotide, there is nucleotide sequence identity approximately 60% of the nucleotide bases, usually approximately 70%, more usually about 80%, preferably about 90%, and more preferably about 95 to 98% of the nucleotide bases. Alternatively, substantial homology (or similarity) exists when a polynucleotide or fragment thereof will hybridize to another under polynucleotide under selective hybridization conditions. Selectivity of hybridization exists under hybridization conditions which allow one to distinguish the target polynucleotide of interest from other polynucleotides. Typically, selective hybridization will occur when there is approximately 55% similarity over a stretch of about 14 nucleotides, preferably approximately 65%, more preferably approximately 75%, and most preferably approximately 90%. See Kanehisa (1984) Nucl. Acids Res.. 12.:203-213. The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of about 17 to 20 nucleotides, and preferably about 36 or more nucleotides.

The hybridization of polynucleotides is affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing polynucleotides, as will be readily appreciated by those skilled in the art. Stringent temperature conditions will generally include temperatures in excess of 30°C, typically in excess of 37°C, and preferably in excess of 45°C. Stringent salt conditions vr.ll ordinarily be less than 1 M, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter.

Wetmur and Davidson (1968) J. Mol. Biol.. 3_l:349-370.

An "isolated" or "substantially pure" polynucleotide is a polynucleotide which is substantially separated from other polynucleotide sequences which naturally accompany a native proteinase sequence. The term embraces a polynucleotide sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide of a fragment thereof. The anti-sense strand of such a polynucleotide is also said to encode the sequence.

A polynucleotide sequence is operably linked when it is placed into a functional relationship with another polynucleotide sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Generally, operably linked means that the sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, it is well known that certain genetic elements, such as enhancers, may be operably linked even at a distance, i.e., even if not contiguous.

The term "recombinant" polynucleotide refers to a polynucleotide which is made by the combination of two otherwise separated segments of sequence accomplished by the artificial manipulation of isolated segments of polynucleotides by genetic engineering techniques or by chemical synthesis. In so doing one may join together polynucleotide segments of desired functions to generate a desired combination of functions.

Polynucleotide probes include an isolated polynucleotide attached to a label or reporter molecule and may be used to identify and isolate other proteinase sequences. Probes comprising synthetic oligonucleotides or other polynucleotides may be derived from naturally occurring or recombinant single or double stranded nucleic acids or be chemically synthesized. Polynucleotide probes may be labelled by any of the methods known in the art, e.g., random hexamer labeling, nick translation, or the Klenow fill-in reaction.

Large amounts of the polynucleotides may be produced by replication in a suitable host cell. Natural or synthetic DNA fragments coding for a proteinase or a fragment thereof will be incorporated into recombinant polynucleotide constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the construct will be suitable for replication in a unicellular host, such as yeast or bacteria, but a multicellular eukaryotic host may also be appropriate, with or without integration within the genome of the host cells. Commonly used prokaryotic hosts include strains of Escherichia coli , although other prokaryotes, such as Bacillus subtilis or Pseudo.rno.nas may also be usec Mammalian or other eukaryotic host cells incluαe yeast, filamentous fungi, plant, insect, amphibian or avian species. Such factors as ease of manipulation, ability to appropriately glycosylate expressed proteins, degree and control of protein expression, ease of purification of expressed proteins away from cellular contaminants or other factors may determine the choice of the host cell.

The polynucleotides may also be produced by chemical synthesis, e.σ., by the phosphoramidite method described by Beaucage nd Carruthers (1981) Tetra.

Letts.. 22.:1859-1862 or the triester method according to Matteucci et al. (1981) J. Am. Chem. Soc.. 103:3185. and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained ^•?:rom the single stranded product of chemical synthesis Either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence. DNA constructs prepared for introduction into a prokaryotic or eukaryotic host will typically comprise a replication system recognized by the host, including the intended DNA fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment. Expression systems (expression vectors) may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Signal peptides may also be included where appropriate from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.

The selection of an appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al. (1989); Ausubel et al. (1987); and Metzger et al. (1988) Nature, 134:31-36. Many useful vectors for expression in bacteria, yeast, mammalian, insect, plant or other cells are well known in the art and may be obtained from such vendors as Stratagene, New England Biolabs, Promega Biotech, and others. In addition, the construct may be joined to an amplifiable gene (e.g., DHFR) so that multiple copies of the gene may be made. For appropriate enhancer and other expression control sequences see also Enhancers and Eukaryotic Gene Expression. Cold Spring Harbor Press, N.Y. (1983) . While such expression vectors may replicate autonomously, they may less preferably replicate by being inserted into the genome of the host cell.

Expression and cloning vectors will likely contain a selectable marker, that is, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector. Although such a marker gene may be carried on another polynucleotide sequence co-introduced into the host cell, it is most often contained on the cloning vector. Only those host cells into which the marker gene has been introduced will survive and/or grow under selective conditions. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxic substances, e.g. ampicillin, neomycin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media. The choice of the proper selectable marker will depend on the host cell; appropriate markers for different hosts are known in the art.

The vectors containing the polynucleotides of interest can be introduced (transformed, transfected) into the host cell by any of a number of appropriate means, including electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is an infectious agent, such as a retroviral genome) . The choice of such means will often depend on the host cell. Large quantities of the polynucleotides and polypeptides of the present invention may be prepared by transforming suitable prokaryotic or eukaryotic host cells with proteinase-encoding polynucleotides of the present invention in compatible vectors or other expression vehicles and culturing such transformed host cells under conditions suitable to attain expression of the proteinase-encoding gene. The proteinase may then be recovered from the host cell and purified.

In another embodiment, polyclonal and/or monoclonal antibodies capable of specifically binding to a proteinase or fragments thereof are provided. The term antibody is used to refer both to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities. Monoclonal or polyclonal antibodies specifically reacting with the proteinases may be made by methods known in the art. See, e.g.. Harlow and Lane (1988) Antibodies: A Laboratory Manual. CSH Laboratories; Goding (1986)

Monoclonal Antibodies: Principles and Practice. 2d ed, Academic Press, New York; and Ausubel et al. (1987) . Also, recombinant immunoglobulins may be produced by methods known in the art, including but not limited to the methods described in U.S. Patent No. 4,816,567. Monoclonal antibodies with affinities of 10⁸ M^"1, preferably 10⁹ to 10¹⁰ or more, are preferred.

Antibodies specific for proteinases may be useful, for example, as probes for screening cDNA expression libraries or for detecting the presence of a proteinase in a test sample. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or noncovalently, a substance which provides a detectable signal. Suitable labels include but are not limited to radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. United States Patents describing the use of such labels include but are not limited to Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

Antibodies specific for proteinases and capable of inhibiting proteinase activity may be useful in treating animals including man suffering from the effects of pollen proteinases. Such antibodies can be obtained by the methods described above and subsequently screening the pollen proteinase specific antibodies for their ability to inhibit proteinase activity.

In another embodiment, compositions and vaccine preparations comprising substantially purified proteinase(s) derived from a pollen(s) and a suitable carrier therefor are provided. Such vaccines are useful, for example, in immunizing an animal, including humans, against an allergic response to pollens. The vaccine preparations comprise an immunogenic amount of at least one proteinase or immunogenic fragments or subunits thereof. Such vaccines may comprise one or more proteinases, or a proteinase in combination with another protein or other immunogen. By "immunogenic amount" is meant an amount capable of eliciting the production of antibodies directed against the proteinase in an individual to which the vaccine has been administered.

Immunogenic carriers may be used to enhance the immunogenicity of the proteinases. Such carriers include, but are not limited to, proteins and polysaccharides, liposomes, and bacterial cells and membranes. Protein carriers may be joined to the proteinases to form fusion proteins by recombinant or synthetic means or by chemical coupling. Useful carriers and means of coupling such carriers to polypeptide antigens are known in the art.

The vaccines may be formulated by any of the means known in the art. Such vaccines are typically prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also, for example, be emulsified, or the protein encapsulated in liposomes. The active immunogenic ingredients are often mixed with excipients or carriers which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. The concentration of the immunogenic polypeptide in injectable formulations will usually be in the range of 0.2 to 5 mg/ml.

In addition, if desired, the vaccines may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include, but are not limited to: aluminum hydroxide; N- acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) ; N- acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP) ; N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2- (1' -2' -dipalmitoyl-sn-glycero- 3-hydroxyphosphoryloxy) -ethylamine (CGP 19835A, referred to as MTP-PE) ; and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against the immunogen resulting from administration of the immunogen in vaccines which are also comprised of the various adjuvants. Such additional formulations and modes of administration as are known in the art may also be used. The proteinases and fragments thereof may be formulated into vaccines as neutral or salt forms. Pharmaceutically acceptable salts include but are not limited to the acid addition salts (formed with free amino groups of the peptide) which are formed with inorganic acids, e.g., hydrochloric acid or phosphoric acids; and organic acids, e.g., acetic, oxalic, tartaric, or maleic acid. Salts formed with the free carboxyl groups may also be derived from inorganic bases, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases, e.g., isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, and procaine.

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity tn be administered, which is generally in the range of - out 100 to 1,000 μg of protein per dose, more generally in the range of about 5 to 500 μg of protein per dose, depends on the subject to be treated, the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of the active ingredient required to be administered may depend on the judgment of the physician and may be peculiar to each individual, but such a determination is within the skill of such a physician.

The vaccine may be given in a single dose or multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may include 1 to 10 or more separate doses, followed by other doses administered at subsequent time intervals as required to maintain and or reinforce the immune response, e.g., at l to 4 months for a second dose, and if needed, a subsequent dose(s) after several months. In another embodiment, a method of monitoring the exposure of an animal to a proteinase is provided. Such monitoring methods are useful, for example, in determining whether pollen spores from which the proteinases of the present invention are derived are responsible for an allergic condition, or for monitoring the progress of a therapy designed to lessen the symptoms of such an allergic condition.

In general, a biological sample obtained from the animal (e.g., blood, saliva, tissue) is incubated with a proteinase or portions thereof under conditions suitable for antibody-antigen interactions. The detection of the formation of such interactions is indicative of prior exposure of the animal and the subsequent development of an immune response to the proteinase. Examples of such tests include but are not limited to radioallergosorbent tests (RAST) and enzyme- linked immunosorbent assays (ELISA) .

Alternatively, the subject may be exposed to a proteinase and the subsequent reaction monitored. Such exposure may be cutaneously (e.g., by application to the skin via pricking or scratching), intracutaneously (e.g., via intracutaneous injection) or introduced in the form of an aerosol (generally an aqueous aerosol) into the nasal or bronchial passages (nasoprovocation or bronchoprovocation, respectively) , using methods well known in the art. Typical reactions, e.g., a wheal and erythema in skin testing, or asthma or rhinitis in naso- or bronchoprovocation, indicate an immunological response to the proteinase. See, e.g., Basic and Clinical Immunology. 6th ed., Stites et al., eds., (Appleton & Lange, 1987) , pp. 436-438, for a general description.

The proteinases may also be used in methods of identifying agents that modulate proteinase activity, whether by acting on the proteinase itself or preventing the interaction of a proteinase with a cell of an allergic individual. One such method comprises the steps of incubating a proteinase with a putative therapeutic agent; determining the activity of the proteinase incubated with the agent; and comparing the activity obtained with the activity of a control sample of proteinase that has not been incubated with the agent.

