WO1998008863A1 - Enzyme and dna sequence encoding same - Google Patents

Abstract

The present invention provides nucleic acid and corresponding amino acid sequences of a protein that has been found to be useful in numerous medical and cosmetic contexts.

Description

ENZYME AND DNA SEQUENCE ENCODING SAME

The present invention relates to purified nucleic acids encoding a kπll-deπved enzymes such as proteinases, which can be a multifunctional protein, and to puπfied polypeptides A protein having "multifunctional activity," is defined herein as including at least one of a chymotrypsm. trypsin. collagenase, elastase or exo peptidase activity, or asialo GM \ ceramide binding activity

Multifunctional proteins are useful for multiple purposes, including treating viral infections such as herpes outbreaks, fungal, bacterial or parasitic mfections, including the pπmarv and secondary infections of leprosy, colitis, ulcers, hemorrhoids, corneal scarring, dental plaque, acne, cystic fibrosis. blood clots, wounds, immune disorders including autoimmune disease, such as lupus erythematosus and multiple sclerosis, and cancer Puπfied polypeptides having protemase or multifunctional activity and puπfied nucleic acids encoding such polypeptides are desirable to provide pharmaceutically useful products Other uses for proteinases are well recognized m the art and include digesting protemaceous mateπal for a vaπety of purposes including cleaning and creating improved feeds for animals or bacteriology SUMMARY OF THE INVENTION

Until now, sequences encoding kπll-deπved proteinases similar to those set forth in the Sequence Listmg have not been identified The ammo acid sequence included in SEQ ID NOS 4 5. 6. 10, 20 22 or 24 or other isoforms thereof or chimeπc polypeptides thereof are examples of such enzymes In particular, in many cases the invention is specified in terms of a reference protein sequence which is AA64-300 of SEQ ID NO 4 or a sequence differing therefrom bv at least one of the residue differences found m SEQ ID NOS 5. 20. 22. or 24 In an embodiment of the invention, the refenence reference sequence further has the differences relative to SEQ ID NO 4 that are found in SEQ ID NO 8 These differences are illustrated in Figures 6 and 7

One preferred embodiment of the present invention is a substantially pure nucleic acid co pπsing a nucleic acid encoding a polypeptide having at least about 70% homology (such as identity or similanty) to a kπll-deπved multifunctional protein or a reference protein, such as the polypeptide of SEQ ID NOS 4, 5, 6. 8. 10. 20. 22 or 24 and in one embodiment especially SEQ ID NOS 4, 6. 10 or 22. and more preferably, at least about 80% homology. and most preferabK . at least about 90% homology Preferably. In another embodment embodiment, the homology is with reference to SEQ ID NOS 4. 5. 20. 22 or 24 Even more preferably, the nucleic acid comprises a nucleic acid encoding a polypeptide sharing at least about 70% amino acid identitv with a reference protein or a kπll-deπved multifunctional protein and yet more preferably, at least about 80% identity, and still more preferably, at least about 90% identity. Yet still more preferably, the homology or identity is at least about 95% These levels of homology (such as identity) apply to all embodiments of the invention.

In certain preferred embodiments, the substantially pure nucleic acid compπses an engineered nucleic acid variant encoding a polypeptide differing from a reference protein or a krill- derived multifunctional protein or its isoforms by no more than about 33 ammo acid substitutions, and more preferably, no more than about 20 ammo acid substitutions. Preferably, the engineered substitutions cause a conservative substitution in the amino acid sequence of a reference sequence or a multifunctional protein. Preferred substantially pure nucleic acids also include nucleic acid analogs. In certain preferred embodiments, the nucleic acid comprises the open reading frames of SEQ ID NOS:l. 2. 3, 7, 9, 19, 21 or 23 and more preferably, SEQ ID NOS 1. 3, 9 or 22 In another aspect, the nucleic acid includes base pairs 1 0-900 of SEQ ID NO 1. base pairs 2-556 of SEQ ID NO 2. base pairs 190-900 of SEQ ID NO.3. base pairs 215-925 of SEQ ID NO 9, base pairs 1-633 of SEQ ID NO: 19, base pairs 196-906 of SEQ ID NO.21. or base pairs 215-535 of SEQ ID NO 23 (in one embodiment, 1 -900 of SEQ ID NO.1 , base pairs 2-556 of SEQ ID NO:2. base pairs 1-900 of SEQ ID NO:3, base pairs 2-925 of SEQ ID NO.9. base pairs 1 -633 of SEQ ID NO 19, base pairs 1-906 of SEQ ID NO:21. or base pairs 2-535 of SEQ ID NO.23), or more preferably base pairs 190-900 of SEQ ID NO.1, base pairs 2-556 of SEQ ID NO.2. base pairs 1-633 of SEQ ID NO.19. base pairs 196-906 of SEQ ID NO.21 or base pairs 215-535 of SEQ ID N0.23 . The invention additionally includes vectors capable of reproducing in a cell (such as a eukaryotic or prokaryotic cell) having a nucleic acid described above as well as transformed cells having such nucleic acid. Further, the invention includes a substantially pure nucleic acid comprising a nucleic acid that hybridizes, under stringent conditions, to a nucleic acid encoding a polypeptide having at least about 70% homology to a reference sequence or a kπll-deπved multifunctional protein, such as the polypeptide of SEQ ID NOS.4, 5, 6, 8, 10, 20, 22 or 24 and more preferably. SEQ ID NOS:4, 6, 10 or 22.

In one embodiment, the nucleic acid is a reference nucleic acid sequence including the open reading frame sequence of SEQ ID NO: 1 (or prfcrably preferably the portion from the NH₂- IleValGlyGlvMet sequence through to the end) or a sequence diffenng therefrom by at least one of the bp differences found in SEQ ID NOS:2, 19, 21 or 23 (or prferably preferably the portion from the NHb-IleValGlyGly sequence through to the end). In one embodiment, the nucleic acid includes a sequence with about 50%, about 60%. about 70%. about 80%, about 90% or about 95% identity to a reference sequence. Another preferred embodiment is a transformed cell (such as a prokaryotic or eukaryotic cell) compπsing a nucleic acid encoding a polypeptide having at least about 70% homology to a reference sequence or a kπll-deπved multifunctional protein Preferably, the transformed cell expresses one of the enzymes descπbed herem Yet another preferred embodiment is a vector capable of reproducing in a cell such as a eukaryotic or prokaryotic cell, the vector compπsing a nucleic acid encoding a polypeptide having at least about 70% homology to a reference sequence or a kπll-deπved multifunctional protein Preferably, the inventive vector codes for expression, mtracellularly or extracellularlv, of one of the multifunctional proteins descnbed herem Another embodiment of the present invention is a polypeptide comprising a substantially pure lsoform of a reference sequence or a kπll-deπved multifunctional protein or engineered vaπant thereof, and preferably, a polypeptide compπsing SEQ ID NOS 4. 5. 6. 8. 10. 20. 22 or 24 and especially. SEQ ID NOS 4. 6, 10 or 22 In one embodiment, the lsoform is a polypeptide comprising SEQ ID NOS 4, 5, 20. 22 or 24 The invention further provides a pharmaceutical composition for treating an a mal compπsing an effective amount of such a polypeptide together with a pharmaceutically acceptable earner

Yet another prefened embodiment is a method of prepaπng an enzyme such as a multifunctional protein, wherein the protem has at least about 70% homology to a reference sequence or a kπll-deπved multifunctional protein Such method comprises (a) transforming an appropriate eukaryotic or prokaryotic cell with an expression vector for expressing mtracellularly or extracellularlv a nucleic acid encoding the protein,

(b) growing the transformed cell in culture, and

(c) isolating the protein from the transformed cell or the culture medium Yet another preferred embodiment is a pharmaceutical composition for treating an animal compπsing an effective amount of an expression vector compnsing a nucleic acid encoding a multifunctional protein, and a pharmaceutically acceptable earner The invention further provides a pharmaceutical composition for treating an animal compπsing an effective amount of a nucleic acid encoding a polypeptide havmg at least about 70% homology to a reference sequence or a knll-deπved multifunctional protein

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the DNA sequence of a first isoform ("p62") (SEQ ID NO 1) of a kπll- denved multifunctional protein aligned with the DNA sequence of a second isoform ("p31") (SEQ ID NO 2) Figure 2 shows the ammo acid sequence of the first isoform ("p62") (SEQ ID NO 4) of a kπll-deπved multifunctional protein aligned with the amino acid sequence of the second isoform ("p31") (SEQ ID N0 5)

Figure 3 shows the DNA sequence of a third isoform ("p5 la") (SEQ ID NO 7) of a kπll- derived multifunctional protein aligned with the DNA sequence of the first isoform ("p62") (SEQ ID NO 1)

Figure 4 shows the amino acid sequence of the third isoform ("p5 la") (SEQ ID NO 8) aligned with the ammo acid sequence of the first isoform ("p62") (SEQ ID NO 4) and the ammo acid sequence of the second isoform ("p31") (SEQ ID NO 5) Figure 5 shows the ammo acid sequences of several proteins, namely, Factor VII, thrombin, kalhkrein. a Limulus pro-clotting enzyme, plasmin, hepsm and Factor XII. aligned with the amino acid sequence of the first isoform ("p62") (SEQ ID NO 1)

Figure 6 shows sequence comparisons between the nucleic acid sequences for p62 (SEQ ID NO l), pl3 (SEQ ID NO 19), p912 (SEQ ID NO 21), p5 lb (SEQ ID NO 23) and p31 (SEQ ID NO 2), as well as sequence compaπsions comparisons for the aligned peptide sequences (SEQ ID NOS 4. 20, 22, 24 and 5, respectively) Nuchc Nucleic acid sequence differences m the open reading frames relative to p62 are indicated by underlining, and differences in amino acid sequence are indicate with recitals of the diffenng residues

Figure 7 shows sequence comparisons between the polypeptide sequences encoded by p62 (SEQ ID NO 4), pl3 (SEQ ID NO 20), p912 (SEQ ID NO 22), p5 lb (SEQ ID NO 24) and p31 (SEQ ID NO 5)

Figure 8 illustrates a sequence alignment between the polypeptide sequences encoded by p62 (SEQ ID NO 4). p912 (SEQ ID NO 22), p5 la (SEQ ID NO 8) and p31 (SEQ ID NO 5) DETAILED DESCRIPTION For the purposes of this application, the terms listed below shall have the following meaning

* enzymatically active segment

A segment of a multifunctional protein having activity comprising at least one of a chymotrypsin. trypsin. collagenase, elastase or exo peptidase activity • hydrolase

An enzyme that degrades bonds formed by dehydration reactions such as amide, ester, or ether bonds The term encompasses, but is not limited to, proteases such as trypsin and chymotrypsin • isoform

A naturally occurπng sequence vaπant of a substantially homologous protein within the same organism Preferably, the isoform shares at least about 80% identity, and more preferably, at least about 85% identity with SEQ ID NO 4 • krill-derived multifunctional protein

A multifunctional protein having the same sequence as a protein isolated from kπll having the properties of the protein descπbed in the section entitled "Preferred Characteristics of the Multifunctional Protein " This protein is also referred to as the "kπll-deπved multifunctional hvdrolase" and includes all isoforms of the protein The ammo acid sequence included in SEQ ID NO 4. SEQ ID NO 5, SEQ ID NO 6. SEQ ID NO 8 or SEQ ID NO 10 or other isoforms thereof or chimeπc polypeptides thereof are examples of kπll-deπved multifunctional prote s ^■ multifunctional protein

A protein having activity compπsing at least one of a chymotrypsin. trypsin. collagenasc elastasc or exo peptidase activity or asialo GM _j ceramide binding activity, and substantial homology to at least a segment of a kπll-dcπved multifunctional protein

• nucleic acid

The nucleic acid sequence embodiments of the invention are preferably deoxyπbonucleic acid sequences, preferably double-stranded deoxyπbonucleic acid sequences However, they can also be πbonucleic acid sequences, or nucleic acid mimics, meaning compounds designed to preserve the hydrogen bonding and base-paiπng properties of nucleic acid but which differ from natural nucleic acid m. for example, susceptibility to nucleases

• reference protein or sequence

A reference protein sequence is AA64-300 of SEQ ID NO 4 or AA 1-300 of SEQ ID NO 4 or a sequence diffeπng therefrom by at least one of the residue differences found in SEQ ID NOS 5. 20. 22. or 24 A reference protein is a protein having the reference protein sequence With reference to Figure 6, examples of reference proteins are (a) a protein with sequence of AA64-300 of SEQ ID NO 4 except that residue 128 is seπne or (b) a protein with sequence of SEQ ID NO 4 except that before Leu¹ is senne In a preferred embodiment, the N-terminal additions from SEQ ID NOS 22 and 24 are NH Ala. NH HeAla, NH₂-ArgIleAla. NH₂-SerArgIleAla. NH_:- ArgSerArglleAla, NH₂-GlyArgSerArgIleAla or NH₂-ProGlyArgSerArgIleAla