In another embodiment, immunotherapeutic methods in which the proteinase is repeatedly injected in increasing dosage over a prolonged period of time in order to reduce the symptoms of allergic rhinitis in patients with pollen allergies are provided. See, e.g., Stites et al. (1987), p. 442, for a general description. In another embodiment, methods of treating or ameliorating the affects of pollen proteinases on animals including humans are provided. Such methods include administering to the animal an effective amount of a physiologically acceptable pollen proteinase inhibitor. Known proteinase inhibitors are generally not physiologically acceptable, acceptable inhibitors will include agents that inhibit the pollen proteinases but do not affect, or affect only marginally, the activity of endogenous proteinases. Such inhibitors can be obtained from a variety of sources includir^, but not limited to, inhibitory antibodies and small mc ecules. The inhibitors can be administered by a variety of methods including, but not limited to, topically, via aerosol to the nasal passages or lungs, gastrointestinally, subdermally, intravenously and intraperitoneally. The inhibitors can be administered as needed, particularly when applied topically or via aerosol. These methods of administration are known in the art and will not be described in detail herein.

The foregoing discussion and following examples illustrate but do not limit the invention. Persons of ordinary skill in the art will appreciate that the invention can be implemented in other ways and is defined solely by the claims.

Exa ple 1 Purification Scheme for the Isolation of a Trypsin-Like

Proteinase From Mesquite

In order to determine whether proteinases are present in pollens, the following protein purification procedure was performed for the mesquite proteinase. Using the same purification technique, proteinases from this novel family have also been purified from ragweed, almond and typha.

50 grams of mesquite pollen were mixed with 200 ml of 0.02 M Bis-Tris buffer, pH 6.5, containing 5 mM CaCl₂- The mixture was stirred in the cold room (4°C) for 42-48 hr. The extract was then centrifuged for 30 min at 48,200 x g.

The supernatant was brought to 30% saturation with solid ammonium sulfate, allowed to sit for 10 min, and then centrifuged at 48,200 x g for 20 min. The supernatant was then brought to 60% saturation with solid ammonium sulfate, allowed to sit for 10 min, and centrifuged as above.

The precipitate was dissolved in 100 ml of 0.02 M Bis-Tris, pH 6.5, containing 5 mM CaCl₂, and dialyzed with two changes of the same buffer for four hours. The dialyzed protein solution was brought to pH 4.5 with 0.4 M sodium acetate buffer, pH 3.8, and centrifuged at 48,200 x g for 15 min. The supernatant was brought to pH 6.5 with 1.0 M Tris-HCl, pH 8.0 and dialyzed against 0.02 M Bis-Tris, pH 6.5, containing 5 mM CaCl^ for 24 hr with two changes of buffer.

The dialysate was put onto a Cibacron Blue Sepharose column (3.0 x 27.0 cm (190 ml volume)) equilibrated with 0.02 M Bis-Tris buffer, pH 6.5, containing 5 mM CaCl2 and the column was washed with the same buffer. Collection was started immediately because the proteinase elutes in the void volume. Assays for proteinase and amidase activity were performed, as described in Example 5, on individual fractions and the peak of activity retained. The active fractions were pool.d and applied to a DEAE-Sephacel column (3.0 x 18.0 cm (127 ml vol.)) and washed until the ^A28₀nm ^{was less} than 0.050. Elution was then performed with a linear gradient of from zero to 0.5 M NaCl in 0.02 M Bis-Tris, pH 6.5, 5 mM CaCl₂. Active fractions were collected, pooled, and solid NaCl added to make a final concentration of 2.0 M NaCl. This solution was then applied to a Phenyl- Sepharose column (2.0 x 17.2 cm, (54.0 ml column volume)), equilibrated with 0.02 M Bis-Tris, pH 6.5, 5 mM CaCl₂, 2.0 M NaCl. The column was washed with this buffer until the A₂aonm was less than 0.05. Elution of enzyme was then obtained with a linear gradient of from 2.0 M to 0 M NaCl, all in 0.02 M Bis-Tris, pH 6.5, 5 mM CaCl₂. Active fractions eluted from the phenyl

Sepharose column were pooled, concentrated on an Amicon filter to approximately 7.0 ml and applied to a Sephadex G-150 column (2.2 x 90.0 cm (342 ml column volume)), equilibrated with 0.02 M Bis-Tris, pH 6.5, 5 mM CaCl₂, 0.2 M NaCl. The protein was eluted with the same buffer. Active fractions were pooled, concentrated to 5-10 ml, dialyzed against 0.02 M Bis-Tris, pH 7.0, 5 mM CaCl₂ and applied to a Mono-Q-ion-exchange column in association with a Fast Protein Liquid Chromatography (FPLC) system. The column was developed with 0.1 M NaCl in starting buffer, followed by a linear gradient of from 0.1 M NaCl to 0.25 M NaCl in the same buffer. Enzyme was eluted between 0.15 M NaCl and 0.165 M NaCl. Example 2 Proteinase Molecular Weight

The proteinase purified as described in Example 1 has a varying apparent MW, depending on the mechanism used for its measurement.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the tricine-SDS-PAGE protocol; according to the method described by Schaggen and von Jagow (1987) Anal.

Biochem.. 166:368-379. SDS-PAGE gave a MW of between 85 kD and 95 kD.

The MW was also determined by gel filtration using the FPLC system. The MW appeared to be near 67 kD, since the peak of mesquite proteinase eluted in the same position as bovine serum albumin. Thus, the hydrodynamics of the protein are such as to give aberrant MW values, depending on the method used for this measurement.

Example 3 Protein Amino Acid Residue Sequences

The proteinases have a blocked amino terminus, since no amino acid residues could be detected after ten cycles using a protein sequencer. Protein sequencing was thus performed by first cleaving the proteinases into small fragments and sequencing the fragments.

For the tryptic digestion, 400 μg (4.4 nmol) of mesquite pollen proteinase was incubated with 6 M guanidine-HCL at 37°C for 30 minutes. Then 3.2 nmol of dithiothreitol (DTT) was added, flushed with nitrogen, and the mixture incubated for 2 hr followed by addition of 48 nmol of iodoacetamide and further incubation in the dark for 2 hr. The mixture was then dialyzed overnight against 2 1 of 50 mM ammonium bicarbonate pH 7.0. TPCK- treated bovine trypsin (at a ratio of 1:50, trypsin to proteinase) together with 5 mM CaCl was added to the dialyzed proteinase, and the mixture was incubated for 6 hr, then lyophilized. The lyophilized sample was brought up to 1.0 ml with HPLC-grade water and run on a Waters Associates HPLC using a Micro Pak SP column. The peptide peaks were collected and lyophilized for sequencing. For the cyanogen bromide (CNBr) digestion, 200 μg (2.2 nmol) mesquite pollen proteinase was incubated in 700 μl containing 300 μg of CNBr (1:100 molar ratio of number of methionine resides in proteinase to CNBr) and 70% formic acid at 20°C for 20 hrs. in the dark flushing with nitrogen. The sample was diluted ten- fold with HPLC-grade water and lyophilized. The lyophilized sample was solubilized in sample buffer, run on SDS-PAGE followed by electroelution from a PVDP membrane which was stained for protein and the bands cut out for sequencing. Sequencing was performed using the Applied

Biosystems, Inc., Model #4a77A or 470A on line with High Pressure Liquid Chromatography. The procedure was performed according to the manufacturer's instructions and was based entirely on the Edman degradation procedure.

The following amino acid residue sequences were obtained :

(1) TLAGKEYAQYXR (tryptic digest)

(2 ) KGPYXYXYYX (tryptic diges.) ( (33)) TLAGKEYAQYXR (tryptic digest)

(4 ) FNFYLDVSKPEEGLKVLTPXR (tryptic digest)

( 5 ) ENGLPNIIVYHLPPIGGPL (tryptic digest)

( 6 ) MEGIDTTVSHR (tryptic digest)

( 7 ) IKEDDTTAPLR (tryptic digest) (8) DEILRPDXAXLXRLGTEQX (cyanogen bromide digest)

(9) XXFYFYMXSYXP (V-8 proteinase digest) In all instances X represents an amino acid residue that was not identified.

Example 4 cDNA Encoding mesquite Pollen Proteinase

The cDNA encoding the mesquite pollen proteinase was obtained as described herein, subcloned and expressed in yeast. Unless otherwise mentioned, molecular biology techniques followed the methods described by Sambrook et al. Molecular Cloning 2ed. Cold Spring Harbor Laboratory Press (1989) . PolyA÷ RNA was isolated from fresh budding mesquite anthers harvested in Jerome, AZ and used to generate first strand cDNA for PCR analysis. Briefly, RNA was isolated from non-defatted Mesquite pollen according to the AGPC method described by Chomczynski and Sacchi (1987) Anal. Biochem. 162:156 and was subjected to oligo d(T) chromatography. 10 μg of the PolyA+ RNA generated was used to make a λZAPII (Stratagene, La Jolla, CA) cDNA library of 2 x 10⁶ complexity according to the method described by Zapf et al. (1990) J. Biol. Chem. 265:14892.

The cDNA library was used to generate a probe by amplifying a sequence with PCR using degenerate primers based on the known amino acid sequences of tryptic peptide sequences of mesquite pollen proteinase. The primers were made on ABI Model No. 394 DNA/RNA synthesizer by phosphoramidite chemistry in accordance with the manufacturer's instructions. The following nucleotide, MK-39-50, a 256-fold degenerate primer, corresponds to the coding strand. - 3.1 -

PheAsnPhe Tyr LeuAsn ValSer 5' AG^ATCTGAATTCTTTAATTT^C/_TTA^C/_TTTIGA /_TGGI^A/_T ^G/_CI

Lys ProGlu Glu Gly AA^A/_GCCIGA^A/_QGA^A/_GGG 3'

The following nucleotide, MK-42-44, a 32-fold degenerate primer corresponds to the non-coding strand.

TTAAGTCTAGA 5'

Both primers include inosines at wobble positions encoded by 4 nucleotides as well as EcoRl restriction endonuclease sites to facilitate cloning of PCR fragments.

Hot start PCR was employed to reduce non¬ specific annealing as follows. 100 pmol each primer plus dNTPs at a final concentration of 200 μM, 2.5 mM MgCl₂ and IX PCR Buffer were heated to 80°C with one Ampliwax bead. The reaction was cooled to 25°C and the remaining 5 μl first strand cDNA template, 2 units Taql Polymerase and water to 100 μl were added. The reaction was cycled 10 times at 94°C, 1 min then 32°, 10 min followed by 25 cycles at 94°C, 1 min; 55°C, 1 min; 72°C, 1 min with a 7 min 72°C final polish.

A PCR product of 300 bp was identified by southern blot analysis with the following probe, HG-3-23, a 12-fold degenerate non-coding strand oligomer based on a tryptic peptide

MetGlu Glylle Asp ThrThrVal 3' TACCT^C/_TCCITA^A/_G/_TCT^A/_GTGITGICA 5'

This 300 bp PCR fragment was cloned into the EcoRI site of pCR-script (Stratagene) using the EcoRI sites generated by the PCR primers, according to the manufacturer's instructions. This clone, designated A6, was identified by dot and southern blot with probe HG-3- 23. Sequence analysis showed this clone to contain the 3 regions used as primers and probe. The mesquite pollen library was screened with the 300 bp EcoRI fragment from clone A6 to identify a partial cDNA clone. A 2.1 Kb cDNA was identified. Figure 2 shows the restriction endonuclease map of this clone, designated MPP1-1 (1-1) . As MPP1-1 is not full length, the 300 bp Bglll/Hindlll 5' fragment was 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 restriction endonuclease map of MPP8-3. This clone appears to have been internally primed in an A rich region and, as shown in Figure 2, extends through the putative initiator methionine at the 5' end and overlaps MPPl-1 at a unique EcoRI site close to the 3' end.