• sequence identity

"Identity." as known in the art. is a relaUonship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences, particularly, as determined b\ the match between stπngs of such sequences "Identity" is readily calculated by known methods {Computational Molecular Biology, Lesk, A M , ed Oxford University Press. New York, 1988. Bwcomputmg Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data. Part 1. Griffin, A M . and Gπffin, H G . eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Heinje, G , Academic Press, 1987, and Sequence Analysis Prime/, Gnbskov, M and Devereuv J , eds , M Stockton Press, New York, 1991) While there exist a number of methods to measure identity between two sequences, the term is well known to skilled artisans Methods commonK employed to determine identity between sequences include, but are not limited to those disclosed in Canllo. H , and Lipman, D , SIAM J Applied Math 48 1073 ( 1 88) or, preferably, in Needleman and Wunsch, J Mol Biol , 48 443-445. 1 70, wherein the parameters are as set in version 2 of DNASIS (Hitachi Software Engineering Co San Bruno, CA) Computer programs for determining identity are publicly available Computer program methods to determine identiU between two sequences include, but are not limited to GCG program package (Devereuv J . et al , Nucleic Acids- Research 12(1) 387 (1984)), BLASTP. BLASTN and FASTA (Atschul. S F et al . J Molec Biol 215: 403-410 (1990)) The BLAST X program is publicly available from NCBI

nlm nih gov)and other sources {BIΛS1 Manual Altschul, S . et al NCBI NLM NIH Bethesda. MD 20894. Altschul, S . et al Mol Biol 215 403-410 ( 1990)) * substantial homology At least about 60% sequence homology, for example 60% sequence identity

The present invention provides DNA and corresponding amino acid sequences of a kπll- deπved enzymes such as proteinases, which can be multifunctional proteins Crustaceans including antarctic kπll, are useful sources for the multifunctional protein of the invention A protein having "multifunctional activity." is defined herein as including at least one of a chymotrypsin. trypsin, collagenase. elastase or exo peptidasc activity, or asialo GMj ceramide bmd g activity For purification of kπll-deπved multifunctional protein, see, for example. U S patent application Senal No 08/600.273 (filed February 8. 1996) deFaire et al inventors entitled "Multifunctional Enzyme "

The present invention provides nucleic acids (such as πbonucleic acids or deoxyπbonucleic acids) and polypeptides and analogs thereof including nucleic acids that bind to a multifunctional protein encoding nucleic acid, as well as pharmaceutical compositions, gene therapy and antibodies and antisera against the multifunctional protein Some of the nucleic acids and polypeptides are naturally occurring variants (isoforms) whereas others are non-natural occurring (engineered) variants 1. Nucleic Acids

The nucleic acid embodiments of the invention are preferably deoxyπbonucleic acids (DNAs), both single- and double-stranded, and most preferably double-stranded deoxyπbonucleic acids However, they can also be. without limitation, πbonucleic acids (RNAs), as well as hybrid RNA DNA double-stranded molecules

Nucleic acids encoding a multifunctional protein include all multifunctional protc - encoding nucleic acids, whether native or synthetic, RNA DNA or cDNA, that encode a multifunctional protein, or the complementary strand thereof, including but not limited to nucleic acid found m a multifunctional protein-expressing organism For recombinant expression purposes, codon usage preferences for the organism in which such a nucleic acid is to be expressed are advantageously considered in designing a synthetic multifunctional protein-encoding nucleic

The nucleic acid sequences of the invention can encode, for example one of several isoforms of a kπll-deπved protein SEQ ID NOS 4, 5. and 8 represent three isoforms that share about 88-89% identity with each other in overlapping amino acids See, for example. Figure 1 which compares the DNA sequence of the first isoform. SEQ ID NO 1, with the DNA sequence of the second isoform, SEQ ID NO 2. which share about 88% identical nucleotides See also, for example. Figure 3, which provides a comparison of the DNA sequence of the third isoform (SEQ ID NO 7) and the first isoform (SEQ ID NO 1). which share about 89% identical nucleotides These isoforms all lack the initiation codon methionine Further, two of these three isoforms contain a hydrophobic sequence which may function as a signal sequence, na eh . LLLALVAAASA, which is amino acid residues 1-1 1 in the first isoform. SEQ ID NO 4. and PGRSRIALLLALVAATASA. which is ammo acid residues 1-19 in the third isoform. SEQ ID NO 8 These two isoforms additionally contain a pro-protein segment The pro-protein segment is the segment of the protein, other than the hydrophobic segment, that is present in the precursor protein but absent in the mature protein Without being limited to a particular theory it is possible that at least a part of the pro-protem segment may still be attached to the mature protein Further it is believed that kπll-deπved multifunctional proteins may have two chains linked by a disulfide bond For example, a cysteine in the pro-protem segment may participate in a disulfide bond in the mature protein

In the first isoform, the pro-protein segment has the following sequence, which corresponds to amino acid residues 12-63 in the first isoform, SEQ ID NO 4 AEWRWQFRHPTVTPNPRAKNPFRVTKSSPVQPPAVRGTKAVENCGPVAPRNK The third isoform has a pro-protein segment with the following sequence, which corresponds to amino acid residues 20-71 in SEQ ID NO 8 SEWRWQFRHPTVTPNPRANNPFRPSKVAPVQPPAV- RGTKAVENCGPVAPKNK The remaining ammo acid sequences of these polypeptides (other than the hydrophobic segment and the pro-protem segment) represent the mature protein See Figure 2. which provides a compaπson of the amino acid sequence of the first isoform and the second isoform. which share about 89% identical am o acids Additionally, see Figure 4 which provides a comparison of the amino acid sequences of all three isoforms

Further embodiments of the invention include nucleic acid sequences that encode polypeptides that are preferably present in the protein The following examples are derived from the pro-protcin segment of SEQ ID NO 4, and are polypepudes that are preferably present in the mature protein Without being limited to a particular theory, these polypeptides may form at least part of a first amino acid chain that is linked via a disulfide bond to a second amino acid chain which can be. for example, the mature protein For instance, in certain preferred embodiments, the nucleic acid further encodes a polypeptide sequence such as AVENCGPVAPR (SEQ ID NO 11 ). AVENCGPVAPRNK (SEQ ID NO 12), GTKAVENCGPVAPR (SEQ ID NO 13).

GTKAVENCGPVAPRNK (SEQ ID NO 14), SSPVQPPAVRGTKAVENCGPVAPR (SEQ ID NO 15). SSPVQPPAVRGTKAVENCGPVAPRNK (SEQ ID NO 16). or AVENCGPVA (SEQ ID NO 25). or a sequence diffeπng therefrom as indicated in the corresponding sequence fragments of SEQ ID NOS 21 or 24 Without being limited to a particular theory, the above-listed polypeptides (SEQ ID NO 1 1-16, 25) may be linked to the remainder of the mature kπll-deπved multifunctional protein via a disulfide bond as follows For example, the cysteme residue in one of these sequences (SEQ ID NO 1 1-16, 25) may participate in a disulfide bond with, for example, a cysteine in the mature protein, such as a cysteine corresponding the cysteine at residue 171 of SEQ ID NO 4 At least one of these sequences (SEQ ID NO 1 1 -16), 25) are therefore present in preferred embodiments of the invenUon See, for example. Figure 5. which shows the amino acid sequences of several proteins, namely, Factor VII. thrombin. kalhkrein, a Limulus pro-clotting enzyme from the Japanese horshoe horsehoe crab (Tachypleus tridentatus), plasmm. hepsin and Factor XII. aligned with the amino acid sequence of SEQ ID NO 4 All of the proteins aligned with the krill-deπved multifunctional protein, except for the Limulus protein and Hepsin. are involved m the human blood coagulation pathway

Without being limited to any particular theory, it is believed that kπll-denved multifunctional proteins include a larger N -terminus than that found in the first, second or the third isoform, SEQ ID NO 4, SEQ ID NO 5 or SEQ ID NO 8. respectively NOS 4, 5 or 8. 20. 22 or 24 The nucleic acids of the invention can encode engineered multifunctional proteins based on forming chimeπc polypeptides from the above isoforms. for example The hydrophobic sequence or the pro-protein segment of one naturally occurπng isoform can optionally be matched with the mature protein sequences of another naturally occurring isoform or isoforms For example, the mature protein segment of SEQ ID NO 4 is ammo acids 64-300 SEQ ID NO 5, for mstance. is a partial sequence of the second isoform, which has a mature protein sequence of about 75% of the length of the mature protein segment of SEQ ID NO 4 Therefore, certain embodiments of the mvention include a chimeπc polypeptide in which the N-terminus of the polypeptide of SEQ ID NO 5 is linked to the remaining 25% of the length of the mature protein sequence found in SEQ ID NO 4, namely ammo acids 64-116 In another embodiment of the invention, a hypothetical chimeπc sequence includes the first 63 amino acids of the protein of SEQ ID NO 4 together with the amino acid sequence of SEQ ID NO 5 See SEQ ID NO 6. which is a composite of the proteins of SEQ ID NO 4 and SEQ ID NO 5 See, for example. Figure 2, which aligns SEQ ID NO 4 with SEQ ID NO 5 The nucleic acid sequence corresponding to the amino acid sequence of SEQ ID NO 6 is provided in SEQ ID NO 3, which provides the first 344 nucleic acids of SEQ ID NO 1 together with the nucleic acid sequence of SEQ ID NO 2

Thus, the nucleic acids of the invention include nucleic acids that code for the mature protein, the protein including the pro-protem segment or the protein including the hydrophobic segment and the pro-protem segment, or portions thereof For example, the nucleic acid of the first isoform. SEQ ID NO 1. or the chimeπc molecule. SEQ ID NO 3, are nucleic acids encoding the pro-protein, including the hydrophobic sequence and the pro-protem segment The chimeπc molecule. SEQ ID NO 3, represents the first 344 nucleotides of SEQ ID NO 1. coding for the hydrophobic sequence and the pro-protein segment of the protein and the first 25% of the mature protein, together with the 599 nucleotides of SEQ ID NO 2, coding for the remaining 75% of the mature protein

Further, for example, the N-terminus of SEQ ID NO 8 can be attached to the mature protein sequences of SEQ ID NO 5, thereby forming a chimeπc polypeptide, shown in SEQ ID NO 10 The corresponding DNA sequence can be found SEQ ID NO 9

Alternatively, for example, ammo acid sequences of several isoforms can be used to create an engineered polypeptide For example, the chimeπc polypeptide of SEQ ID NO 6 can be further modified by adding to the N-terminus of the protein the amino acid sequence PGRSRIA, which is ammo acid residues 1-7 from the N-terminus of the third isoform. SEQ ID NO 10

Without being bound to a particular theory, it is believed that there are at least about 4 5 isoforms. each having a different ammo acid at die position corresponding to amino acid residue 68 of SEQ ID NO 4, including glutamine, methionine. lysine and asparagine Such isoforms and other homologous polypeptides can be isolated using the techniques descπbed under Section 5 below, entitled "Means for Identifying Polypeptides with Multifunctional Activity "

To construct engineered vaπants of multifunctional protein-encoding nucleic acids, the native sequences of any of the isoforms can be used as a starting point and modified to suit particular needs. For example, in certain embodiments, the nucleic acid sequence need not include the sequences encoding the 5' portion of the ammo acid sequence that is absent in the mature protein, including ammo acids 1-63 of SEQ ID NO 4 Thus, in certain embodiments of the invention, the encoded polypeptide is homologous to or has the sequence of the mature protein only, and not the segments corresponding to the N-terminal portions that are removed during cellular processing, namely, the hydrophobic sequence and the pro-protein segment

Nonetheless, in preferred embodiments of the nucleic acids of the invention, the sequences encoding the N-terminal portion of the ammo acid sequence that is absent in the mature protein including amino acids 1-63 of SEQ ID NO 4. are included m the nucleic acid sequences The amino acid sequence forming a synthetic multifunctional protein preferably includes an enzymatically active segment of a kπll-deπved multifunctional protein, such as amino acids 64- 300 of SEQ ID NO 4, particularly including the histidine at residue 104. the aspartic acid at residue 151 and the seπne at residue 246, which are implicated in the catalytic mechanism of seπnc proteases Thus, the protein need not include the I drophobic sequence or pro-protem segment that are present in a kπll-deπved protein before cellular processing occurs, although the hydrophobic sequence and the pro-protein segment are preferably present

Preferably, the nucleic acids will encode polypeptides having at least about 70% homology, more preferably, at least about 80% homolog\ . even more preferably, at least about 85% homology, yet more preferably at least about 90% homology. and most preferably at least about 95% homology to a reference protein or a krill-deπved multifunctional protem, such as the polypeptides of SEQ ID NO 4, SEQ ID NO 5. SEQ ID NO 6. SEQ ID NO 8 or SEQ ID NO 10. NOS 4, 5. 6. 8. 10, 20. 22 or 24 or amino acid sequences 64-300 of SEQ ID NO 4. or other naturally occurring isoforms Even more preferably, the nucleic acids will encode polypeptides sharing at least about 70% identity, more preferably, at least about 80% identity, yet more preferably, at least about 85% identity, still more preferably at least about 90% identity, and most preferably at least about 95% identity with a kπll-deπved multifunctional protein

Additionally, the invention includes a substantial ly pure nucleic acid comprising a nucleic acid that binds to a nucleic acid encoding a polypeptide having at least about 70% homology to a reference protem or a krill-deπved multifunctional protein Even more preferably, the nucleic acid binds to a nucleic acid encoding a polypeptide having at least about 80% homology. and more preferably, at least about 90% homology to a kπll-deπved mulϋfunctional prote Yet more preferably the nucleic acid binds to a nucleic acid encoding a polypeptide shaπng at least about 70% amino acid identity, and more preferably, at least about 80% ammo acid identity, and yet more preferably, at least about 90% amino acid identity with a krill-deπved multifunctional protem, such as the polypeptide of SEQ ID NO 4, SEQ ID NO 5. SEQ ID NO 6, SEQ ID NO 8 or SEQ ID NO 10 and especially, SEQ ID NO 4. SEQ ID NO 6 or SEQ ID NO 10 A nucleic acid that binds to a nucleic acid that encodes a polypeptide homologous to a krill-deπved multifunction protein can be used as a probe, for example, to identify additional multifunctional proteins or to determine multifunctional protein expression

Numerous methods for determining percent homology (such as similarity or identity) are known in the art One preferred method is to use version 6 0 of the GAP computer program for making sequence comparisons The program is available from the University of Wisconsin Genetics Computer Group and utilizes the alignment method of Needleman and Wunsch ./ Mol Biol 48 443 1970. as revised by Smith and Waterman Adv Appl Math 2 482, 1981

Numerous methods for determining percent identity are also known in the art. and a preferred method is to use the FASTA computer program, which is also available from the University of Wisconsin Genetics Computer Group

The mature protein of the polypeptide of SEQ ID NO 4 is about 61% identical to the chymotrypsin-Iike serine protemase in the shrimp Penaeus vannamei according to the sequence provided by Genbank (Mountain View, CA), database acquisition no X6641 . and about 60% identical to the collagenolytic seπne protemase in the fiddler crab Uca pugilator. according to the sequence provided by Genbank, database acquisition no U49931 The amino acid sequence of the pro-protein of SEQ ID NO 4 is about 53% identical to the precursor of the chymotrypsin-hke seπne protemase in the shπmp Penaeus vannamei, and about 51% identical to the precursor of the collagenolytic seπne protemase in the fiddler crab Uca pugilator Preferably, the nucleic acids encoding polypeptides having multifunctional activity are less than about 70% lden cal to the above-identified proteinases of Penaeus vannamei or Uca pugilator

In addition to nucleic acids encoding a multifunctional protein, the present invention includes nucleic acids encoding polypeptides that are homologous to a reference protein or a knll- deπved multifunctional protem or that share a percentage identity with a reference protem or a kπll-deπved multifunctional protein Further, the present invention includes nucleic acids that encode a portion of a multifunctional protem or a variant thereof, such as the enzymaticallv acUve portion of the protein or the portion of the protein that provides asialo GM_j ceramide binding activity

The invention also is directed to a nucleic acid encoding a kπll-deπved multifunctional protem that has at least one of the following activities chymotrypsin, trypsin. collagenase. elastase and exopeptidase activity or asialo GM_j ceramide binding activity Preferably, die encoded polypeptide will be effective to remove or inactivate a cell-surface adhesion molecule, and most preferably, the encoded polypeptide will be pharmaceutically effective

For identifying the active segment or segments of multifunctional protein, one approach is to take a multifunctional protem cDNA and create deletional mutants lacking segments at either the 5' or the 3' end by, for instance, partial digestion with S 1 nuclcase, Bal 31 or Mung Bean nuclease (the latter approach descπbed in literature available from Stratagcne. San Diego. CA in connection with a commercial deletion cloning kit) Alternatively the deletion mutants are constructed by subcloning restriction fragments of a multifunctional protein cDNA The deletional constructs are cloned into expression vectors and tested for their multifunctional activity These structural genes can be altered by mutagcnesis methods such as that described

Adelman et al , DNA. 2 183. 1983 or through the use of synthetic nucleic acid strands The products of mutant genes can be readily tested for multifunctional activity

The nucleic acid sequences can be further mutated for example, to incorporate useful restriction sites See Maniatis ct al Molecular Cloning a Laboratory Manual (Cold Spring Harbor Press, 1989) Such restriction sites can be used to create "cassettes", or regions of nucleic acid sequence that are facilely substituted using restriction enzymes and hgation reactions The cassettes can be used to substitute synthetic sequences encoding mutated multifunctional protein ammo acid sequences

The multifunctional protein-encoding sequence can be. for instance, substantially or fully synthetic See. for example. Goeddel et al , Proc Nail Acad Sci USA, 76, 106-1 10. 1979 For recombinant expression purposes, codon usage preferences for the organism in which such a nucleic acid is to be expressed are advantageously considered in designing a synthetic multifunctional protein-encoding nucleic acid Since the nucleic acid code is degenerate, numerous nucleic acid sequences can be used to create the same amino acid sequence Further, with an altered amino acid sequence, numerous methods are known to delete sequence from or mutate nucleic acid sequences that encode a polypeptide and to confirm the function of the polypeptides encoded by these deleted or mutated sequences Accordingly, the invention also relates to a mutated or deleted version of a multifunctional prote nucleic acid that encodes a polypeptide that retains multifunctional protein activity

Conservative mutations of the naturally occurring isoforms are preferred for engineered vaπants Such conservative mutations include mutations that switch one ammo acid for another within one of the following groups

1 Small aliphatic, nonpolar or slightly polar residues Ala. Ser, Thr. Pro and Gly .

2 Polar, negatively charged residues and their amides Asp, Asn, Glu and Gin.

3 Polar, positively charged residues His, Arg and Lys,

4 Large aliphatic, nonpolar residues Met. Leu, He. Val and Cys: and

5 Aromatic residues Phe. Tyr and Tip

A preferred listing of conservative substitutions is the following

The types of substitutions selected may be based on the analysis of the frequencies of ammo acid substitutions between homologous proteins of different species developed by Schulz et al . Principles of Protein Structure, Spπnger-Verlag. 1978, pp 14-16, on the analyses of structure- forming potentials developed by Chou and Fasman. Biochemistry 13, 21 1, 1974 or other such methods reviewed by Schulz et al. Principles in Protein Structure, Springer- Verlag. 1978, pp 108-130, and on the analysis of hydrophobicity patterns in proteins developed by Kyte and Doohttle, J Mol Biol 157 105-132, 1982 2. Polypeptides Polypeptides of the invention include all polypeptides having multifunctional activin whether native or synthetic, including but not limited to poKpeptidcs purified from a multifuncUonal protein-expressing organism A preferred embodiment of the invention provides a polypeptide comprising a substantially pure isoform of a reference protein or a kπll-deπved multifunctional protein or engineered variant thereof, and more preferably, a polypeptide comprising SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 SEQ ID NO 8 or SEQ ID NO 10

NOS 4. 5. 6. 8, 10, 20, 22 or 24 and especially , SEQ ID NO 4 SEQ ID NO 6 or SEQ ID NO 10 NOS 4, 6 1 or 22 Further, polypeptides of the invention preferably comprise at least one of the ammo acid sequences of SEQ ID NO NOS 1 1-16 and 25

In addition to the a reference protein or a multifunctional protein, and its their isoforms and portions thereof, the present invention includes poly peptides that are homologous to a reference protein or a krill-deπved multifunctional protein or that share a percentage ldentif. with a reference protein or a krill-deπved multifunctional protein Further, the present invention includes portions of the a reference protein or a multifunctional protein or a variant thereof, such as the enzymatically active portion of the protein or the portion of the protein that provides asialo GM j ceramide binding activity

Additionally, the present invention includes engineered vaπants of multifunctional proteins that retain multifunctional activity In certain embodiments, these engineered variants lack, for example, no more than about 63 amino acid residues at the N-termmal end of SEQ ID NO 4

Preferably, the vaπants will have at least about 70% homology, more preferably, at least about 80% homology. even more preferably, at least about 85% homology, still more preferably at least about 90% homology. and most preferably at least about 95% homology to a kπll-denved multifunctional protein, such as the polypeptides of SEQ ID NO 4. SEQ ID NO 5. SEQ ID NO 6. SEQ ID NO 8, SEQ ID NO 10, or other isoforms, or amino acid sequences 64-300 of SEQ ID NO 4 Even more preferably, the analogs will share at least about 70% identity, more preferably, at least about 80% identity, yet more preferably, at least about 85% identity, still more preferably at least about 90% identity, and most preferably at least about 95% identity with a krill-deπved multifunctional protein

Preferably, the polypeptide has the sequence of a contiguous stretch of at least about 237 ammo acids of the following mature proteins in SEQ ID NO 4, amino acid residues 64-300. in SEQ ID NO 6, amino acid residues 64-300, and in SEQ ID NO 10. ammo acid residues 72-308

Amino acid analogs of the above-descπbed polypeptides are also included m the present invention

Additionally, the present invention provides a pharmaceutical composition for treating an animal compnsing an effective amount of a polypeptide compπsing a substantially pure isoform of a krill-deπved multifunctional protem or engineered variant thereof and a pharmaceutically acceptable carrier More preferably, the polypeptide comprises SEQ ID NO 4, SEQ ID NO 5 SEQ ID NO 6. SEQ ID NO 8 or SEQ ID NO 10, and even more preferably, SEQ ID NO 4, SEQ ID NO 6 or SEQ ID NO 10, and the polypeptide preferably compπses at least one of the amino acid sequences of SEQ ID NO 1 1-16 3. Methods of Synthesizing Polypeptides

In one embodiment, the polypeptides of the invention are made as follows, using a gene fusion For example, fusion to maltose-binding protein ("MBP") can be used to facilitate the expression and purification of a multifunctional protein in a prokarvote such as E coli The hvbπd protem can be purified, for example, using affinity chromatography using the binding protein's substrate See, for example, Gent? 67 21-30 (1988) When using a fusion protein that includes maltose binding protein, a cross-linked amylose affinity chromatography column can be used to punfy the protein

The cDNA specific for a given multifunctional protein or analog thereof can also be linked using standard means to a cDNA for glutathione s-transferase ("GST"), found on a commercial vector, for example The fusion protein expressed by such a vector construct includes the multifunctional protem or analog and GST. and can be treated for puπfication Should the MBP or GST portion of the fusion protein interfere with function, it is removed by partial proteolytic digestion approaches that preferentially attack unstructured regions such as the linkers between MBP or GST and the multifunctional protem The linkers are designed to lack structure, for instance using the rules for secondary structure-forming potential developed by Chou and Fasman. Biochemistry 13. 21 1. 1974 The linker is also designed to lncorporatc protease target amino acids, such as trypsm. arginme and lysine residues To create the linkers, standard synthetic approaches for making oligonucleotides are employed together with standard subcloning methodologies Other fusion partners other than GST or MBP can also be used Additionally, the multifunctional proteins can be directly synthesized from nucleic acid (by the cellular machinery) without use of fusion partners For instance, nucleic acids having the sequence of SEQ ID NO 1, SEQ ID NO 3 or SEQ ID NO 9 are subcloned into an appropπate expression vector having an appropπate promoter and expressed m an appropriate organism Antibodies against the kπll multifunctional protein can be employed to facilitate purification Additional purifications techniques are applied as needed, including without limitation, preparative electrophoresis, FPLC (Pharmacia, Uppsala. Sweden). HPLC (e g . using gel filtration, reverse-phase or mildly hydrophobic columns), gel filtration, differential precipitation (for instance, "salting out" precipitations), ion-exchange chromatography and affinity chromatography (including affinity chromatography using the RE1 duplex nucleotide sequence as the affinity gand).