The 5' Bglll/EcoRI fragment from MPP8-3 and the 3' EcoRl/Xhol fragment from MPP1-1 were ligated into BamHI/XhoI digested pBluescript (Stratagene) vector. This recloned fragment contains the full open reading frame of mesquite pollen proteinase plus 80 bp of 5' untranslated sequence; the Xhol site immediately follows the stop codon at the 3' end. This composite full length clone is designated MPP.

Both strands of the full length cDNA were sequenced by the Sanger dideoxy method after first subcloning into M13.

The following nucleic acid sequence was obtained. The amino acid residues encoded are shown directly beneath the sequence. 10 20 30 40 50 60 * * * * * * _{* *} * _* * * 'CGACTCTAGAGGATCTACΛGGAAAACATt-ΛGAGAAAGAGTAAAAATAAAGGAAAAGTGTA

70 80 90 100

_* * * * _* * * _* *

TTAAATAAAGAATAAAGAA ATG GTA CTT TCT TCA TCT CTG TCC TTC ATT Met Val Leu Ser Ser Ser Leu Ser Phe Ile> 10 120 130 140 150

* * * * * * * * *

TGC GCG GAC ATT CAC CGA TTT TTC ACC ATT CCG CTG ACT CTC ACT Cys Ala Asp lie His Arg Phe Phe Thr lie Pro Leu Thr Leu Thr>

160 170 180 190

* * * * * * * * * ^{GTG GTG} C"¹"^{1, τcc GCT GGG} CT CCT CCA CCT TTT CTT GCC TCT GCC Val Val Leu Ser Ala Gly Leu Pro Pro Pro Phe Leu Ala Ser Ala> 00 210 220 230 240

* * * * * * * * *

TCT CGG TTC TCT CAT CAA CAC CGC GTC GCT TCC AAG TCA GTT CGG Ser Arg Phe Ser His Gin His Arg Val Ala Ser Lys Ser Val Arg>

250 260 270 280

* * * * * * * * *

TCT TTG TCC TCG TCG GCG ATG GCT TTC TCC CAG TCT CAA TAT CCG Ser Leu Ser Ser Ser Ala Met Ala Phe Ser Gin Ser Gin Tyr Pro 90 300 310 320 330

* * * * * * * * *

CCT CCT CCG GTG GCT AAG AAA GTG GAA CAC CCA ATG GAG ATG TTC Pro Pro Pro Val Ala Lys Lys Val Glu His Pro Met Glu Met Phe> 340 350 360 370

* * * * * * * * *

GGT GAC GTG AGG ATC GAC AAC TAT TAC TGG CTT CGG GAC GAT TCT Gly Asp Val Arg lie Asp Asn Tyr Tyr Trp Leu Arg Asp Asp Ser> 80 390 400 410 420

* * * * * * * * *

CGC ACC AAT CCC GAT GTC CTC TCA TAC CTC CGT CAA GAA AAT GCA Arg Thr Asn Pro Asp Val Leu Ser Tyr Leu Arg Gin Glu Asn Ala>

430 440 450 460

* * * * * * * * *

TAC ACT GAC TCC ATT ATG AAA GGG ACC AAG GAA TTT GAA GAT AAG Tyr Thr Asp Ser lie Met Lys Gly Thr Lys Glu Phe Glu Asp Lys> 70 480 490 500 510

* * * * * * * * *

CTT TTT GCT GAG ATA AGA GGA AGG ATT AAG GAG GAT GAT ACC ACT Leu Phe Ala Glu lie Arg Gly Arg He Lys Glu Asp Asp Thr Thr>

520 530 540 550

* * * * * * * * *

GCA CCT TTA CGA AAG GGG CCT TAC TAT TAT TAT GAG AGA ACT CTG Ala Pro Leu Arg Lys Gly Pro Tyr Tyr Tyr Tyr Glu Arg Thr Leu> 560 570 580 590 600

* _* * _* * * * _{* *}

GCG GGG AAG GAG TAT GCT CAA TAT TGT CGG CGT CCA GTA CCT GAC Ala Gly Lys Glu Tyr Ala Gin Tyr Cys Arg Arg Pro Val Pro Asp>

610 620 630 640

* * * * * * * * * GAC AAG GCA ACA CCA TCT ATT TAT GAT ACT GTT CCG ACT GAA CCT Asp Lys Ala Thr Pro Ser He Tyr Asp Thr Val Pro Thr Glu Pro>

650 660 670 680 690

* _* * * * * * * *

GAT GCA CCT GAG GAG CAT GTT ATT TTG GAT GAG AAT ATC AAG GCT Asp Ala Pro Glu Glu His Val He Leu Asp Glu Asn He Lys Ala>

700 710 720 730

* * * * * _* * * *

CAA AAT CAT GAA TAC TAC AAT ATC GGT GCT TTT AAG GTT AGT CCA Gin Asn His Glu Tyr Tyr Asn He Gly Ala Phe Lys Val Ser Pro>

740 750 760 770 780

* * * * * * * * *

AAT AAT AAG TTA GTA GCA TAT GCA GAG GAC ACT AAA GGT GAT GAA Asn Asn Lys Leu Val Ala Tyr Ala Glu Asp Thr Lys Gly Asp Glu> 790 800 810 820

* * * * * * * * *

ATT TAT ACT ATT TAT GTC ATA GAT GCT GAA ACT CAA GAT CCT ATA He Tyr Thr He Tyr Val He Asp Ala Glu Thr Gin Asp Pro Ile>

830 840 850 860 870

* * * * * * * * *

GGA GAG CCT CTT CAT AAT GTA ACA TCA TAT ATT GAA TGG GCT GGC Gly Glu Pro Leu His Asn Val Thr Ser Tyr He Glu Trp Ala Gly>

880 890 900 910

* * * * * _* * _* *

GAT GAA GCT TTG GTT TAT ATC ACA ATG GAT GAG ATT CTC AGG CCT Asp Glu Ala Leu Val Tyr He Thr Met Asp Glu He Leu Arg Pro>

920 930 940 950 960

* * * * * * * * *

GAT AAG GCA TGG TTG CAC AGG TTG GGA ACA GAA CAG TCA AAG GAT Asp Lys Ala Trp Leu His Arg Leu Gly Thr Glu Gin Ser Lys Asp>

970 980 990 1000

* * * _* * _* * _* *

ATA TGT CTT TAT GTG GAA AAG GAT GAT AAA TTT TCT TTG GAT CTA He Cys Leu Tyr Val Glu Lys Asp Asp Lys Phe Ser Leu Asp Leu>

1010 1020 1030 1040 1050

* * * * * * * * * CAA GCT TCT GAG AGC AAG AAA TAT TTG TTT GTA GCA TCA GAA AGT Gin Ala Ser Glu Ser Lys Lys Tyr Leu Phe Val Ala Ser Glu Ser>

1060 1070 1080 1090

* * * * * * * * *

AAA AAT ACA AGG TTT AAT TTT TAT CTT GAT GTT TCC AAA CCT GAA Lys Asn Thr Arg Phe Asn Phe Tyr Leu Asp Val Ser Lys Pro Glu> 1100 1110 1120 1130 1140

* * * * * * * _* *

GAG G-5A CTT AAA GTT TTG ACA CCA CGC ATG GAG GGT ATT GAT ACA Glu Gly Leu Lys Val Leu Thr Pro Arg Met Glu Gly He Asp Thr>

1150 1160 1170 1180

* * * * * * * * * ACT GTT AGC CAT CGA GGA AAT CAT TTT TTC ATT AAA AGG AGG AGT Thr Val Ser His Arg Gly Asn His Phe Phe He Lys Arg Arg Ser>

1190 1200 1210 1220 1230

* * * * * * * * *

GAT GAG TTT TTT AAT TCA GAA GTA GTA GCT TGC CCG GTT GAT AAT Asp Glu Phe Phe Asn Ser Glu Val Val Ala Cys Pro Val Asp Asn>

1240 1250 1260 1270

* * * * * * * * *

ACC TCC TCT ACT ACA GTT CTT CTT CCC CAC AGG GAA AGT GTT AAA

Thr Ser Ser Thr Thr Val Leu Leu Pro His Arg Glu Ser Val Lys>

1280 1290 1300 1310 1320

ATT CAG GAG ATT CAG CTT TTT ATT GAT CAC CTT GTT GCA TAT GAG He Gin Glu He Gin Leu Phe He Asp His Leu Val Ala Tyr Glu>

1330 1340 1350 1360

* * * * * * * * *

AGA GAA AAT GGT CTA CCA AAT ATA ATA GTT TAT CAC CTT CCT CCC Arg Glu Asn Gly Leu Pro Asn He He Val Tyr His Leu Pro Pro>

1370 1380 1390 1400 1410

* * * * * * * * *

ATT GGA GAA CCA CTA AGG AGC CTT GGA GAT GGT CAT GCT GTT AAT ^{Ile G1}y ^{Glu Pr}° ^{Leu Ar}9 ^{Ser Leu G1}y As ^G1y ^H*-*^s A**-³* ^Val Asn>

1420 1430 1440 1450

* * * * * * * * *

TTT GCT GAT CCA GTA TAT TCA GTG GAA TCT TCG GAG TCA GAA TTT Phe Ala Asp Pro Val Tyr Ser Val Glu Ser Ser Glu Ser Glu Phe>

1460 1470 1480 1490 1500

* * * * * * * * * TCC TCA AAT ATT TTG CGG TTT TCA TAC AGT TCC TTG AAG ACT CCT Ser Ser Asn He Leu Arg Phe Ser Tyr Ser Ser Leu Lys Thr Pro>

1510 1520 1530 1540

* * * * * * * * *

TCC TCT GTA TAT GAT TAT GAT ATG AAT TCA AGC ATT TCT GCT TTG Ser Ser Val Tyr Asp Tyr Asp Met Asn Ser Ser He Ser Ala Leu>

1550 1560 1570 1580 1590

* * * * * * * * *

AAG AAG ATT GAC TCA GTA TTG GGT GGT TTT GAT GCG GCA CAA CAT Lys Lys He Asp Ser Val Leu Gly Gly Phe Asp Ala Ala Gin His>

1600 1610 1620 1630

* * * * * _* «• * *

GTT ACT GAT AGG CTG TGG GCA CCT GGT T. 3AT GGA ACT TTG ATT Val Thr Asp Arg Leu Trp Ala Pro Gly Le... Asp Gly Thr Leu He> 1640 1650 1660 1670 1680