A polypeptide or nucleic acid is "isolated" in accordance with the invention in that the molecular cloning of the nucleic acid of interest, for example, involves taking a multifunctional protem nucleic acid from a cell, and isolating it from other nucleic acids This isolated nucleic acid may then be inserted into a host cell, which may be yeast or bacteria, for example A polypeptide or nucleic acid is "substantially pure" in accordance with the invention if it is predominantly free of other polypeptides or nucleic acids, respectively A macromolecule. such as a nucleic acid or a polypeptide. is predominantly free of other polypeptides or nucleic acids if it constitutes at least about 50% by weight of the given macromolecule in a composition Preferably . the polypeptide or nucleic acid of the present invention constitutes at least about 60% by weight of the total polypeptides or nucleic acids, respectively, that are present in a given composition thereof, more preferably about 80%. still more preferably about 90%, yet more preferably about 95%. and most preferably about 100% Such compositions are referred to herein as being polypeptides or nucleic acids that are 60% pure. 80% pure. 90% pure. 95% pure, or 100% pure. any of which are substantially pure 4. Preferred Characteristics of the Multifunctional Protein

Knll. including without limitation kπll of the genuses Euphasia (such as superba. crystallorphias.frigida. triacantha. vellantmi. lougirostns. lucens. similis. spimfera. recurva and the like). Meganyctiphanes (such as norvegica and the like) and Tysanoessa (such as macurura. vicma, gregana and the like), are a preferred source of kπll-deπved multifunctional proteins

Preferably, the protein has a molecular weight between about 20 kd and about 40 kd. and more preferably from about 26 kd to about 32 kd, and most preferably about 29 kd, as determined by sodium dodecyl sulfate ("SDS") polyacrylamide gel electrophoresis ("PAGE") Further, the protem preferably has substantial homology to a kπll-deπved multifunctional protein Preferred proteins are hydrolases. and preferably, proteases Preferably, the protein is selectively reactive with cell-surface receptors such as polypeptides or glycolipids

Protease activity can be determined by incubating a protein preparation with casein (concentration 1 % w/v) at 30 C for 20 hours and measuring the release of amino acids or peptides (which can be measured by the increase in colorometπcally determinablc amino groups) Isolated multifunctional protein of 95% puπty will typically have a specific activity of at least about 25 Casein Units per mg Casein Units are defined in Biochem J , 173 291-298, 1978 (using azocasein as the substrate) Alternatively, tryptic protease activity can be measured against tyrosine-arginine-methyl- ester ("TAME") The multifunctional protein (of at least about 95% purity) will preferably have specific activity of at least about 60 TAME Units per mg Or, tryptic activity can be measured using Benzoyl-Val-Gly-Arg-p-Nθ2-anιlιde as the substrate Using this substrate and the method of Biochemical J . 185 423-433. 1980. the multifunctional protein will preferably have specific activity of at least about 210 Units per mg Chymotryptic activity can be measured using

Succmyl-Ala-AIa-Pro-Phe-p-Nθ2-anιlιde as the substrate Using this substrate and the method of

./ Biol Chem . 269 19565-19572, 1994. the multifunctional protein will preferably have specific activity at least about 260 Units per mg Elastase activity can be measured using Boc-Ala-Ala- Pro-Ala-p-Nθ2-anιhde as the substrate Using this substrate and the method of J Biol Chem . 269 19565-19572, 1994, the multifunctional protein will preferably have specific activity of at least about 270 Units per mg

Generally, the multifunctional protein will be sufficiently stable so that at least about 50% of the proteolytic activity is retained after incubation at 50° C for 24 hours at pH 7 0 at a concentration of 5 mg/ml Preferably at least about 50% of the proteolytic activity is retained after incubation at 60° C for 5 hours at pH 7 0 at a concentration of 5 mg/ml

Preferably, the pH optimum of the multifunctional protein is substrate dependent For the substrate azocasein. the pH optimum is preferably from about 3 5 to about 6 5. more preferably from about 4 0 to about 6 0 For the substrate Benzoyl-Val-GK-Arg-p-nitroanihde. the pH optimum is preferably in excess of about 8 0, more preferably in excess of about 9 0 For the substrate Boc-Ala-Ala-Pro-Ala-p-nitroanihdc. the pH optimum is preferably between about 6 0 and about 7 0, more preferably about 7 0

Using Benzoyl-Val-Gly-Arg-p-nitroanilide as the substrate, the K_m at about pH 9 5 in the presence of 2 mM Ca^⁺ is preferably between about 200 and about 240 μM Using Succ yl- Ala-Ala-Pro-Phe-p-nitroanihde as the substrate, the K_m at pH 9 5 in the presence of 2 mM Ca^⁺ is preferably between about 250 and about 290 μM

Preferably, the multifunctional protein has a temperature optimum for activity against casein of between about 45° C and about 60° C Generally the protein retains at least about 50% of its activity when incubated at 5 mg/ml for 18 hours at a pH ranging from about 5 0 to about 9 5 at 25 C

When HL60 cells are pretreated with kπll-deπved multifunctional hydrolase. their binding to TNF stimulated endothehal cells is inhibited by more than about 60% Preferably, treatment of HL60 or endothehal cells with the multifunctional protein of the invention will inhibit HL60 cell binding to TNF stimulated endothehal cells by at least about 20%. more preferably at least about 40%. still more preferably at least about 60%. yet more preferably at least about 80% Alternately, the multifunctional protein will preferably have at least about 30% of the adhesion- lnhibiting activity of the krill-deπved multifunctional hydrolase More preferably, the multifunctional protein shall have at least about 60% of the adhesion inhibiting activity of the kπll- deπved multifunctional hydrolase, still more preferably at least about 80%, yet more preferably at least about 100%

The multifuncUonal protein of the invention effectively removes or inactivates certain cell- surface adhesion molecules, such as ICAM-1 (t e , CD 54). ICAM-2. VCAM-1, CD4. CD8. CD28, CD31, CD44 and the asialo GM_j ceramide. without affecting cell viability This adhesion site removal or inactivation phenomenon is believed to provide at least a partial explanation for the protein's effectiveness against many, though probably not all. of the indications against which the multifunctional protem is effective as a treatment or preventative agent Other cell surface receptors have neen found to be substantially resistant to removal or inactivation by the multifunctional protem, such as the T-cell receptor, the Class I major histocompatibility complex or the mtegπns CD 1 1 and CD 18 5. Means for Identifying Polypeptides with Multifunctional Activity

In one aspect, the present invention provides methods for identifying polypeptides that are homologous to the multifunctional protein Such polypeptides may be found, for example, in fish and crustaceans The method by which multifunctional protein cDNA was isolated illustrates how readily multifunctional proteins are identified For instance, see Example 1 The same methodology can be used to identify other sequences from other sources that have multifunctional activity

Additionally, probes for multifunctional protein expression can be used, for example, to detect the presence of a multifunctional protem Such probes include antibodies directed against multifunctional protein or fragments thereof, nucleic acid probes that hybπdize to multifunctional protein mRNA under stπngent conditions, and oligonuclcotides that specifically prime a PCR amplification of multifunctional protein mRNA Nucleic acid molecules that bind to a multifunctional protein-encoding nucleic acid under high stringency conditions are identified functionally, or by using the hybπdization rules reviewed in Sambrook et al . Molecular Cloning A Laboratory Manual, 2nd ed . Cold Spπng Harbor Press. 1989

Many deletional or mutational analogs of nucleic acid sequences for a multifunctional protein are effective hybridization probes for multifunctional protein-encoding nucleic acid Accordingly, the present invention relates to nucleic acids that hybridize with such multifunctional prote -encodmg nucleic acids under stringent conditions Preferably, the nucleic acid of the present invention hybridizes with at least a segment of the nucleic acid described as SEQ ID NO 1. SEQ ID NO 2, SEQ ID NO 3. SEQ ID NO 7 or SEQ ID NO 9 NOS 1. 2, 3, 7. 9. 19. 21 or 23 under stπngent conditions

"Stπngent conditions" refers to conditions that allow for the hybridization of substantially related nucleic acids, where relatedness is a function of the sequence of nucleotides in the respective nucleic acids For mstance. for a nucleic acid of 100 nucleotides. such conditions will generally allow hybridization thereto of a second nucleic acid havmg at least about 85% homology. and more preferably having at least about 90% homology Such hybridization conditions are descπbed by Sambrook et al . Molecular Cloning A -Laboratory Manual. 2nd ed . Cold Spr g Harbor Press, 1989 PCR (polymerase chain reaction) can be used to detect nucleic acids having multifunctional protein sequences through amplification of such sequences using multifunctional protein nucleic acid primers PCR methods of amplifying nucleic acids utilize at least two pπmers one pπmer and often at least two One of these pπmers is capable of hybridizing to a first strand of the nucleic acid to be amplified and of pπming protem-dπven nucleic acid synthesis in a first direction The other is capable of hybridizing the reciprocal sequence of the first strand (if the sequence to be amplified is smgle stranded, this sequence is initially hypothetical, but is synthesized in the first amplification cycle) and of pπming nucleic acid synthesis from that strand in the direction opposite the first direction and towards the site of hybridization for the first pπmer Conditions for conducting such amplifications, particularly under preferred high stringency conditions, are well known See, for example, PCR Protocols, Cold Spring Harbor Press, 1991

Antibodies against multifunctional proteins can also be used to identify polypeptides that are homologous to multifunctional protein Antigens for eliciting the production of antibodies against the a reference protein or a multifunctional protem can be produced recombmantly by expressing all of or a part of the nucleic acid of a reference prote or a multifunctional protein in a prokaryote such as bacteria or a eukaryote such as yeast In one embodiment, the recombinant protein is expressed as a fusion protein, with the non-multifunctional functional protein portion of the protein serving either to facilitate purification or to enhance the immunogenicity of the fusion protein For mstance, the non-multifunctional functional protein portion comprises a protein for which there is a readily-available binding partner that is utilized for affinity puπfication of the fusion protem The antigen includes an "antigenic determinant," I e . a minimum portion of ammo acids sufficient to bind specifically with an anti-multifunctional protein antibody

Antisera to a reference protein or a reference protein or a multifunctional protein can be made, for example, by creating a multifunctional protein antigen by linking a portion of the cDNA for human multifunctional protein to a cDNA for glutathione s-transfcrase ("GST") found on a commercial vector The resulting vector expresses a fusion protein containing an antigenic segment of multifunctional protein and GST that is readily purified from the expressing bacteria using a glutathione affinity column The purified antigenic fusion protein is used to immunize rabbits The same approach is used to make antigens based on other segments of the multifunctional protem Procedures for making antibodies and for identifying antigenic segments of proteins are well known See. for instance, Harlow, Antibodies. Cold Spring Harbor Press. 1989 6. Gene Therapy

The invention also encompasses the use of gene therapy approaches to insert a gene expressing a multifunctional protem or a polypeptide with multifunctional protein activity For gene therapy, medical workers prefer to incorporate, into one or more cell types of an organism, a DNA vector capable of directing the synthesis of a polypeptide missing from the cell or useful to the cell or organism when expressed in greater amounts The methods for introducing DNA to cause a cell to produce a new polypeptide or a greater amount of a polypeptide are called "transfection" methods See, generally, Sambrook et al , Molecular Cloning A Laboratory Manual, 2nd cd , Cold Spring Harbor Press, 1989

For viral gene therapy vectors, dosages are generally from about 1 g to about 1 mg of nucleic acid per kg of body mass For non-infective gene therapy vectors, dosages are generally from about 1 g to about 100 mg of nucleic acid per kg of body mass 7. Routes of Administration

The multifunctional protein polypeptides and nucleic acid compositions of the invention can be administered orally, topically, rectally, vaginally. by instillation (for instance into the urinary tract or into fistulas), by pulmonary route by use of an aerosol, by application of drops to the eye. or systemically. such as parenterally, including, for example, intramuscularly, subcutaneously, intraperitoneally. lntraarteπally or intravenously The multifunctional protein composition can be administered alone, or it can be combined with a pharmaceutically-acceptable earner or excipient according to standard pharmaceutical practice For the oral mode of administration, the multifunctional protein composition can be used in the form of tablets. capsules, lozenges, chewing gum. troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like In the case of tablets, carriers that is used include lactose, sodium citrate and salts of phosphoric acid Various dismtegrants such as starch, and lubricating agents such as magnesium stearatc and talc, are commonly used in tablets For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols If desired, certain sweetening and/or flavoring agents are added For parenteral administration sterile solutions of the multifunctional protein are usually prepared, and the pHs of the solutions arc suitably adjusted and buffered For intravenous use. the total concentration of solutes should be controlled to render the preparation isotonic For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or polyvinyl alcohol, preservatives such as sorbic acid EDTA or benzylchromum chlonde, and the usual quantities of diluents and/or carriers For pulmonary administration, diluents and or carriers will be selected to be appropriate to allow the formation of an aerosol For topical administrations, the multifunctional protein is typically administered m aqueous form or m a hydrogel A preferred hydrogel comprises an aqueous suspension of from about 1% (w/v) to about 10% of low molecular weight hydrolyzed starch

Suppository forms of the multifunctional protein are useful for vaginal, urethral and rectal administrations Such suppositories will generally be constructed of a mixture of substances that is solid at room temperature but melts at body temperature The substances commonly used to create such vehicles include theobroma oil, glyceπnated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weighty and fatty acid esters of polyethylene glycol. See. Remington's Pharmaceutical Sciences, 16th Ed.. Mack Publishing. Easton. PA. 1980, pp. 1530-1 33 for further discussion of suppository dosage forms Analogous gels or cremes can be used for vaginal, urethral and rectal administrations

Numerous administration vehicles will be apparent to those of ordinary skill in the art. including without limitation slow release formulations, hposomal formulations and polymenc matrices.

For topical treatments, a suitable dose of multifunctional protein per application ranges from about 0.1 μg/cm^ to about 1 mg/cm preferably from about 1 μg/cm^ (for example, using about 10 μg/ml) to about 1 mg/cm^ (for example, using about 10 mg/ml). more preferably from about 5 μg cm^ (for example, using about 50 μg/ml) to about 100 μg/cm^ (for example, using about 1 mg/ml), yet more preferably from about 1 μg/cm^ to about 250 μg/cm^. still yet more preferably from about 10 μg/cm^ (for example, using about 100 μg/ml) to about 50 μg/cm^ (for example, about 500 μg/ml). For systemic treatments, dosages ill generally be selected to maintain a serum level of multifunctional protein between about 0 1 μg/lOOcc and about 5 μg/lOOcc. preferably between about 0.5 μg lOOcc and about 2 0 μg/l OOcc In an alternative measure of preferred systemic administration amounts, preferably from about 0 1 mg/kg to about 10 mg/kg, more preferably about 1 mg/kg. will be administered (although toxicology in animal models suggests that in excess of 25 mg/kg is acceptable) For ocular treatments, a suitable dose of multifunctional protein per application ranges from about 0 01 mg per eye to about 5 mg per eye, preferably from about 0.1 mg per eye to about 2 0 mg per eye. For vaginal and urinary tract treatments, suitable flushing/ instillation solutions of the multifunctional protein will generally have concentrations from about 1 μg/ml to about 15 mg/ml. preferably from about 100 μg/ml to about 3 mg/ml. For oral treatments, suitable mouthwash solutions will generally have concentration of multifunctional protein from about 1 mg/ml to about 15 mg/ml preferably from about 2 mg ml to about 10 mg/ml. Lozenges will typically contain from about 100 μg to about 1 mg of multifunctional protein. Aerosols will generally be made from solutions having protein concentrations from about 0.1 mg/ml to about 15 mg/ml. preferably from about 1 mg ml to about 10 mg/ml. Generally, from about 0.1 ml to about 2 ml of aerosol will be applied to the airways of the patient, preferably from about 0.5 ml to about 1.0 ml For scar and keloid treatments, generally between about 0.1 mg and about 5 mg of multifunctional protein will be injected into each enr of the lesion, preferably from about 0.5 mg to about 3 mg For treating adhered connective tissue or joints, generally between about 0 5 mg and about 10 mg of multifunctional protein will be injected interstitial ly at the adhesion, preferably between about 1 mg and about 5 mg For all treatments, the protein composition will generally be applied from about 1 to about 10 times per day, preferably from about 2 to about 5 times per day These values, of course, will vary with a number of factors including the type and seventy of the disease, and the age. weight and medical condition of the patient, as will be recognized by those of ordinary skill in the medical arts It is beiieved that substantially higher doses can be used without substantial adverse effect

For treating or preventing infection, the multifunctional protein can be administered systemically or in a manner adapted to target the affected tissue For preventing cold or influenza transmission, the composition is preferably applied to the lungs or airways For treating immune disorders, the composition may be applied systemically or in a manner adapted to target the affected tissue For treating the primary and secondary mfections of leprosy, the primary administration route will generally be the topical route For treating scar or keloid tissue, generally the composition will be injected into the scar or keloid. except that for corneal scars the composition will generally be applied ocularly without injection For cancer treatment, the composition will generally be administered systemically by a route or m a manner adopted to target the affected tissue For treating atherosclerosis, the composition will generally be administered systemically. although the site of administration mav be chosen to administer the highest dosages to the portion of the circulatory system most at πsk For asthma, the general route of administration will be pulmonary For treating pseudomonas infections, the infection will typically be a lung infection and the administration route pulmonary For reperfusion injury, the composition will generally be administered systemically. although the site of administration may be designed to administer the highest dosages to the portion of the body that suffered an ischemic event For treating the painful symptoms of malaria, the administration mode will generally by systemic

For wound healing, the multifunctional protein is preferably be applied more often than simply the time at which the wound is first dressed Preferably, the multifunctional protein is applied at least about every time the wound dressing is changed The multifunctional protein can also be applied at least about every other day, more preferably, every day In one embodiment, the multifunctional protem is administered to a wound substantially free of necrotic tissue The phrase "substantially free of necrotic tissue" shall mean sufficiently lacking in necrotic tissue so that an ordinarily-skilled pathologist would consider any residue of necrotic tissue to be irrelevant to determining a wound-healing prognosis For organ transplants, the organ to be transplanted will preferably be bathed in a solution of the multifunctional protem for between about 10 minutes and about 5 hours The protein solution will preferably contain between about 0 01 mg/ml and about 25 mg ml of the multifunctional protein, more preferably, between about 0 5 mg/ml and about 5 mg/ml After transplantation, the multifunctional protem will preferably be administered systemically us g the conditions descπbed above

For cleaning contact lenses in situ the solutions descπbed above for ocular treatments are preferred, For ex vivo treatments, higher concentrations of protein will generally be used Cleaning incubations of from about 5 to about 30 minutes at from about 20 °C to about 50 °C are also preferred For ex vivo treatments, the higher end of the temperature range is preferred

For leprosy, many of the associated infections will be appropπately treated with a topical application of the multifunctional protein For CF or COPD. the multifunctional protein can be used to treat (a) the build up of viscous fluids in the lungs and (b) associated pulmonary infections Preferably, treatments of CF and COPD patients include pulmonary treatments with an aerosol of the multifunctional protein, but can include other routes of administration including systemic administrations

Particularly important among the diseases relevant to the transmission inhibitory embodiment of the invention are sexually-transmitted diseases, such as Candida, gonorrhea, chlamydia. syphilis, tπchomonas. chancroid, HIV, herpes or hepatitis mfections Among these viral diseases are particularly preferred targets for transmission prevention. HIV is a still more preferred target For this use. the body cavity involved in sexual activity is generally rinsed or flushed with a composition containing the multifunctional protem. or a creme, gel or suppository designed to localize the composition to the body cavity is used The composition can be used soon before, in conjunction with, or soon after, sexual activity, although prior or concurrent use is preferred

For herpes infections, the viral targets include HSV-1. which pπmanly manifests as oral herpes, HSV-2, which pπmanly manifests as genital herpes, and herpes zoster

For autoimmune diseases or diseases with autoimmune components, treatment targets include without limitation rheumatoid arthritis, multiple sclerosis, primary biliary cirrhosis, active chronic hepatitis, ulcerative colitis, rheuma c arthπtis. scleroderma. systemic lupus erythematosus, Hashimoto's thyroiditis, pπmary myxedema. thyroroxicosis, pernicious anemia Addison's disease, premature onset of menopause, autoimmune male infertility, insulin-dependent diabetes, type B insulin resistance of acanthosis nigπcans. alopic allergy, myasthenia gravis. Lambert-Eaton syndrome. Goodpasture's syndrome, pemphigus vulgaπs, pemphigoid phacogenic uveitis, sympathetic ophthalmia, autoimmune hemolytic anemia, ldiopathic thrombocytopenic puφura, Sjogren's syndrome, discoid lupus erythematosus. dermatomyositis and mixed connective tissue disease

For adhesion disorders, the cells or viruses involved can include, without limitation. endothehal cells, lymphocytes, including T-cells, tumor cells, microbial cells, viruses, including HIV and herpes Adhesion processes are believed to be involved in tissue invasion, for instance. by immune cells, microbes, and tumor cells

For many of diseases for which the multifunctional protein of the invention is useful as a prophylactic treatment- including those not caused by microbes, a patient's medical history, lifestyle or genetic background will often indicate a predisposition to acquire the disease This is true, for instance, of atherosclerosis

Generally, the multifunctional protein will be administered in an effective amount An effective amount is an amount effective to either ( 1 ) reduce the symptoms of the disease sought to be treated, (2) induce a pharmacological change relevant to treating the disease sought to be treated, (3) inhibit or prevent infection or re-infection by an infective agent, or (4) prevent the occurrence of a non-mfectious disease (for instance a disease treatable by blocking a cell adhesion phenomenon) For cancer, an effective amount further includes an amount effective to prevent or limit metastasis, for instance, to reduce the level of metastasis, reduce the size of a tumor, slow the growth of a tumor, and increase the life expectancy of the affected animal For wound treatment. one aspect, an effective amount includes an amount which, if regularly applied, prevents the occurrence of infection In another aspect, for wound healing, an effective amount includes an amount effective to reduce the average time it takes for a wound to heal

Humans are the preferred subjects for treatment However, the multifunctional protein can be used in many veteπnary contexts to treat animals, preferably to treat mammals, as will be recognized by those of ordinary skill in light of the present disclosure

The present mvention is further exemplified by the following non-limiting examples Example 1. Cloning of PHIM Polypeptide

The PHIM polypeptide was puπfied and the polypeptide was partially sequenced, as descπbed in U S patent application Seπal No 08/600.273 (filed February 8, 1996). deFaire et al . inventors, entitled "Multifunctional Enzyme " Degenerate ohgonucleotide pπmers were constructed based on the partial ammo acid sequence The pπmers had the following sequences CACGCCTACCCITGGCA (SEQ ID NO 17) and GTGTTGGACTCGATCCAGATC (SEQ ID NO- 18) The pπmers were used to screen a kπll cDNA library that was constructed in lambda zap. using the lambda zap cDNA synthesis kit (Stratagene. San Diego. CA) Three positive clones were identified through screening with a PCR fragment as a probe The PCR fragment used as a probe was sequences 217 to 881 of SEQ ID NO 1 , with the following changes at 219, T to C. at 222. T to C, at 228, C to G, at 270, T to A, at 330, G to A. at 417. C to A, at 534. T to C. at 741, C to T, and at 825. C to G The three positive clones were sequenced, the first clone resulting in SEQ ID NO.1. the second clone resulting in SEQ ID NO 2 and the third clone resulting in SEQ ID NO 7 These isoforms all lack the initiation codon methionine Example 2. Expression of Recombinant Multifunctional Protein Enzyme

A recombinant multifunctional protein was expressed in an E cob as follows, using the BamHI and Xho I sites of a pET23c vector provided by Novagen (Abingdon. Oxford. U K ) The pET23c vector includes a gene 10 tag for facilitating puπfication of the expressed recombinant protein Further, the pET vector places the recombinant multifunctional protein under the control of bactcπophage T7 transcription and translation signals Once established in a non-expression host. E coli MC 1061. the plasmid was then transferred to an expression host. E coli BL21 (DE3 ) pLYS S having a chromosomal copy of the T7 polymerase gene under lacUV5 control Expression was induced by the addition of 1 mM IPTG at an optical density of 0 5 at wavelength 600 The cells were harvested after 2 hours at an optical density of 1 0 The recombinant protein was insoluble in the lysate and after harvesting, it was washed and dissolved in 6 M urea Refolding of the recombinant protem was carried out by 200-fold dilution using a buffer containing 100 mM tπs HCl pH 9 5, 100 mM CaCl2 0 3 mM oxidized glutathione and 3 mM reduced glutathione. followed by stirπng overnight at 4 C Example 3.