* * * * _* * * * *

CCC ATT TCA ATT GTC TAC CGG AAG GAC CTT GTT AAA CTT GAT GGA

Pro He Ser He Val Tyr Arg Lys Asp Leu Val Lys Leu Asp Gly>

1690 1700 1710 1720

* * _* * * * * * * TCT GAT CCT TTA CTA CTT TAT GGC TAT GGG TCT TAT GAG ATT TCC

Ser Asp Pro Leu Leu Leu Tyr Gly Tyr Gly Ser Tyr Glu He Cys>

1730 1740 1750 1760 1770

* * * * * * * _* *

ATA GAT CCC AGT TTC AAG TCA TCA AGG CTG TCA TTG TTA GAT CGA

He Asp Pro Ser Phe Lys Ser Ser Arg Leu Ser Leu Leu Asp Arg>

1780 1790 1800 1810

* * * * * * * * *

GGT TTT ATA TTT GCA ATT GCT CAT ATT CGC GGA GGT GGT GAA ATG Gly Phe He Phe Ala He Ala His He Arg Gly Gly Gly Glu Met>

1820 1830 1840 1850 1860

* * * * * * _* * *

GGA AGG CAG TGG TAT GAG AAT GGG AAG TTC TTG AAA AAA AAG AAC Gly Arg Gin Trp Tyr Glu Asn Gly Lys Phe Leu Lys Lys Lys Asn> 1870 1880 1890 1900

* * * * * * * * *

ACT TTT ACA GAT TTT ATT GCT TGT GCT GAA TAT TTG ATT GAT CAA Thr Phe Thr Asp Phe He Ala Cys Ala Glu Tyr Leu He Asp Gln>

1910 1920 1930 1940 1950

* * * * * * * * *

AAA TAT TCT TCA AAG GAA AGA CTG TGC ATC AAT GGT CGA AGT GCA Lys Tyr Ser Ser Lys Glu Arg Leu Cys He Asn Gly Arg Ser Ala>

1960 1970 1980 1990

* * * * * * * * *

GGG GGT TTA CTA ATT GGT GCA GTC CTT AAT ATG AGG CCT GAC TTG Gly Gly Leu Leu He Gly Ala Val Leu Asn Met Arg Pro Asp Leu>

2000 2010 2020 2030 2040

* * * * * * * * *

TTC AAG GCT GCT GTT GCG GGT GTA CCT TTT GTG GAT GTT GTG ACA Phe Lys Ala Ala Val Ala Gly Val Pro Phe Val Asp Val Val Thr>

2050 2060 2070 2080

* * * * _* * * * *

ACC ATG CTT GAT CCA TCT ATA CCT CTG ACA ACT TCA GAA TGG GAG Thr Met Leu Asp Pro Ser He Pro Leu Thr Thr Ser Glu Trp Glu>

2090 2100 2110 2120 2130

* * * * * * * * * GAA TGG GGA GAT CCT AGG AAG GAG GAG TTC TAC TTC TAC ATG AAG Glu Trp Gly Asp Pro Arg Lys Glu Glu Phe Tyr Phe Tyr Met Lys>

2140 2150 2160 2170

* * * * * * * * *

TCA TAT TCT CCG GTT GAT AAT GTG AAG GCA CAA GAT TAT CCT CAC Ser Tyr Ser Pro Val Asp Asn Val Lys Ala Gin Asp Tyr Pro His> 2180 2190 2200 2210 2220

* * * _* * * * * *

ATT CTT GTT ACT GCT GGC TTA AAT GAT CCA CGT GTT CTG TAT TCT lie Leu Val Thr Ala Gly Leu Asn Asp Pro Arg Val Leu Tyr Ser>

2230 2240 2250 2260

* * * _{* *} * * _{* *} GAA CCT GCT AAG TTT GTG GCA AAA CTG AGG GAT ATG AAG ACC GAT Glu Pro Ala Lys Phe Val Ala Lys Leu Arg Asp Met Lys Thr Asp>

2270 2280 2290 2300 2310

* * * * * * * * *

GAT AAC ATT CTA CTG TTT AAA TGC GAG CTT GGT GCA GGG CAT TTT Asp Asn lie Leu Leu Phe Lys Cys Glu Leu Gly Ala Gly His Phe>

2320 2330 2340 2350

* * * * * * * * *

TCA AAG TCT GGG AGG TTC GAG AAG CTC CAA GAA GAT GCC TTC ACC Ser Lys Ser Gly Arg Phe Glu Lys Leu Gin Glu Asp Ala Phe Thr>

2360 2370 2380 2390 2400

* * * * * * * * * *

TAT GTC TTT ATT TTG AAG GCC CTG AAT ATG ATT AGT TAG CTCGAGAC Tyr Val Phe lie Leu Lys Ala Leu Asn Met lie Ser ***>

2410 2420 2430 2440 2450 2460

* * * * * * _* * _* * * *

CCGTGGGCAACTCAACTCGAGTCTGGAGGTTC-A^^

2470 2480 2490 2500 2510 2520

* * * * * * * * * * * *

CΆC_ATGGATCGTTTATTCΆCCATGGGTTAATCTAT 2530 2540 2550 2560 2570 2580

* * * * * * * * _* * * *

TAGTTAACITTGTGATTCGTCTC-ATAATC-AGT^

2590 2600 2610 2620 2630 2640

* * * * * * * * * * * *

TGCCGGAC.AGCCAAGAGAGAACATTATGATTGTAACC-AAAAAA

2650 2660 2670 2680 2690 2700

_* * * * * * * * * * * *

ATAATAAAATTTATTGC-AAGCTO-AAAAAAAAAAAAAAA^

2710

*

GAGCTC 3'

As shown in Figure 2, the MPP cDNA encodes one open reading frame of 772 amino acid residues. The predicted molecular weight of the full length protein is 88 kD with a predicted pi of 5.3. There are 3 potential N-linked glycosylation sites, a potential signal sequence of 29 amino acid residues at the amino terminus and a 200 amino acid residue stretch at the carboxy terminus with 63% identity to E. coli Protease II and decreased homology to the prolyl endopeptidase family of serine proteases as well as the same catalytic triad amino acid residue order of Ser, Asp, His. This particular arrangement of amino acid residues places this enzyme in a separate class of enzymes from the subtilisin and chymotrypsin families.

In order to express the protein encoded by the recombinant gene, the full length MPP sequence was cloned into the yeast expression vector pYT in a two-step process as follows. pYT is a 15.2 Kb vector containing the yeast 2 μm sequence for autonomous replication, URA3 and LEU2d genes for selection in yeast, and the pBR322 sequences containing both the E. coli origin of replication and the ampicillin resistance gene. pYT also contains the a-factor terminator required for translation termination downstream from BamHI and Sail unique cloning sites. Due to the lack of unique cloning sites in such a large vector, an initial subcloning was required for in- frame insertion of a promoter and/or other signal sequences with the gene of interest. For MPP expression, the glucose regulatable alcohol dehydrogenase II (ADH2) gene (Shuster (1989) Yeast Genetic Engineering. Butterworths, Massachusetts, p.83-107) was used. The ADH2 promoter was cloned by PCR and ligated into pBluescript Notl/Xhol restriction endonuclease sites. Also included in the PCR fragment is the cloning site Xbal at the 3' end of the promoter, for in-frame insertion of the gene to be expressed. The entire cassette can be excised at the BamHI site 5' of the ADH2 promoter and the Xhol site at the 3' end of MPP and cloned into pYT at the BamHI/Sall sites to obtain plasmid MPPpYT. The pBluescript construct is shown in Figure 3, and the PYT construct is shown in Figure 4. The MPPpYT plasmid was used to transform the yeast strain designated LXR1 ( [cir°] , MATa,leu2,trpl, ura3-52, prbl-1122,pep4-3, prcl-407) , an endogenous plasmid cured strain of BJ2168 (on deposit at the Berkeley Type Culture Collection) by the spheroplast method using uracil selection as described by Barr et al. (1987) Biotech. 5:486. Transformants were maintained o leucine minus plates. Transformants were grown in YEPD media for 48-72 hours at which point the glucose is depleted and the ADH2 promoter is derepressed to express the recombinant MPP.

Example 5 Proteinase Inhibitor Profile

In order to characterize the proteinases, the inhibitor profile of the protein purified as described in Example 1 was determined as follows.

The inhibition of trypsin-like activity, a control was employed, consisting of an incubation mixture of pollen extract, buffer (0.1 M sodium phosphate, pH 7.4, 0.15 M NaCl), and Bz-L-Arg-pNA (substrate) in a total volume of 1.0 ml. The mixture was incubated for 10 min at room temperature. 50 μl glacial acetic acid was added to stop the reaction, after which readings were made a.~ 405 nm to determine the appearance of yellow pigmentation indicating proteolytic cleavage. Release of P-nitroaniline can be quantified to indicate the extent of hydrolysis. To test the inhibition of proteinase by each inhibitor sample, various amounts of each inhibitor were added to the basic incubation mixture, minus Bz-L-Arg- pNA, preincubated for 5-10 min, substrate was then added and the mixture treated as above. Inhibition of the proteinase for each sample was measured as the difference between values for the control and the value for each sample containing inhibitor.

All of the inhibitors were purchased from Sigma Chemical Co., with the exception of EDTA (Baker); leupeptin (Calbiochem) ; aprotonin (Bayer) ; and ovomucoid (Worthington) . Tomato and potato inhibitors were gifts of Dr. C. Ryan. The concentrations of the inhibitors used were 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%; rhuslancia/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 mg/ml) ; SBTI (0.5 mg/ml) ; BTI (0.5 mg/ml); and 2-1-PI (1.2 nmol). In order to measure the inhibition of caseinolytic activity, the assay consisted of incubation mixtures of 0.4 ml of pollen extract, 0.1 ml of buffer (0.1 M sodium phosphate, pH 7.4, 0.15 M NaCl) and 0.25 ml 3% azocasein. The mixture was incubated for either 24 or 48 hrs at 37°C, at which time 0.75 ml 10% trichloroacetic acid (TCA) was added. Inhibitor (5 mM, 0.4 ml) was added to the basic incubation mixture for 5 to 10 min prior to addition of the azocasein solution. Controls of azocasein alone and extracts alone were also performed. When proteolytic cleavage occurs, indicating enzyme activity, soluble yellow color remains in the supernatant and casein and uncleaved azocasein remain in the precipitates.

The data obtained are presented in Tables l and 2. Table 1 depicts the inhibition spectra of trypsin- like activity in selected pollens and Table 2 depicts the inhibition spectra of caseinolytic activity in pollen. Table 1

Inhibitor Percent of Control

Mesqu: Lte Rhuslancia Ragweed

PMSF 110 80 83

EDTA 115 88 83

Cysteine 113 111 98 Benzamidine 33 21 32

Antipain 28 29 51

Leupeptin 34 21 38

TLCK 12 4 24

TPCK 99

E-64 88 78 78 p-aminobenzamidine 103 105 104

Aprotonin 104 99 109 Ovomucoid 114 104 113

SBTI 119 117 109

LBTI 113 121 102 o.-1-PI 94 90 97

Table 2

Inhibitor Percent of Control Mesquite Rhuslancia Ragweed

PMSF 57 75 24 EDTA 83 76 69 Cysteine 127 76 74

The proteinases are not affected by any of the common proteinase inhibitors. These include α-l-PI, soybean trypsin inhibitor, lima bean trypsin inhibitor, tomato trypsin inhibitors I, II and PCI, potato trypsin inhibitor PCT1 and tobacco inhibitor Til, tosyl-L- phenylalanine chloromethyl ketone (TPCK) , E-64 (trans- EpoxySuccinyl-leucylamido- (4-guanido) butaine) PMSF, ethylenediaminetetraacetic acid EDTA, benzamidine, p- aminobenzamidine and the Kunitz basic pancreatic trypsin inhibitor. The tomato, potato and tobacco inhibitors were tested later on the purified proteinases and found to be inactive.

Inhibition was found with PMSF, DFP, benzamidine, antipain, leupeptin, and TLCK. Hence, the proteinase is unlikely to be a member of either the metalloproteinase or cysteine proteinase class of enzymes. This indicates that the enzyme is a member of the serine class of proteinases.

Example 6

Proteinase Activity Assays of a Variety of Crude Pollen Extracts

In order to determine whether other pollens contain proteinases within the family described herein, crude purifications were carried out on the pollens listed in Table 3, and their activities were assayed. In Table 3 the plants labeled with the asterisks were found to be weakly or non-allergenic.

Table 3

POLLEN SAMPLES TESTED FOR PROTEOLYTIC ACTIVITY

Almond Jojoba

Amarantha* Mesquite Baccharia Mistletoe*

Corn Rhuslancia

Cottonwood Pine

Creosote Ragweed

Dandelion* Sahauro*

Desert Broom Sunflower

Ephedra Texas Ranger Fall Composition Typha

Fan Palm Wolfberry Japanese cedar* The crude preparations were made as follows: 1.0 g pollen were mixed with 12.0 ml 0.1 M Tris-HCl, pH 8.0; and 0.15 M NaCl, in the presence of 3.0 g 0.5 mm glass beads. The sample was vortexed for 10 min, followed by centrifugation for 20 min at 48,000 x g.

The crude fractions were then tested for activity in the following systems: azocasein degradation, hide powder, elastin esterase, cathepsin G esterase and trypsin-like activity. The azocasein degradation assay was performed as described in Example 5 to measure the inhibition of caseinolytic activity described. The results obtained are presented in Table 4. In Table 4 the plants labeled with the asterisks are weakly or non-allergenic.

Table 4 AZOCASEIN DEGRADING ACTIVITY IN POLLEN

Most Active Least Active

1. Ragweed 1. Dandelion*

2. Wolfberry 2. Texas Ranger

3. Sunflower 3. Fall Composite

4. Jojoba 4. Typha

5. Almond 5. Creosote

6. Mesquite 6. Mistletoe* & Sahauro*

The hide powder assay was performed as follows: 10 mg of blue hide powder was mixed with 0.8 ml buffer (1.0 M Tris-HCl, pH 8.0, 0.15 M NaCl, 1% Brij) and 0.4 ml of pollen extract. Controls of hide powder or pollen extracts alone were also used. Incubation was for 2.5, 5.0, and 24 hrs at 37°C with the sample being continuously shaken. At the end of a given time period the material was allowed to settle and the color development in the supernatant was read at 595 nm. The results obtained are presented in Table 5. In Table 5 the plants marked with the asterisks are weakly or non- allergenic. The blue hide powder is insoluble but, if the enzyme has activity, there will be proteolytic release of peptides bound to the dye, i.e., soluble peptide fragments containing the dye will be quantita¬ tively measurable as an indication of the effectiveness of the proteinase being tested.

Table 5

HIDE POWDER DEGRADING ACTIVITY IN POLLEN

Most Active Least Active

1. Texas Ranger 1. Sahauro*

2. Mesquite 2. Mistletoe*

3. Ragweed 3. Amarantha*

4. Sunflower 4. Creosote

5. Typha 5. Fan Palm

6. Cottonwood 6. Dandelion*

The elastin esterase assay was performed as follows: Zero to 0.5 ml of pollen extract or purified enzyme was incubated in 0.1 M sodium phosphate buffer, pH 7.4, 0.15 M NaCl and 0.04 ml of Suc-Ala-Ala-Ala-pNA (5 mM) (Sigma) for 20 min. The absorbance at 405 nm was then read as a measure of substrate digestion as described above. The results obtained are presented in Table 6. In Table 6 the plants marked with the asterisks are weakly or non-allergenic.

Table 6

ELASTIN ESTERASE ACTIVITY IN POLLEN

Most Active Least Active

1. Mistletoe* 1. Mesquite

2. Typha 2. Almond

3. Sahauro* 3. Ragweed . Sunflower 4. Pine

5. Cottonwood 5. Dandelion*

6. Amarantha* 6. Wolfberry The cathepsin G esterase activity assay was performed as follows: Zero to 0.5 ml of pollen extract or purified enzyme was incubated in 0.1 M sodium phosphate buffer, pH 7.4, 0.15 M NaCl and 0.01 ml of Suc- Ala-Ala-Pro-Phe-pNA (50 mM) for 10 min. The absorbance at 405 nm was then read as a measure of substrate digestion as described above. The results obtained are presented in Table 7. In Table 7, the plants marked with the asterisks are weakly or non-allergenic

Table 7 CATHEPSIN G ESTERASE ACTIVITY IN POLLEN

Most Active Least Active

1. Cottonwood 1. Amarantha*

2. Wolfberry 2. Mistletoe

3. Typha 3. Dandelion*

4. Sunflower 4. Sahauro*

5. Almond 5. Rhuslancia

6. Ephedra 6. Fan Palm

The trypsin-like and plasma kallikrein activity assay was performed as follows: Zero to 0.5 ml of pollen extract or purified enzyme was incubated in 0.1 M sodium phosphate buffer, pH 7.4, 0.15 M NaCl and 40 μl of Pro- Phe-Arg-pNA (5 mM) (Sigma) for 10 min. The absorbance at 405 nm was then read as a measure of substrate digestion. The results obtained are presented in Table 8. In Table 8 the plants marked with the asterisks are weakly or non- allergenic

Table 8

TRYPSIN-LIKE ACTIVITIES IN POLLEN

Most Active Least Active

1. Mesquite 1. Sahauro*

2. Rhuslancia 2. Pine

3. Almond 3. Cottonwood

4. Typha 4. Fall Composite

5. Sunflower 5. Amarantha*

6. Ragweed 6. Dandelion*

Example 7

Activity Assays of Purified Proteins

The mesquite pollen proteinase purified from pollen was tested to determine its amino acid residue specificity in order to more fully characterize its activity. The protocol to measure the amino acid residue specificity used peptides of known sequence and was as follows. 1.0 ml of 25 mM ammonium bicarbonate buffer, pH 7.8; 5 mM calcium chloride; and 0.125% sodium azide; was added to 250 μg of either insulin; mellitin; or dynorphin fragment 1-13 and mixed well. 80 μl was removed at time zero while 80 μl was also incubated overnight at 37°C. Then, 14 μl of purified mesquite proteinase (10.08 μg) (1:25 molar ration of proteinase to peptide substrate) purified as described in Example 1, was added, mixed well, and incubated at 37°C. At each time point (zero, 0.25, 1.0, 5.0 and 24 hr) a 166 μl sample was removed, frozen quickly in dry ice and stored frozen until analyzed. Samples were thawed, peptide fragments separated by HPLC, and peptide peaks collected and analyzed for amino acid composition.

The protocol for activation of prothrombin was as follows: 0.74, 1.48 or 2.22 μg of mesquite proteinase purified as described in Example 1 was incubated with 10 or 20 μg of prothrombin (Calbiochem) in 0.1 M Tris-Hcl, pH 8.0; 0.15 M NaCl; and 5 mM calcium chloride in a final volume cf 0.2 ml for up to 90 min. Then, 0.8 ml of the same buffer and H-D-Phe-Plp-Arg-pNA (Kabi Pharmaceuticals) , a substrate for thrombin, were added. After 10 min incubation, the reaction was stopped with the addition of acetic acid and the absorbance measured at 405 nm. Since the mesquite proteinase also cleaves this substrate, although much more slowly, controls without prothrombin and also controls without proteinase were run and the rates of hydrolysis subtracted from that found for the mixture of proteinase and prothrombin.

Active proteinase fractions were found to hydrolyze both synthetic substrates and proteins specifically after arginine residues and, to a much lesser extent, lysine residues. Proteins tested, including mellitin and the oxidized insulin B chain, were not hydrolyzed by the enzyme. Nor was there any activation of trypsinogen which requires cleavage at a Lys-lie bond. However, the small peptide fragment of dynorphin (1-13) was cleaved between an Arg-Arg residue and an Arg-lie residue. Furthermore, there was activation of prothrombin which requires cleavage between an Arg-lie peptide bond, as well as prekallikrein (PK) conversion which also requires cleavage at the same type of peptide bond. A number of synthetic substrates:

D-pro-phe-arg-p-nitroanilide (Sigma) ; and Bz-L-Arg-pNA, (Kabi Pharmaceuticals) were readily hydrolyzed by the enzyme as well.

Example 8

Pollen Proteinase Activation of Prekallikrein

In order to determine if the proteinases exert a physiological effect on the human immune system, the crude extracts were tested to determine their ability to activate PK. PK is activated to kallikrein which in turn activates kininogen to bradykinin, a vasodilator. Bradykinin is responsible for some allergic reaction symptoms. As shown in Table 9, the proteinases activate PK in Hageman factor-deficient plasma. Thus, the enzyme may be indirectly responsible for the generation of the vasoagent bradykinin.

The assay was performed by adding 60 μl of a 1:25 dilution of the crude pollen extracts prepared as described in Example 5 to 60 μl of PK (a gift from Dr. Bruce Zoran, Scripps Research Institute, LaJolla, California) incubated for 30 min at 37°C in a buffer containing 0.1 M NaP0₄ buffer, pH 7.4; 0.15 M NaCl. The samples were then split into two equal aliquots and one half was treated with 0.3 μg SBTI (Sigma) and the other half with buffer.

Each sample was then assayed for kallikrein activity using D-Pro-Phe-Arg-pNA (S-2302, Kabi Pharmaceuticals) . The results obtained are presented in Table 9.

Table 9

PK ACTIVATION BY POLLEN EXTRACTS

Pollen Relative Kallikrein Activity

Mesquite + PK + SBTI 1.0 Mesquite + PK 1.9

Rhuslancia + PK + SBTI 1.0

Rhuslancia + PK 2.1

Ragweed + PK + SBTI 1.0

Ragweed + PK 1.5 Pine + PK + SBTI 1.0

Pine + PK 1.3

Purified mesquite proteinase +

PK + SBTI 1.0

Purified mesquite proteinase +

PK 1.6

Example 9 Activation of Prothrombin by Mesquite Pollen Proteinase

Mesquite proteinase, purified as described in Example 1, was tested for its ability to activate prothrombin. The activity assay was performed as described in Example 8. The data are presented in Table 10 where the proteinase was purified and the concentration was measured as described in Example 1.

Table 10 Prothrombin Activation by Purified Mesquite Proteinase Proteinase Concentration Thro bin Activity Released 0.74 17

1.48 30

2.22 62 The data presented in Table 10 show that prothrombin is converted to thrombin by the mesquite proteinase. Hence, activation of the coagulation system by this enzyme could occur in vivo. Experiments are now in progress to determine whether the enzyme(s) can directly release bradykinin from kininogen in plasma, or whether the indirect mechanism is the only pathway.