A fragment from the p62 clone that encodes ammo acids 56 to 300 and the stop codon (see SEQ ID NOs 1 an 4) was excised and inserted it into a pET-23c expression vector Note that this clone excludes the cysteine required to form a disulfide link to a light chain The encoded protem was expressed in Ecoli strain BL21(DE3)pLysS. which yielded an insoluble material The insoluble material was dissolved m 6 M urea, and re-folded by a 200-fold dilution into an aquesous solution buffered at pH 9 5. containing 0 1 mM CaCl and oxidized reduced glutathione The resulting solution was concentrated to recover the recombinant protein

The recombinant protein was shown to cleave the model substrate succinyl-ala-ala-pro- phe- ?-nιtroanihde, thereby demonstrating its proteolytic actιvιt\ The proteolytic activity was inhibited by the protease inhibitor Eghn

The nucleic acid or amino acid sequences referred to herein by SEQ ID NOs are as follows

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(A) APPLI CATION NUMBER :

( B ) FILING DATE :

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/705,875

(B) FILING DATE: 28-AUG-1996

(A) APPLICATION NUMBER: US 08/768,318

(B) FILING DATE: 17-DEC-1996 (vm) ATTORNEY/AGENT INFORMATION:

(A) NAME: Bloom, Allen

(B) REGISTRATION NUMBER: 29,135

(C) REFERENCE/DOCKET NUMBER: 314572-102WO (lx) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 609-520-3214

(B) TELEFAX: 609-520-3259

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO:l: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 943 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CTCTTACTCG CCCTTGTGGC TGCTGCTAGT GCCGCAGAAT GGCGCTGGCA GTTTCGTCAC 60

CCTACAGTGA CCCCCAACCC TAGGGCTAAG AACCCCTTCA GAGTCACCAA AAGCTCTCCA 120

GTCCAACCAC CAGCAGTCAG AGGAACAAAG GCTGTTGAGA ACTGTGGACC AGTAGCACCA 180

AGGAACAAGA TTGTAGGAGG CATGGAGGTG ACTCCCCATG CTTACCCCTG GCAGGTGGGA 240

CTTTTCATTG ATGATATGTA CTTCTGTGGT GGATCAATCA TCTCCGACGA ATGGGTCCTT 300

ACAGCTGCTC ACTGTATGGA TGGTGCTGGG TTTGTTGAGG TTGTGATGGG TGCTCACAGT 360

ATCCATGACG AAACTGAGGC CACACAGGTC CGTGCCACAT CAACTGATTT CTTCACCCAC 420

GAGAACTGGA ACTCCTTCAC CCTCTCCAAT GATCTTGCTC TCATTAAGAT GCCAGCACCA 480

ATTGAATTCA ACGATGTGAT CCAGCCTGTC TGCCTACCAA CCTATACTGA TGCTAGTGAT 540

GATTTTGTTG GTGAATCAGT CACTCTTACT GGATGGGGTA AACCATCTGA CTCTGCTTTT 600

GGCATCGCTG AACAACTTCG TGAGGTTGAT GTGACAACAA TCACTACTGC TGACTGCCAG 660

GCATACTACG GCATTGTCAC TGACAAAATC CTCTGCATCG ACTCCGAAGG AGGCCATGGT 720

TCCTGCAATG GTGATTCCGG CGGGCCAATG AACTATGTAA CTGGTGGTGT TACTCAGACC 780

CGTGGTATTA CCTCTTTCGG ATCCTCTACC GGCTGCGAGA CTGGCTACCC TGATGGTTAC 840

ACACGAGTCA CCAGCTATCT GGACTGGATT GAATCTAACA CTGGCATTGC CATTGATCCA 900

TAAATACAAT TCTAGCAAAA ATACAATAAA TTATACTTAA ATG 943

(2) INFORMATION FOR SEQ ID NO: 2: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 599 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

GATGGGTGCT CACAGTATCC ATGACGATAC TGAGGCCTCT CGCGTCAGTG CCACATCAAC 60

TGATTTCTTC ACCCACGAGA ACTGGAACTC CTTCACCCTC ACCAATGATC TTGCTCTCAT 120

TAAGATGCCA GCACCAATTG AATTCACACC TGAAATTCAA CCTGTCTGCC TACCAAGCTA 180

CACTGATGCT GCTGATGATT TCATTGGTGA ATCTGTTGTC CTTACTGGAT GGGGCCGTGA 240

TTCTGATGCT GCTTCCGGCA TCTCTGAACT ACTCCGTGAG GTTCATGTGA CCACAATCTC 300

CACTGCCGAC TGCCAGGCAT ACTACGGCAT TGTCACTGAC AAAATCCTCT GCATTTCCTC 360

TGAAGACGGA CATGGTTCTT GTAATGGTGA TTCCGGTGGG CCAATGAACT ATGTAACTGG 420

TGGTGTTACT CAGACCCGTG GTATTACCTC CTTCGGATCC TCTACCGGGT GTGAGACTGG 480

CTACCCTGAT GGTTACACAC GTGTCACCAG CTATCTGGAC TGGATTGAAT CTAACACTGG 540

CATTGCCATT GATGCTTGAA TATAATACTA GATATGTAAT CAAATAAATT TCATGAATT 599

(2) INFORMATION FOR SEQ ID NO: 3: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 943 base pairs

(B) TYPE: nucleic ac d

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

CTCTTACTCG CCCTTGTGGC TGCTGCTAGT GCCGCAGAAT GGCGCTGGCA GTTTCGTCAC 60

CCTACAGTGA CCCCCAACCC TAGGGCTAAG AACCCCTTCA GAGTCACCAA AAGCTCTCCA 120

GTCCAACCAC CAGCAGTCAG AGGAACAAAG GCTGTTGAGA ACTGTGGACC AGTAGCACCA 1B0

AGGAACAAGA TTGTAGGAGG CATGGAGGTG ACTCCCCATG CTTACCCCTG GCAGGTGGGA 240

CTTTTCATTG ATGATATGTA CTTCTGTGGT GGATCAATCA TCTCCGACGA ATGGGTCCTT 300

ACAGCTGCTC ACTGTATGGA TGGTGCTGGG TTTGTTGAGG TTGTGATGGG TGCTCACAGT 360

ATCCATGACG ATACTGAGGC CTCTCGCGTC AGTGCCACAT CAACTGATTT CTTCACCCAC 420

GAGAACTGGA ACTCCTTCAC CCTCACCAAT GATCTTGCTC TCATTAAGAT GCCAGCACCA 480

ATTGAATTCA CACCTGAAAT TCAACCTGTC TGCCTACCAA GCTACACTGA TGCTGCTGAT 540

GATTTCATTG GTGAATCTGT TGTCCTTACT GGATGGGGCC GTGATTCTGA TGCTGCTTCC 600

GGCATCTCTG AACTACTCCG TGAGGTTCAT GTGACCACAA TCTCCACTGC CGACTGCCAG 660

GCATACTACG GCATTGTCAC TGACAAAATC CTCTGCATTT CCTCTGAAGA CGGACATGGT 720

TCTTGTAATG GTGATTCCGG TGGGCCAATG AACTATGTAA CTGGTGGTGT TACTCAGACC 780

CGTGGTATTA CCTCCTTCGG ATCCTCTACC GGGTGTGAGA CTGGCTACCC TGATGGTTAC 840

ACACGTGTCA CCAGCTATCT GGACTGGATT GAATCTAACA CTGGCATTGC CATTGATGCT 900

TGAATATAAT ACTAGATATG TAATCAAATA AATTTCATGA ATT 943

(2) INFORMATION FOR SEQ ID NO: 4: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 300 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Leu Leu Leu Ala Leu Val Ala Ala Ala Ser Ala Ala Glu Trp Arg Trp

1 5 10 15

Gin Phe Arg H s Pro Thr Val Thr Pro Asn Pro Arg Ala Lys Asn Pro

20 25 30

Phe Arg Val Thr Lys Ser Ser Pro Val Gin Pro Pro Ala Val Arg Gly

35 40 45

Thr Lys Ala Val Glu Asn Cys Gly Pro Val Ala Pro Arg Asn Lys lie

50 55 60

Val Gly Gly Met Glu Val Thr Pro His Ala Tyr Pro Trp Gin Val Gly

65 70 75 80

Leu Phe lie Asp Asp Met Tyr Phe Cys Gly Gly Ser lie lie Ser Asp

85 90 95 Glu Trp Val Leu Thr Ala Ala His Cys Met Asp Gly Ala Gly Phe Val

100 105 110

Glu Val Val Met Gly Ala His Ser He His Asp Glu Thr Glu Ala Thr

115 120 125

Gin Val Arg Ala Thr Ser Thr Asp Phe Phe Thr His Glu Asn Trp Asn

130 135 140

Ser Phe Thr Leu Ser Asn Asp Leu Ala Leu He Lys Met Pro Ala Pro 145 150 155 160

He Glu Phe Asn Asp Val He Gin Pro Val Cys Leu Pro Thr Tyr Thr

165 170 175

Asp Ala Ser Asp Asp Phe Val Gly Glu Ser Val Thr Leu Thr Gly Trp

180 185 190

Gly Lys Pro Ser Asp Ser Ala Phe Gly He Ala Glu Gin Leu Arg Glu

195 200 205

Val Asp Val Thr Thr He Thr Thr Ala Asp Cys Gin Ala Tyr Tyr Gly

210 215 220

He Val Thr Asp Lys He Leu Cys He Asp Ser Glu Gly Gly His Gly 225 230 235 240

Ser Cys Asn Gly Asp Ser Gly Gly Pro Met Asn Tyr Val Thr Gly Gly

245 250 255

Val Thr Gin Thr Arg Gly He Thr Ser Phe Gly Ser Ser Thr Gly Cys

260 265 270

Glu Thr Gly Tyr Pro Asp Gly Tyr Thr Arg Val Thr Ser Tyr Leu Asp

275 280 285

Trp He Glu Ser Asn Thr Gly He Ala He Asp Pro 290 295 300

(2) INFORMATION FOR SEQ ID NO : 5 : (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 185 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

Met Gly Ala His Ser He His Asp Asp Thr Glu Ala Ser Arg Val Ser

1 5 10 15

Ala Thr Ser Thr Asp Phe Phe Thr His Glu Asn Trp Asn Ser Phe Thr

20 25 30

Leu Thr Asn Asp Leu Ala Leu He Lys Met Pro Ala Pro He Glu Phe

35 40 45

Thr Pro Glu He Gin Pro Val Cys Leu Pro Ser Tyr Thr Asp Ala Ala

50 55 60

Asp Asp Phe He Gly Glu Ser Val Val Leu Thr Gly Trp Gly Arg Asp 65 70 75 80

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

85 90 95

Thr Thr He Ser Thr Ala Asp Cys Gin Ala Tyr Tyr Gly He Val Thr

100 105 110

Asp Lys He Leu Cys He Ser Ser Glu Asp Gly His Gly Ser Cys Asn

115 120 125

Gly Asp Ser Gly Gly Pro Met Asn Tyr Val Thr Gly Gly Val Thr Gin

130 135 140

Thr Arg Gly He Thr Ser Phe Gly Ser Ser Thr Gly Cys Glu Thr Gly 145 150 155 160

Tyr Pro Asp Gly Tyr Thr Arg Val Thr Ser Tyr Leu Asp Trp He Glu

165 170 175

Ser Asn Thr Gly He Ala He Asp Ala 180 185

(2) INFORMATION FOR SEQ ID NO: 6: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 300 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :

Leu Leu Leu Ala Leu Val Ala Ala Ala Ser Ala Ala Glu Trp Arg Trp

1 5 10 15

Gin Phe Arg His Pro Thr Val Thr Pro Asn Pro Arg Ala Lys Asn Pro

20 25 30

Phe Arg Val Thr Lys Ser Ser Pro Val Gin Pro Pro Ala Val Arg Gly

35 40 45

Thr Lys Ala Val Glu Asn Cys Gly Pro Val Ala Pro Arg Asn Lys He

50 55 60

Val Gly Gly Met Glu Val Thr Pro His Ala Tyr Pro Trp Gin Val Gly 65 70 75 80

Leu Phe He Asp Asp Met Tyr Phe Cys Gly Gly Ser He He Ser Asp

85 90 95

Glu Trp Val Leu Thr Ala Ala His Cys Met Asp Gly Ala Gly Phe Val

100 105 110

Glu Val Val Met Gly Ala His Ser He His Asp Asp Thr Glu Ala Ser

115 120 125

Arg Val Ser Ala Thr Ser Thr Asp Phe Phe Thr His Glu Asn Trp Asn

130 135 140

Ser Phe Thr Leu Thr Asn Asp Leu Ala Leu He Lys Met Pro Ala Pro 145 150 155 160

He Glu Phe Thr Pro Glu He Gin Pro Val Cys Leu Pro Ser Tyr Thr

165 170 175

Asp Ala Ala Asp Asp Phe He Gly Glu Ser VaJ Val Leu Thr Gly Trp

180 185 190

Gly Arg Asp Ser Asp Ala Ala Ser Gly He Ser Glu Leu Leu Arg Glu

195 200 205

Val His Val Thr Thr He Ser Thr Ala Asp Cys Gin Ala Tyr Tyr Gly

210 215 220

He Val Thr Asp Lys He Leu Cys He Ser Ser Glu Asp Gly His Gly 225 230 235 240

Ser Cys Asn Gly Asp Ser Gly Gly Pro Met Asn Tyr Val Thr Gly Gly

245 250 255

Val Thr Gin Thr Arg Gly He Thr Ser Phe Gly Ser Ser Thr Gly Cys

260 265 270

Glu Thr Gly Tyr Pro Asp Gly Tyr Thr Arg Val Thr Ser Tyr Leu Asp

275 280 285

Trp He Glu Ser Asn Thr Gly He Ala He Asp Ala 290 295 300

(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 536 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