Example 10

Protocol for Testing Vascular Permeability Enhancement Reactions in Guinea Pigs

In order to determine whether the proteinases exert a physiological effect, their ability to increase vascular permeability was determined. Guinea pigs were anaesthetized using 80 mg/kg of ketamine, after which they were injected with Evans Blue Dye (30 mg/kg) in a metatarsal vein. Proteinase solutions were first subject to a 30 min incubation with human plasma. The preincubated proteinase solutions were then injected intradermally into the shaved back of the guinea pig. After 15 min the guinea pigs were euthanized by exsanguination using a carotid artery cut-down procedure while still under the Metofane (methoxyflurane) anaesthesia. The back skin was peeled and the injected sites were obtained using a 15 mm punch biopsy instrument. The dye from the skin was extracted using formamide and measured spectrophotometrically at 620 nm. The results obtained are presented in Table 11. Table 11 Induction of Vascular Permeability bv Mesquite Proteinase

Enzvme Concentration (M) Dye Released (μg)

0.8 x 10^"6 5.68 2.5 x 10^"7 0.0

0.8 x 10^"7 0.0

A time course study showed 30 min to be the peak of production for vascular permeability enhancing factors. Direct injection of the proteinase into the guinea pig skin yielded very little/no vascular permeability reaction. As a comparison, 100 ng of bradykinin caused the release of 46 μg of dye.

Example 11 Analysis of Recombinant Mesquite Proteinase Produced in Yeast

1. Activity Assays

Yeast control and MPP expressing cultures (rMPP) were centrifuged and the cell pellets lysed in TE + 0.1% Triton X-100 by vortexing with 0.5 mM glass beads. The clarified lysates were assayed for proteolytic activity against 5 mM pyro-GluGlyArgpNA (pNA) in 5 mM K₂HP0₄, 15 mM NaCl, pH 8 according to the method described in Example 6. Samples were measured in a spectrophotometer at 405 nm in a kinetic assay to measure the rate of pNA conversion or simply incubated at 37° and a single reading taken. The rMPP cultures grown at various pH levels showed 5-8.5 times more activity than the yeast control. The results obtained are shown in Figure 5.

2. Inhibitor Binding. The lysates were assayed for their ability to irreversibly bind a chloromethyl ketone inhibitor. The inhibitor TyrAlaLysArgC-K was labeled with ^{1 5}ι and added to 4-10 μg lysates from both MPPpYT transformed and untransformed LXR1 yeast obtained as described above. A 5 μg crude mesquite pollen extract obtained as described in Example 1 was also labeled with the inhibitor as a control. The proteins were separated by 15% SDS-PAGE, coomassie stained and the dried gel was exposed to X-ray film. The results obtained indicate that the mesquite pollen crude extract contains a proteinase of the same molecular weight as rMPP. Both the mesquite pollen extract and the extract from the yeast transformed with MPPpYT, but not that of the untransformed yeast, contain a protein of the same apparent molecular weight which bind the inhibitor.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention, which is delineated by the appended claims.

Claims

Claims

1. A composition comprising a substantially pure proteinase having a molecular weight of about 85-95 kD as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and about 67 kD as determined by Fast Protein Liquid Chromatography; resistance to inhibition by o.-2-macroglobulin, α-1-proteinase inhibitor, trypsin inhibitors; and sensitivity to inhibition by phenyl methane sulfonyl fluoride, difluorophenol, benzamidine, antipain, leupeptin and tosyl-L-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, Japanese cedar, almond, typha or pine.

4. The proteinase according to claim 1 wherein the proteinase is a serine proteinase .

5. The proteinase according to claim l having substrate specificity for Arg-Arg and Arg- lie .

6. The proteinase according to claim 1 having the amino acid residue sequence :

Met Val Leu Ser Ser Ser Leu Ser Phe lie Cys Ala Asp lie His Arg Phe Phe Thr lie Pro Leu Thr Leu Thr

Val Val Leu Ser Ala Gly Leu Pro Pro Pro Phe Leu Ala Ser Ala

Ser Arg Phe Ser His Gin His Arg Val Ala Ser Lys Ser Val Arg

Ser Leu Ser Ser Ser Ala Met Ala Phe Ser Gin Ser Gin Tyr Pro

Pro Pro Pro Val Ala Lys Lys Val Glu His Pro Met Glu Met Phe Gly Asp Val Arg lie Asp Asn Tyr Tyr Trp Leu Arg Asp Asp Ser Arg Thr Asn Pro Asp Val Leu Ser Tyr Leu Arg Gin Glu Asn Ala Tyr Thr Asp Ser lie Met Lys Gly Thr Lys Glu Phe Glu Asp Lys Leu Phe Ala Glu lie Arg Gly Arg lie Lys Glu Asp Asp Thr Thr Ala Pro Leu Arg Lys Gly Pro Tyr Tyr Tyr Tyr Glu Arg Thr Leu Ala Gly Lys Glu Tyr Ala Gin Tyr Cys Arg Arg Pro Val Pro Asp Asp Lys Ala Thr Pro Ser lie Tyr Asp Thr Val Pro Thr Glu Pro Asp Ala Pro Glu Glu His Val He Leu Asp Glu Asn He Lys Ala Gin Asn His Glu Tyr Tyr Asn He Gly Ala Phe Lys Val Ser Pro Asn Asn Lys Leu Val Ala Tyr Ala Glu Asp Thr Lys Gly Asp Glu He Tyr Thr He Tyr Val He Asp Ala Glu Thr Gin Asp Pro He Gly Glu Pro Leu His Asn Val Thr Ser Tyr He Glu Trp Ala Gly

Asp Glu Ala Leu Val Tyr He Thr Met Asp Glu He Leu Arg Pro

Asp Lys Ala Trp Leu His Arg Leu Gly Thr Glu Gin Ser Lys Asp

He Cys Leu Tyr Val Glu Lys Asp Asp Lys Phe Ser Leu Asp Leu

Gin Ala Ser Glu Ser Lys Lys Tyr Leu Phe Val Ala Ser Glu Ser

Lys Asn Thr Arg Phe Asn Phe Tyr Leu Asp Val Ser Lys Pro Glu Glu Gly Leu Lys Val Leu Thr Pro Arg Met Glu Gly He Asp Thr

Thr Val Ser His Arg Gly Asn His Phe Phe He Lys Arg Arg Ser

Asp Glu Phe Phe Asn Ser Glu Val Val Ala Cys Pro Val Asp Asn

Thr Ser Ser Thr Thr Val Leu Leu Pro His Arg Glu Ser Val Lys

He Gin Glu He Gin Leu Phe He Asp His Leu Val Ala Tyr Glu

Arg Glu Asn Gly Leu Pro Asn He He Val Tyr His Leu Pro Pro

He Gly Glu Pro Leu Arg Ser Leu Gly Asp Gly His Ala Val Asn

Phe Ala Asp Pro Val Tyr Ser Val Glu Ser Ser Glu Ser Glu Phe

Ser Ser Asn He Leu Arg Phe Ser Tyr Ser Ser Leu Lys Thr Pro Ser Ser Val Tyr Asp Tyr Asp Met Asn Ser Ser He Ser Ala Leu

Lys Lys He Asp Ser Val Leu Gly Gly Phe Asp Ala Ala Gin His

Val Thr Asp Arg Leu Trp Ala Pro Gly Leu Asp Gly Thr Leu He

Pro He Ser He Val Tyr Arg Lys Asp Leu Val Lys Leu Asp Gly

Ser Asp Pro Leu Leu Leu Tyr Gly Tyr Gly Ser Tyr Glu He Cys Ile Asp Pro Ser Phe Lys Ser Ser Arg Leu Ser Leu Leu Asp Arg Gly Phe He Phe Ala He Ala His He Arg Gly Gly Gly Glu Met Gly Arg Gin Trp Tyr Glu Asn Gly Lys Phe Leu Lys Lys Lys Asn

Thr Phe Thr Asp Phe He Ala Cys Ala Glu Tyr Leu He Asp Gin

Lys Tyr Ser Ser Lys Glu Arg Leu Cys He Asn Gly Arg Ser Ala

Gly Gly Leu Leu He Gly Ala Val Leu Asn Met Arg Pro Asp Leu

Phe Lys Ala Ala Val Ala Gly Val Pro Phe Val Asp Val Val Thr

Thr Met Leu Asp Pro Ser He Pro Leu Thr Thr Ser Glu Trp Glu Glu Trp Gly Asp Pro Arg Lys Glu Glu Phe Tyr Phe Tyr Met Lys

Ser Tyr Ser Pro Val Asp Asn Val Lys Ala Gin Asp Tyr Pro His

He Leu Val Thr Ala Gly Leu Asn Asp Pro Arg Val Leu Tyr Ser

Glu Pro Ala Lys Phe Val Ala Lys Leu Arg Asp Met Lys Thr Asp

Asp Asn He Leu Leu Phe Lys Cys Glu Leu Gly Ala Gly His Phe

Ser Lys Ser Gly Arg Phe Glu Lys Leu Gin Glu Asp Ala Phe Thr

Tyr Val Phe He Leu Lys Ala Leu Asn Met He Ser

7. A substantially pure polynucleotide encoding a proteinase according to claim 1 obtained by a method comprising the steps of: probing a cDNA or genomic library with at least one oligonucleotide having a sequence capable of encoding a peptide fragment of the proteinase or having a high likelihood of hybridizing to a native polynucleotide encoding the proteinase; identifying the presence of the polynucleotide in the library; and isolating and purifying the polynucleotide.

8. A polynucleotide encoding a proteinase according to claim 1 in substantially purified and isolated form obtained by a method comprising the steps of: synthesizing an oligonucleotide having a sequence capable of encoding a peptide fragment of the proteinase or having a high likelihood of hybridizing to a native polynucleotide encoding the proteinase; selectively amplifying a proteinase-encoding polynucleotide present in genomic DNA or mRNA, or in a cDNA or genomic library by the polymerase chain reaction (PCR) , employing the oligonucleotide as a primer; and purifying and isolating said proteinase- encoding polynucleotide.