CCCGGGCAGG TCCAGGATCG CCCTCTTACT TGCCCTTGTG GCTGCTACAG CTAGTGCTTC 60

AGAATGGCGC TGGCAGTTCC GTCACCCCAC TGTGACCCCC AACCCCAGAG CTAACAACCC 120

CTTCAGACCC AGTAAAGTCG CTCCAGTCCA ACCACCAGCA GTCAGAGGAA CAAAGGCTGT 180

TGAGAACTGT GGACCAGTAG CACCAAAGAA CAAGATTGTA GGAGGGCAAG AAGTGACTCC 240

CCATGCTTAC CCCTGGCAGG TGGGACTCTT CATCGATGAC ATGTACTTCT GCGGTGGATC 300

CATCATCTCA GAGGACTGGG TGCTTACAGC TGCTCACTGT GTGGATGGTG CTGGTTTTGT 360

CGAAGTTGTG ATGGGTGCTC ACAGTATCCA TGACGATACT GAGGCCTCTC GCATCAGTGC 420

CACATCAACT GATTTCTTCA CCCACGAGAA CTGGAACTCC TTCACCCTCA CCAATGATCT 480

TGCTCTCATT AAGATGCCAG CACCCATTGA GTTCACACCT GAAATTCAAC CTGTCT 536 (2) INFORMATION FOR SEQ ID NO : 8 : (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 178 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Pro Gly Arg Ser Arg He Ala Leu Leu Leu Ala Leu Val Ala Ala Thr

1 5 10 15

Ala Ser Ala Ser Glu Trp Arg Trp Gin Phe Arg His Pro Thr Val Thr

20 25 30

Pro Asn Pro Arg Ala Asn Asn Pro Phe Arg Pro Ser Lys Val Ala Pro

35 40 45

Val Gin Pro Pro Ala Val Arg Gly Thr Lys Ala Val Glu Asn Cys Gly

50 55 60

Pro Val Ala Pro Lys Asn Lys He Val Gly Gly Gin Glu Val Thr Pro 65 70 75 80

His Ala Tyr Pro Trp Gin Val Gly Leu Phe He Asp Asp Met Tyr Phe

85 90 95

Cys Gly Gly Ser He He Ser Glu Asp Trp Val Leu Thr Ala Ala His

100 105 110

Cys Val Asp Gly Ala Gly Phe Val Glu Val Val Met Gly Ala His Ser

115 120 125

He His Asp Asp Thr Glu Ala Ser Arg He Ser Ala Thr Ser Thr Asp

130 135 140

Phe Phe Thr His Glu Asn Trp Asn Ser Phe Thr Leu Thr Asn Asp Leu 145 150 155 160

Ala Leu He Lys Met Pro Ala Pro He Glu Phe Thr Pro Glu He Gin

165 170 175

Pro Val

(2) INFORMATION FOR SEQ ID NO: 9: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 968 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

( i ) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

CCCGGGCAGG TCCAGGATCG CCCTCTTACT TGCCCTTGTG GCTGCTACAG CTAGTGCTTC 60

AGAATGGCGC TGGCAGTTCC GTCACCCCAC TGTGACCCCC AACCCCAGAG CTAACAACCC 120

CTTCAGACCC AGTAAAGTCG CTCCAGTCCA ACCACCAGCA GTCAGAGGAA CAAAGGCTGT 180

TGAGAACTGT GGACCAGTAG CACCAAAGAA CAAGATTGTA GGAGGGCAAG AAGTGACTCC 240

CCATGCTTAC CCCTGGCAGG TGGGACTCTT CATCGATGAC ATGTACTTCT GCGGTGGATC 300

CATCATCTCA GAGGACTGGG TGCTTACAGC TGCTCACTGT GTGGATGGTG CTGGTTTTGT 360

CGAAGTTGTG ATGGGTGCTC ACAGTATCCA TGACGATACT GAGGCCTCTC GCGTCAGTGC 420

CACATCAACT GATTTCTTCA CCCACGAGAA CTGGAACTCC TTCACCCTCA CCAATGATCT 480

TGCTCTCATT AAGATGCCAG CACCAATTGA ATTCACACCT GAAATTCAAC CTGTCTGCCT 540

ACCAAGCTAC ACTGATGCTG CTGATGATTT CATTGGTGAA TCTGTTGTCC TTACTGGATG 600

GGGCCGTGAT TCTGATGCTG CTTCCGGCAT CTCTGAACTA CTCCGTGAGG TTCATGTGAC 660

CACAATCTCC ACTGCCGACT GCCAGGCATA CTACGGCATT GTCACTGACA AAATCCTCTG 720

CATTTCCTCT GAAGACGGAC ATGGTTCTTG TAATGGTGAT TCCGGTGGGC CAATGAACTA 780

TGTAACTGGT GGTGTTACTC AGACCCGTGG TATTACCTCC TTCGGATCCT CTACCGGGTG 840

TGAGACTGGC TACCCTGATG GTTACACACG TGTCACCAGC TATCTGGACT GGATTGAATC 900

TAACACTGGC ATTGCCATTG ATGCTTGAAT ATAATACTAG ATATGTAATC AAATAAATTT 960

CATGAATT 968

(2) INFORMATION FOR SEQ ID NO: 10: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 308 ammo acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Pro Gly Arg Ser Arg He Ala Leu Leu Leu Ala Leu Val Ala Ala Thr

1 5 10 15

Ala Ser Ala Ser Glu Trp Arg Trp Gin Phe Arg His Pro Thr Val Thr

20 25 30

Pro Asn Pro Arg Ala Asn Asn Pro Phe Arg Pro Ser Lys Val Ala Pro

35 40 45

Val Gin Pro Pro Ala Val Arg Gly Thr Lys Ala Val Glu Asn Cys Gly

50 55 60

Pro Val Ala Pro Lys Asn Lys He Val Gly Gly Gin Glu Val Thr Pro 65 70 75 80

His Ala Tyr Pro Trp Gin Val Gly Leu Phe He Asp Asp Met Tyr Phe

85 90 95

Cys Gly Gly Ser He He Ser Glu Asp Trp Val Leu Thr Ala Ala His

100 105 110

Cys Val Asp Gly Ala Gly Phe Val Glu Val Val Met Gly Ala His Ser

115 120 125

He His Asp Asp Thr Glu Ala Ser Arg Val Ser Ala Thr Ser Thr Asp

130 135 140

Phe Phe Thr His Glu Asn Trp Asn Ser Phe Thr Leu Thr Asn Asp Leu 145 150 155 160

Ala Leu He Lys Met Pro Ala Pro He Glu Phe Thr Pro Glu He Gin

165 170 175

Pro Val Cys Leu Pro Ser Tyr Thr Asp Ala Ala Asp Asp Phe He Gly

180 185 190

Glu Ser Val Val Leu Thr Gly Trp Gly Arg Asp Ser Asp Ala Ala Ser

195 200 205

Gly He Ser Glu Leu Leu Arg Glu Val His Val Thr Thr He Ser Thr

210 215 220

Ala Asp Cys Gin Ala Tyr Tyr Gly He Val Thr Asp Lys He Leu Cys 225 230 235 240

He Ser Ser Glu Asp Gly His Gly Ser Cys Asn Gly Asp Ser Gly Gly

245 250 255

Pro Met Asn Tyr Val Thr Gly Gly Val Thr Gin Thr Arg Gly He Thr

260 265 270

Ser Phe Gly Ser Ser Thr Gly Cys Glu Thr Gly Tyr Pro Asp Gly Tyr

275 280 285

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

290 295 300

Ala He Asp Ala 305

(2) INFORMATION FOR SEQ ID NO: 11: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO : 11 :

Ala Val Glu Asn Cys Gly Pro Val Ala Pro Arg 1 5 10

(2) INFORMATION FOR SEQ ID NO: 12: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 ammo acids

(B) TYPE: ammo acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Ala Val Glu Asn Cys Gly Pro Val Ala Pro Arg Asn Lys 1 5 10

(2) INFORMATION FOR SEQ ID NO: 13: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Gly Thr Lys Ala Val Glu Asn Cys Gly Pro Val Ala Pro Arg 1 5 10

(2) INFORMATION FOR SEQ ID NO: 14: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Gly Thr Lys Ala Val Glu Asn Cys Gly Pro Val Ala Pro Arg Asn Lys 1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 15: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Ser Ser Pro Val Gin Pro Pro Ala Val Arg Gly Thr Lys Ala Val Glu

1 5 10 15

Asn Cys Gly Pro Val Ala Pro Arg

20

(2) INFORMATION FOR SEQ ID NO: 16: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Ser Ser Pro Val Gin Pro Pro Ala Val Arg Gly Thr Lys Ala Val Glu

1 5 10 15 sn Cys Gly Pro Val Ala Pro Arg Asn Lys 20 25

(2) INFORMATION FOR SEQ ID NO: 17: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CACGCCTACC CNTGGCA 17

(2) INFORMATION FOR SEQ ID NO: 18: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

GTGTTGGACT CGATCCAGAT C 21

(2) INFORMATION FOR SEQ ID NO: 19:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 633 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

CACGCCTACC CGTGGCAGGT GGGACTTTTC ATTGATGATA TGTACTTCTG TGGAGGATCA 60

ATCATCTCCG ACGAATGGGT CCTTACAGCT GCTCACTGTA TGGATGGTGC TGGATTTGTT 120

GAGGTTGTGA TGGGTGCTCA CAGTATCCAT GACGAAACTG AGGCCACACA GGTCCGTGCC 180

ACATCAACTG ATTTCTTCAC ACACGAGAAC TGGAACTCCT TCACCCTCTC CAATGATCTT 240

GCTCTCATTA AGATGCCAGC ACCAATTGAA TTCAACGATG TGATCCAGCC TGTCTGCCTA 300

CCAACCTATA CTGATGCCAG TGATGATTTT GTTGGTGAAT CAGTCACTCT TACTGGATGG 360

GGTAAACCAT CTGACTCTGC TTTTGGCATC GCTGAACAAC TTCGTGAGGT TGATGTGACA 420

ACAATCACTA CTGCTGACTG CCAGGCATAC TACGGCATTG TCACTGACAA AATCCTCTGC 480

ATCGACTCCG AAGGAGGCCA TGGTTCCTGC AATGGTGATT CCGGTGGGCC AATGAACTAT 540

GTAACTGGTG GTGTTACTCA GACCCGTGGT ATTACCTCTT TCGGATCCTC TACCGGCTGC 600

GAGACTGGGT ACCCTGATAA TTACACACGA GTC 633

(2) INFORMATION FOR SEQ ID NO: 20: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 211 ammo acids

(B) TYPE: ammo acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

His Ala Tyr Pro Trp Gin Val Gly Leu Phe He Asp Asp Met Tyr Phe

1 5 10 15

Cys Gly Gly Ser He He Ser Asp Glu Trp Val Leu Thr Ala Ala His

20 25 30

Cys Met Asp Gly Ala Gly Phe Val Glu Val Val Met Gly Ala His Ser

35 40 45

He His Asp Glu Thr Glu Ala Thr Gin Val Arg Ala Thr Ser Thr Asp

50 55 60

Phe Phe Thr His Glu Asn Trp Asn Ser Phe Thr Leu Ser Asn Asp Leu 65 70 75 80

Ala Leu He Lys Met Pro Ala Pro He Glu Phe Asn Asp Val He Gin

85 90 95

Pro Val Cys Leu Pro Thr Tyr Thr Asp Ala Ser Asp Asp Phe Val Gly

100 105 110

Glu Ser Val Thr Leu Thr Gly Trp Gly Lys Pro Ser Asp Ser Ala Phe

115 120 125

Gly He Ala Glu Gin Leu Arg Glu Val Asp Val Thr Thr He Thr Thr

130 135 140

Ala Asp Cys Gin Ala Tyr Tyr Gly He Val Thr Asp Lys He Leu Cys 145 150 155 160 Ile Asp Ser Glu Gly Gly His Gly Ser Cys Asn Gly Asp Ser Gly Gly

165 170 175

Pro Met Asn Tyr Val Thr Gly Gly Val Thr Gin Thr Arg Gly He Thr

180 185 190

Ser Phe Gly Ser Ser Thr Gly Cys Glu Thr Gly Tyr Pro Asp Asn Tyr

195 200 205

Thr Arg Val 210

(2) INFORMATION FOR SEQ ID NO: 21: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 967 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

ATCGCCCTCT TACTCGCCCT TGTGGCTGCC ACTGCTAGTG CTTCAGAATG GCGCTGGCAG 60

TTCCGTCACC CCACCGTGAC CCCCAACCCC AGAGCTAACA ACCCCTTCAG ACCAAGTAAA 120

GTTGCTCCAG TCCAACCACC AGCAGTCAGA GGAACAAAGG CTGTACCCAA CTGTGGACAG 180

TCAAAGTCTA CCAAGATTGT AGGAGGTGGT GAGGTAACTC CCCATGCTTA CCCCTGGCAG 240

GTGGGACTTT TCATTGATGA CATGTACTTC TGCGGKGGAT CCATCATCTC AGAGGACTGG 300

GTCCTTACAG CTGCTCACTG TATGGATGGT GCTGGGTTTG TTGAGGTTGT GATGGGTGCT 360

CACAAGATCC ATGATGATAC TGAGGCCTCT CGCGTCAGTG CCATATCAAC TGATTTCTTC 420

ACCCACGAGA ACTGGAACTC CTTCCTTCTC ACCAATGATC TTGCTCTCAT TAAGATGCCA 480

GCACCCATTG CATTCACTGA TGAGATCCAG CCTGTATGCC TGCCAACCTA CACTGACTCC 540

GATGATGATT TTGTTGGTGA ATCAGTCACT CTTACTGGCT GGGGTCGTGC ATCTGACTCT 600

GCTAGCGGCA TCTCTGAAGT ACTTCGTGAG GTTGATGTGA CAACAATAAC TACTGCCGAC 660

TGCCAGGCAT ACTATGGTAT TGTCACTGAC AAAATCCTCT GCATCGACTC AGAAGGAGGT 720

CATGGGTCTT GCAATGGTGA TTCCGGTGGG CCAATGAACT ATGTAACTGG TGGTGTTACT 780

CAGACCCGTG GTATTACCTC CTTCGGATCC TCTACCGGCT GTGAGACTGG CTACCCTGAT 840

GGTTACACAC GAGTCACCAG CTATCTAGAC TGGATTGAAT CTAACACTGG CATTGCCATT 900

GATCCTTGAA TAATATTCTA GCTGAATGAT AATAAATTCA TGATTGATAA TCAAAAAAAA 960

AAAAAAA 967

(2) INFORMATION FOR SEQ ID NO: 22: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 302 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

He Ala Leu Leu Leu Ala Leu Val Ala Ala Thr Ala Ser Ala Ser Glu

1 5 10 15

Trp Arg Trp Gin Phe Arg H s Pro Thr Val Thr Pro Asn Pro Arg Ala

20 25 30

Asn Asn Pro Phe Arg Pro Ser Lys Val Ala Pro Val Gin Pro Pro Ala

35 40 45

Val Arg Gly Thr Lys Ala Val Pro Asn Cys Gly Gin Ser Lys Ser Thr

50 55 60

Lys He Val Gly Gly Gly Glu Val Thr Pro His Ala Tyr Pro Trp Gin 65 70 75 80

Val Gly Leu Phe He Asp Asp Met Tyr Phe Cys Gly Gly Ser He He

85 90 95

Ser Glu Asp Trp Val Leu Thr Ala Ala His Cys Met Asp Gly Ala Gly

100 105 110

Phe Val Glu Val Val Met Gly Ala His Lys He His Asp Asp Thr Glu

115 120 125

Ala Ser Arg Val Ser Ala He Ser Thr Asp Phe Phe Thr His Glu Asn

130 135 140

Trp Asn Ser Phe Leu Leu Thr Asn Asp Leu Ala Leu He Lys Met Pro 145 150 155 160

Ala Pro He Ala Phe Thr Asp Glu He Gin Pro Val Cys Leu Pro Thr

165 170 175

Tyr Thr Asp Ser Asp Asp Asp Phe Val Gly Glu Ser Val Thr Leu Thr

180 185 190

Gly Trp Gly Arg Ala Ser Asp Ser Ala Ser Gly He Ser Glu Val Leu

195 200 205

Arg Glu Val Asp Val Thr Thr He Thr Thr Ala Asp Cys Gin Ala Tyr

210 215 220

Tyr Gly He Val Thr Asp Lys He Leu Cys He Asp Ser Glu Gly Gly 225 230 235 240

His Gly Ser Cys Asn Gly Asp Ser Gly Gly Pro Met Asn Tyr Val Thr

245 250 255

Gly Gly Val Thr Gin Thr Arg Gly He Thr Ser Phe Gly Ser Ser Thr

260 265 270

Gly Cys Glu Thr Gly Tyr Pro Asp Gly Tyr Thr Arg Val Thr Ser Tyr

275 280 285

Leu Asp Trp He Glu Ser Asn Thr Gly He Ala He Asp Pro 290 295 300

(2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 535 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (lx) FEATURE:

(A) NAME/KEY: Coding Sequence

(B) LOCATION: 2...535 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

C CCG GGC AGG TCC AGG ATC GCC CTC TTA CTT GCC CTT GTG GCT GCT ACA 49 Pro Gly Arg Ser Arg He Ala Leu Leu Leu Ala Leu Val Ala Ala Thr

1 5 10 15

GCT AGT GCT TCA GAA TGG CGC TGG CAG TTC CGT CAC CCC ACT GTG ACC 97 Ala Ser Ala Ser Glu Trp Arg Trp Gin Phe Arg His Pro Thr Val Thr 20 25 30

CCC AAC CCC AGA GCT AAC AAC CCC TTC AGA CCC AGT AAA GTC GCT CCA 145 Pro Asn Pro Arg Ala Asn Asn Pro Phe Arg Pro Ser Lys Val Ala Pro 35 40 45

GTT CAA CCA CCA GCA GTC AGA GGA ACA AAG GCT GTT GAG AAC TGT GGA 193 Val Gin Pro Pro Ala Val Arg Gly Thr Lys Ala Val Glu Asn Cys Gly 50 55 60

CCA GTA GCA CCA AAG AAC AAG ATT GTA GGA GGG CAA GAA GTG ACT CCC 241 Pro Val Ala Pro Lys Asn Lys He Val Gly Gly Gin Glu Val Thr Pro 65 70 75 80

CAT GCT TAC CCC TGG CAG GTG GGA CTC TTC ATC GAT GAC ATG TAC TTC 289 His Ala Tyr Pro Trp Gin Val Gly Leu Phe He Asp Asp Met Tyr Phe 85 90 95

TTC GGT GGA TCC ATC ATC TCA GAG GAC TGG GTC GTT ACA GCT CGT CAC 337 Phe Gly Gly Ser He He Ser Glu Asp Trp Val Val Thr Ala Arg His 100 105 110

TGT ATG GAT GGT CGT GGT TTT GTC GAA GTT GTG ATG GGT GCT CAC AGT 385 Cys Met Asp Gly Arg Gly Phe Val Glu Val Val Met Gly Ala His Ser 115 120 125

ATC CTA GAC GAT ACT GAG GCC TCT CGC ATG AGT GCC ACA TCA ACT GAT 433 He Leu Asp Asp Thr Glu Ala Ser Arg Met Ser Ala Thr Ser Thr Asp 130 135 140

TTC TTC ACC CAC GAG AAC TGG AAC TCC TTC ACC CTC ACC AAT GAT CTT 481 Phe Phe Thr His Glu Asn Trp Asn Ser Phe Thr Leu Thr Asn Asp Leu 145 150 155 160

GCT CTC ATT AAG ATG CCA GCA CCC ATT GAG TTC ACA CCT GAA ATT CAA 529 Ala Leu He Lys Met Pro Ala Pro He Glu Phe Thr Pro Glu He Gin 165 170 175

CCT GTC 535

Pro Val

(2) INFORMATION FOR SEQ ID NO: 24: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 178 ammo acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

Pro Gly Arg Ser Arg He Ala Leu Leu Leu Ala Leu Val Ala Ala Thr

1 5 10 15

Ala Ser Ala Ser Glu Trp Arg Trp Gin Phe Arg His Pro Thr Val Thr

20 25 30

Pro Asn Pro Arg Ala Asn Asn Pro Phe Arg Pro Ser Lys Val Ala Pro

35 40 45

Val Gin Pro Pro Ala Val Arg Gly Thr Lys Ala Val Glu Asn Cys Gly

50 55 60

Pro Val Ala Pro Lys Asn Lys He Val Gly Gly Gin Glu Val Thr Pro 65 70 75 80

His Ala Tyr Pro Trp Gin Val Gly Leu Phe He Asp Asp Met Tyr Phe

85 90 95

Phe Gly Gly Ser He He Ser Glu Asp Trp Val Val Thr Ala Arg His

100 105 110

Cys Met Asp Gly Arg Gly Phe Val Glu Val Val Met Gly Ala His Ser

115 120 125

He Leu Asp Asp Thr Glu Ala Ser Arg Met Ser Ala Thr Ser Thr Asp

130 135 140

Phe Phe Thr His Glu Asn Trp Asn Ser Phe Thr Leu Thr Asn Asp Leu 145 150 155 160

Ala Leu He Lys Met Pro Ala Pro He Glu Phe Thr Pro Glu He Gin

165 170 175

Pro Val

(2) INFORMATION FOR SEQ ID NO: 25: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Ala Val Glu Asn Cys Gly Pro Val Ala 1 5 The nucleic acid sequences descπbed herein, and consequently the protem sequences derived therefrom, have been carefully sequenccd However, those of ordinary skill will recognize that nucleic acid sequencing technology can be susceptable susceptible to some inadvertent error Those of ordinary skill in the relevant arts are capable of validating or correcting these sequences based on the ample description herein of methods of isolating the nucleic acid sequences in question, and such modifications that are made readily available by the present disclosure are encompassed by the present invention Furthermore, those sequences reported herein are believed to define functional biological macromolcculcs within the invention whether or not later claπfying studies identify sequencing errors

The application describes a number of nucleic acid sequences, of which the more germane arc summaπzcd below

While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations in the preferred devices and methods may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the claims that follow

Claims

What is claimed

1 An isolated nucleic acid comprising at least one of

(a) a nucleic acid encoding a polypeptide having at least about 70% sequence identity with a protein having a reference sequence which is AA64-300 of SEQ ID NO 4 or AA1-300 of SEQ ID NO 4 or a sequence diffeπng from these by at least one of the residue differences found SEQ ID NOS 5. 20, 22. or 24.

(b) a polynucleotide which is complementary to the polynucleotidc of (a), or

(c) a polynucleotide compπsing at least 15 sequential bases of the polynucleotidc of (a).

(b)

2 The nucleic acid of claim 1. encoding a polypeptide having at least about 80% homology to a reference sequence

3 The substantially pure nucleic acid of claim 1. compπsing base pairs 1 0-900 of SEQ ID NO 1. base pairs 2-556 of SEQ ID NO 2. base pairs 190-900 of SEQ ID NO 3. base pairs 215-925 of SEQ ID NO 9. base pairs 1-633 of SEQ ID NO 19, base pairs 196-906 of SEQ ID NO 21. or base pairs 215-535 of SEQ ID NO 23

4 The substantially pure nucleic acid of claim 3. comprising base pairs 190-900 of SEQ ID NO 1. base pairs 2-556 of SEQ ID NO 2. base pairs 1 -633 of SEQ ID NO 19. base pairs

196-906 of SEQ ID NO 21 or base pairs 215-535 of SEQ ID NO 23

5 A transformed cell comprising the nucleic acid of claim 1

6 An expression vector comprising the nucleic acid of claim 1. wherein the vector is capable of reproducing in a eukaryotic or prokaryotic cell

7 A pharmaceutical composition for treatmg an animal comprising an effective amount of a nucleic acid of claim 1 and a pharmaceutically acceptable earner

8 A polypeptide comprising a substantialh pure isoform of a protemase encoded by a nucleic acid of claim 1 9 The polypeptide of claim 8, compπsing a polypeptide whose sequence includes a portion of SEQ ID NOS 4. 5, 6, 8, 10, 20 or 22 that correspondes to AA64-300 of SEQ ID NO 4

10 The polypeptide of claim 8. compπsing at least one of the amino acid sequences of SEQ ID NOS 1 1-16 or 25

1 1 A method of preparing a multifunctional protein comprising

(a) transforming a cell with an expression vector for expressing mtracellularly or extracellularly a nucleic acid encoding a polypeptide having at least about 70% identity to a reference sequence which is SEQ ID NO 4 or a sequence differing therefrom by at least one of the sequence differences found in SEQ ID NOS 5, 8, 20 or 22.

(b) growing the transformed cell in culture, and

(c) isolating the protein from the transformed cell or the culture medium

12 The method of claim 1 1, wherein the polypeptide has at least about 80% identity to a reference sequence