9. A recombinant polynucleotide sequence comprising the nucleic acid residues:

10 20 30 40 50 60

* * _{* * *} * _* * * _{* * *}

5'CGACrCTAGAGGATCTACAGGAAAACATC-AGAGAAAGAGTAAAAATAAAGGAAAAGTGTA

70 80 90 100

* _* * _* * * * * *

TTAAATAAAGAATAAAGAA ATG GTA CTT TCT TCA TCT CTG TCC TTC ATT

110 120 130 140 150 _{* * * * * * * * *}

TGC GCG GAC ATT CAC CGA TTT TTC ACC ATT CCG CTG ACT CTC ACT

160 170 180 190

_* •* * * * * _* * *

GTG GTG CTT TCC GCT GGG CTT CCT CCA CCT TTT CTT GCC TCT GCC

200 210 220 230 240

* * * * * * * _* * TCT CGG TTC TCT CAT CAA CAC CGC GTC GCT TCC AAG TCA GTT CGG

250 260 270 280

* * * * * * * * *

TCT TTG TCC TCG TCG GCG ATG GCT TTC TCC CAG TCT CAA TAT CCG

290 300 310 320 330

* * * * * * _* * *

CCT CCT CCG GTG GCT AAG AAA GTG GAA CAC CCA ATG GAG ATG TTC

340 350 360 370

* * * * * * * * *

GGT GAC GTG AGG ATC GAC AAC TAT TAC TGG CTT CGG GAC GAT TCT

380 390 400 410 420

* * * * * * _* * *

CGC ACC AAT CCC GAT GTC CTC TCA TAC CTC CGT CAA GAA AAT GCA 430 440 450 460

* * * _* * * _* * *

TAC ACT GAC TCC ATT ATG AAA GGG ACC AAG GAA TTT GAA GAT AAG

470 480 490 500 510

* * * * * * * _{* *}

CTT TTT GCT GAG ATA AGA GGA AGG ATT AAG GAG GAT GAT ACC ACT

520 530 540 550

_{* *} * * _{* *} * _* *

GCA CCT TTA CGA AAG GGG CCT TAC TAT TAT TAT GAG AGA ACT CTG

560 570 580 590 600

* * * * * * * * *

GCG GGG AAG GAG TAT GCT CAA TAT TGT CGG CGT CCA GTA CCT GAC 610 620 630 640

* * * * * * * * *

GAC AAG GCA ACA CCA TCT ATT TAT GAT ACT GTT CCG ACT GAA CCT

650 660 670 680 690

* * * * * * * * *

GAT GCA CCT GAG GAG CAT GTT ATT TTG GAT GAG AAT ATC AAG GCT

700 710 720 730

* * * * * * _* * *

CAA AAT CAT GAA TAC TAC AAT ATC GGT GCT TTT AAG GTT AGT CCA

740 750 760 770 780

* * * * * * * * *

AAT AAT AAG TTA GTA GCA TAT GCA GAG GAC ACT AAA GGT GAT GAA

790 800 810 820

* * * * _* * * * * ^{Aττ TAT ACT ATT TAT} GTC ATA GAT GCT GAA ACT CAA GAT CCT ATA

830 840 850 860 870

* * * * * * * * *

GGA GAG CCT CTT CAT AAT GTA ACA TCA TAT ATT GAA TGG GCT GGC

880 890 900 910

* * * * * * * _* *

GAT GAA GCT TTG GTT TAT ATC ACA ATG GAT GAG ATT CTC AGG CCT

920 930 940 950 960

* * * * * * * * *

GAT AAG GCA TGG TTG CAC AGG TTG GGA ACA GAA CAG TCA AAG GAT

970 980 990 1000

* * * * * * * _* *

ATA TGT CTT TAT GTG GAA AAG GAT GAT AAA TTT TCT TTG GAT CTA 1010 1020 1030 1040 1050

* * * * * * * * *

CAA GCT TCT GAG AGC AAG AAA TAT TTG TTT GTA GCA TCA GAA AGT

1060 1070 1080 1090

* * * * * * * * *

AAA AAT ACA AGG TTT AAT TTT TAT CTT GAT GTT TCC AAA CCT GAA 1100 1110 1120 1130 1140

* _* * _{* * * * *} *

GAG GGA CTT AAA GTT TTG ACA CCA CGC ATG GAG GGT ATT GAT ACA

1150 1160 1170 1180

* * * * * * _* * *

ACT GTT AGC CAT CGA GGA AAT CAT TTT TTC ATT AAA AGG AGG AGT

1190 1200 1210 1220 1230

* * * * * * _{* * *}

GAT GAG TTT TTT AAT TCA GAA GTA GTA GCT TGC CCG GTT GAT AAT

1240 1250 1260 1270

* * _{* *} * * _* * *

ACC TCC TCT ACT ACA GTT CTT CTT CCC CAC AGG GAA AGT GTT AAA 1280 1290 1300 1310 1320

* * * * * * * _* *

ATT CAG GAG ATT CAG CTT TTT ATT GAT CAC CTT GTT GCA TAT GAG

1330 1340 1350 1360

* * * * * * _{* *} *

AGA GAA AAT GGT CTA CCA AAT ATA ATA GTT TAT CAC CTT CCT CCC 1370 1380 1390 1400 1410

* * * _* * * _* * *

ATT GGA GAA CCA CTA AGG AGC CTT GGA GAT GGT CAT GCT GTT AAT

1420 1430 1440 1450

* * * * * * * _* *

TTT GCT GAT CCA GTA TAT TCA GTG GAA TCT TCG GAG TCA GAA TTT

1460 1470 1480 1490 1500

* * * * * * * _* *

TCC TCA AAT ATT TTG CGG TTT TCA TAC AGT TCC TTG AAG ACT CCT

1510 1520 1530 1540

* * * * * * * * *

TCC TCT GTA TAT GAT TAT GAT ATG AAT TCA AGC ATT TCT GCT TTG

1550 1560 1570 1580 1590

* * * * * * * _{* *} AAG AAG ATT GAC TCA GTA TTG GGT GGT TTT GAT GCG GCA CAA CAT

1600 1610 1620 1630

* * * * * * * * *

GTT ACT GAT AGG CTG TGG GCA CCT GGT TTA GAT GGA ACT TTG ATT

1640 1650 1660 1670 1680

* * * * * * _* * _*

CCC ATT TCA ATT GTC TAC CGG AAG GAC CTT GTT AAA CTT GAT GGA

1690 1700 1710 1720

* * * * * * _{* *} *

TCT GAT CCT TTA CTA CTT TAT GGC TAT GGG TCT TAT GAG ATT TCC

1730 1740 1750 1760 1770

* * * _* * * _{* * *}

ATA GAT CCC AGT TTC AAG TCA TCA AGG CTG TCA TTG TTA GAT CGA 1780 1790 1800 1810

* * * * * _{* *} * *

GGT TTT ATA TTT GCA ATT GCT CAT ATT CGC GGA GGT GGT GAA ATG

1820 1830 1840 1850 1860

* * * _* * * * * *

GGA AGG CAG TGG TAT GAG AAT GGG AAG TTC TTG AAA AAA AAG AAC

1870 1880 1890 1900

* * * * * * * * *

ACT TTT ACA GAT TTT ATT GCT TGT GCT GAA TAT TTG ATT GAT CAA

1910 1920 1930 1940 1950

* * * * * * * * *

AAA TAT TCT TCA AAG GAA AGA CTG TGC ATC AAT GGT CGA AGT GCA I960 1970 1980 1990

* * * * * * * * *

GGG GGT TTA CTA ATT GGT GCA GTC CTT AAT ATG AGG CCT GAC TTG

2000 2010 2020 2030 2040

* * * * * * * * *

TTC AAG GCT GCT GTT GCG GGT GTA CCT TTT GTG GAT GTT GTG ACA

2050 2060 2070 2080

* * * * * * * * *

ACC ATG CTT GAT CCA TCT ATA CCT CTG ACA ACT TCA GAA TGG GAG

2090 2100 2110 2120 2130

* * * * * * _* * *

GAA TGG GGA GAT CCT AGG AAG GAG GAG TTC TAC TTC TAC ATG AAG

2140 2150 2160 2170

* * * * * * * _* * TCA TAT TCT CCG GTT GAT AAT GTG AAG GCA CAA GAT TAT CCT CAC

2180 2190 2200 2210 2220

_* * * * * * _* * *

ATT CTT GTT ACT GCT GGC TTA AAT GAT CCA CGT GTT CTG TAT TCT

2230 2240 2250 2260

* * * * * * * _* *

GAA CCT GCT AAG TTT GTG GCA AAA CTG AGG GAT ATG AAG ACC GAT

2270 2280 2290 2300 2310

_* * * * * * * * *

GAT AAC ATT CTA CTG TTT AAA TGC GAG CTT GGT GCA GGG CAT TTT

2320 2330 2340 2350

* * * * * * * _* *

TCA AAG TCT GGG AGG TTC GAG AAG CTC CAA GAA GAT GCC TTC ACC 2360 2370 2380 2390 2400

* * * * * * * * * *

TAT GTC TTT ATT TTG AAG GCC CTG AAT ATG ATT AGT TAG CTCGAGAC

2410 2420 2430 2440 2450 2460

* * * * * * * * * _* * *

CCGTGGGCAACΓC-AACΓCGAGTCTGGAGGTTC-AATT^^ 2470 2480 2490 2500 2510 2520

* * * * * * * * * * * *

C-AC-ATGGATCGTTTATTCACCATGGGTTAATC^*TATTGTTCTGACTATGTGCTTCTGTTAT

2530 2540 2550 2560 2570 2580

* * * * * * * * * * * *

TAGTTAACTTTGTGATTCGTCTCΛTAATC-AGTTTTTTTCCCCCCC-ACTTATTTGTATTAT

2590 2600 2610 2620 2630 2640

* * * * * * * * * * * *

TOCCGGAC-AGCCIAAGAGAGAACATTATGATTGTAACCAAAAAAAAAAAAAATC-AATTTTA

2650 2660 2670 2680 2690 2700

* * * * * * * * * * * *

ATAATAAAATTTATTGO-AG(-TΛAAAAAAAAAAAAAAAACCrTGTAGATCCCCGGGTACC 2710

*

GAGCTC3'

10. An expression system that, when transformed into a host cell, can express the polynucleotide of claim 9.

11. A method of preparing a recombinant proteinase comprising culturing the cells transformed with the expression system according to claim 10 under conditions suitable to attain expression of the gene encoding the proteinase and recovering the proteinase.

12. A composition comprising a substantially purified pollen proteinase and a suitable carrier therefor.

13. The composition according to claim 12 wherein the proteinase is a serine proteinase.

14. The composition according to claim 12 wherein the pollen is derived from mesquite, Rhuslancia, ragweed, Japanese cedar, almond, typha or pine.

15 . The composition according to claim 12 wherein the proteinase has a substrate specificity for Arg-Arg and Arg- lie .

16 . The composition according to claim 12 wherein the proteinase has the amino acid residue sequence :

Met Val Leu Ser Ser Ser Leu Ser Phe lie

Cys Ala Asp lie His Arg Phe Phe Thr lie Pro Leu Thr Leu Thr

Val Val Leu Ser Ala Gly Leu Pro Pro Pro Phe Leu Ala Ser Ala

Ser Arg Phe Ser His Gin His Arg Val Ala Ser Lys Ser Val Arg

Ser Leu Ser Ser Ser Ala Met Ala Phe Ser Gin Ser Gin Tyr Pro

Pro Pro Pro Val Ala Lys Lys Val Glu His Pro Met Glu Met Phe

Gly Asp Val Arg He Asp Asn Tyr Tyr Trp Leu Arg Asp Asp Ser

Arg Thr Asn Pro Asp Val Leu Ser Tyr Leu Arg Gin Glu Asn Ala

Tyr Thr Asp Ser He Met Lys Gly Thr Lys Glu Phe Glu Asp Lys

Leu Phe Ala Glu He Arg Gly Arg He Lys Glu Asp Asp Thr Thr

Ala Pro Leu Arg Lys Gly Pro Tyr Tyr Tyr Tyr Glu Arg Thr Leu

Ala Gly Lys Glu Tyr Ala Gin Tyr Cys Arg Arg Pro Val Pro Asp

Asp Lys Ala Thr Pro Ser He Tyr Asp Thr Val Pro Thr Glu Pro

Asp Ala Pro Glu Glu His Val He Leu Asp Glu Asn He Lys Ala

Gin Asn His Glu Tyr Tyr Asn He Gly Ala Phe Lys Val Ser Pro

Asn Asn Lys Leu Val Ala Tyr Ala Glu Asp Thr Lys Gly Asp Glu

He Tyr Thr He Tyr Val He Asp Ala Glu Thr Gin Asp Pro He

Gly Glu Pro Leu His Asn Val Thr Ser Tyr He Glu Trp Ala Gly

Asp Glu Ala Leu Val Tyr He Thr Met Asp Glu He Leu Arg Pro

Asp Lys Ala Trp Leu His Arg Leu Gly Thr Glu Gin Ser Lys Asp

He Cys Leu Tyr Val Glu Lys Asp Asp Lys Phe Ser Leu Asp Leu

Gin Ala Ser Glu Ser Lys Lys Tyr Leu Phe Val Ala Ser Glu Ser

Lys Asn Thr Arg Phe Asn Phe Tyr Leu Asp Val Ser Lys Pro Glu

Glu Gly Leu Lys Val Leu Thr Pro Arg Met Glu Gly He Asp Thr

Thr Val Ser His Arg Gly Asn His Phe Phe He Lys Arg Arg Ser Asp Glu Phe Phe Asn Ser Glu Val Val Ala Cys Pro Val Asp Asn Thr Ser Ser Thr Thr Val Leu Leu Pro His Arg Glu Ser Val Lys He Gin Glu He Gin Leu Phe He Asp His Leu Val Ala Tyr Glu Arg Glu Asn Gly Leu Pro Asn He He Val Tyr His Leu Pro Pro He Gly Glu Pro Leu Arg Ser Leu Gly Asp Gly His Ala Val Asn Phe Ala Asp Pro Val Tyr Ser Val Glu Ser Ser Glu Ser Glu Phe Ser Ser Asn He Leu Arg Phe Ser Tyr Ser Ser Leu Lys Thr Pro Ser Ser Val Tyr Asp Tyr Asp Met Asn Ser Ser He Ser Ala Leu Lys Lys He Asp Ser Val Leu Gly Gly Phe Asp Ala Ala Gin His Val Thr Asp Arg Leu Trp Ala Pro Gly Leu Asp Gly Thr Leu He Pro He Ser He Val Tyr Arg Lys Asp Leu Val Lys Leu Asp Gly Ser Asp Pro Leu Leu Leu Tyr Gly Tyr Gly Ser Tyr Glu He Cys

He Asp Pro Ser Phe Lys Ser Ser Arg Leu Ser Leu Leu Asp Arg

Gly Phe He Phe Ala He Ala His He Arg Gly Gly Gly Glu Met

Gly Arg Gin Trp Tyr Glu Asn Gly Lys Phe Leu Lys Lys Lys Asn

Thr Phe Thr Asp Phe He Ala Cys Ala Glu Tyr Leu He Asp Gin

Lys Tyr Ser Ser Lys Glu Arg Leu Cys He Asn Gly Arg Ser Ala ^Gly ^G1y ^{Leu Leu Ile G1}y Ala Val Leu Asn Met Arg Pro Asp Leu

Phe Lys Ala Ala Val Ala Gly Val Pro Phe Val Asp Val Val Thr

Thr Met Leu Asp Pro Ser He Pro Leu Thr Thr Ser Glu Trp Glu

Glu Trp Gly Asp Pro Arg Lys Glu Glu Phe Tyr Phe Tyr Met Lys

Ser Tyr Ser Pro Val Asp Asn Val Lys Ala Gin Asp Tyr Pro His

He Leu Val Thr Ala Gly Leu Asn Asp Pro Arg Val Leu Tyr Ser

Glu Pro Ala Lys Phe Val Ala Lys Leu Arg Asp Met Lys Thr Asp

Asp Asn He Leu Leu Phe Lys Cys Glu Leu Gly Ala Gly His Phe

Ser Lys Ser Gly Arg Phe Glu Lys Leu Gin Glu Asp Ala Phe Thr Tyr Val Phe He Leu Lys Ala Leu Asn Met He Ser

17. A vaccine comprising a substantially purified pollen proteinase and a suitable carrier therefor.

18. The vaccine according to claim 17 wherein the proteinase is a serine proteinase.

19. A vaccine according to claim 17 wherein the pollen is derived from mesquite, Rhuslancia, ragweed, Japanese cedar, almond, typha or pine.

20. A vaccine according to claim 17 wherein the proteinase has a substrate specificity for Arg-Arg and Arg-lie.

21. A vaccine according to claim 17 wherein the proteinase has the amino acid residue sequence:

Met Val Leu Ser Ser Ser Leu Ser Phe He Cys Ala Asp He His Arg Phe Phe Thr He Pro Leu Thr Leu Thr

Val Val Leu Ser Ala Gly Leu Pro Pro Pro Phe Leu Ala Ser Ala

Ser Arg Phe Ser His Gin His Arg Val Ala Ser Lys Ser Val Arg

Ser Leu Ser Ser Ser Ala Met Ala Phe Ser Gin Ser Gin Tyr Pro

Pro Pro Pro Val Ala Lys Lys Val Glu His Pro Met Glu Met Phe

Gly Asp Val Arg He Asp Asn Tyr Tyr Trp Leu Arg Asp Asp Ser

Arg Thr Asn Pro Asp Val Leu Ser Tyr Leu Arg Gin Glu Asn Ala

Tyr Thr Asp Ser He Met Lys Gly Thr Lys Glu Phe Glu Asp Lys

Leu Phe Ala Glu He Arg Gly Arg He Lys Glu Asp Asp Thr Thr Ala Pro Leu Arg Lys Gly Pro Tyr Tyr Tyr Tyr Glu Arg Thr Leu

Ala Gly Lys Glu Tyr Ala Gin Tyr Cys Arg Arg Pro Val Pro Asp

Asp Lys Ala Thr Pro Ser He Tyr Asp Thr Val Pro Thr Glu Pro

Asp Ala Pro Glu Glu His Val He Leu Asp Glu Asn He Lys Ala

Gin Asn His Glu Tyr Tyr Asn He Gly Ala Phe Lys Val Ser Pro

Asn Asn Lys Leu Val Ala Tyr Ala Glu Asp Thr Lys Gly Asp Glu

He Tyr Thr He Tyr Val He Asp Ala Glu Thr Gin Asp Pro He

Gly Glu Pro Leu His Asn Val Thr Ser Tyr He Glu Trp Ala Gly

Asp Glu Ala Leu Val Tyr He Thr Met Asp Glu He Leu Arg Pro Asp Lys Ala Trp Leu His Arg Leu Gly Thr Glu Gin Ser Lys Asp He Cys Leu Tyr Val Glu Lys Asp Asp Lys Phe Ser Leu Asp Leu Gin Ala Ser Glu Ser Lys Lys Tyr Leu Phe Val Ala Ser Glu Ser Lys Asn Thr Arg Phe Asn Phe Tyr Leu Asp Val Ser Lys Pro Glu

Glu Gly Leu Lys Val Leu Thr Pro Arg Met Glu Gly He Asp Thr

Thr Val Ser His Arg Gly Asn His Phe Phe He Lys Arg Arg Ser

Asp Glu Phe Phe Asn Ser Glu Val Val Ala Cys Pro Val Asp Asn

Thr Ser Ser Thr Thr Val Leu Leu Pro His Arg Glu Ser Val Lys

He Gin Glu He Gin Leu Phe He Asp His Leu Val Ala Tyr Glu Arg Glu Asn Gly Leu Pro Asn He He Val Tyr His Leu Pro Pro

He Gly Glu Pro Leu Arg Ser Leu Gly Asp Gly His Ala Val Asn

Phe Ala Asp Pro Val Tyr Ser Val Glu Ser Ser Glu Ser Glu Phe

Ser Ser Asn He Leu Arg Phe Ser Tyr Ser Ser Leu Lys Thr Pro

Ser Ser Val Tyr Asp Tyr Asp Met Asn Ser Ser He Ser Ala Leu

Lys Lys He Asp Ser Val Leu Gly Gly Phe Asp Ala Ala Gin His

Val Thr Asp Arg Leu Trp Ala Pro Gly Leu Asp Gly Thr Leu He

Pro He Ser He Val Tyr Arg Lys Asp Leu Val Lys Leu Asp Gly

Ser Asp Pro Leu Leu Leu Tyr Gly Tyr Gly Ser Tyr Glu He Cys He Asp Pro Ser Phe Lys Ser Ser Arg Leu Ser Leu Leu Asp Arg

Gly Phe He Phe Ala He Ala His He Arg Gly Gly Gly Glu Met

Gly Arg Gin Trp Tyr Glu Asn Gly Lys Phe Leu Lys Lys Lys Asn

Thr Phe Thr Asp Phe He Ala Cys Ala Glu Tyr Leu He Asp Gin

Lys Tyr Ser Ser Lys Glu Arg Leu Cys He Asn Gly Arg Ser Ala

Gly Gly Leu Leu He Gly Ala Val Leu Asn Met Arg Pro Asp Leu

Phe Lys Ala Ala Val Ala Gly Val Pro Phe Val Asp Val Val Thr

Thr Met Leu Asp Pro Ser He Pro Leu Thr Thr Ser Glu Trp Glu

Glu Trp Gly Asp Pro Arg Lys Glu Glu Phe Tyr Phe Tyr Met Lys Ser Tyr Ser Pro Val Asp Asn Val Lys Ala Gin Asp Tyr Pro His

He Leu Val Thr Ala Gly Leu Asn Asp Pro Arg Val Leu Tyr Ser

Glu Pro Ala Lys Phe Val Ala Lys Leu Arg Asp Met Lys Thr Asp

Asp Asn He Leu Leu Phe Lys Cys Glu Leu Gly Ala Gly His Phe

Ser Lys Ser Gly Arg Phe Glu Lys Leu Gin Glu Asp Ala Phe Thr Tyr Val Phe He Leu Lys Ala Leu Asn Met He Ser

22. A method of monitoring exposure of an animal to a pollen proteinase, comprising the steps of: _ obtaining a sample from the animal; incubating the sample with a pollen .proteinase or portions thereof under conditions suitable for antibody-antigen interaction; and detecting the presence of antibody-antigen ₀ complexes; wherein the presence of antigen-antibody complexes is indicative of exposure of the animal to the proteinase.

₅ 23. A method of immunizing an animal against an allergic response to a pollen, comprising the steps of: obtaining at least one purified pollen proteinase or immunogenic subunit thereof; ₀ administering an amount of at the least one pollen proteinase or immunogenic subunit thereof effective to elicit an immune response to the proteinase.

24. A method of identifying agents that modulate the effect of a pollen proteinase on animals comprising the steps of a) incubating the pollen proteinase with the agen ; b) exposing animal cells sensitive to the

-₀ proteinase to pollen proteinase incubated with the agent; c) determining the ability of the proteinase incubated with the agent to affect the cells; and d) comparing the effect seen in step c) with the effect of a control sample of proteinase that has not

35 been incubated with the agent on animal cells susceptible to the proteinase; wherein the agents that modulate the effect of the pollen proteinase change the activity of the proteinase compared to the control sample.

25. A method of identifying agents that modulate pollen proteinase activity comprising the steps of: a) incubating a pollen proteinase with the agent; b) determining the activity of the proteinase incubated with the agent; and c) comparing the activity obtained in step b) with the activity of a control sample of proteinase that has not been incubated with the agent.

26. A method of ameliorating the affects of pollen proteinases on an animal affected by the proteinase, comprising administering to the animal an effective amount of a physiologically acceptable pollen proteinase inhibitor.