CA2246737A1 - Esterases - Google Patents

Abstract

Esterase enzymes derived from various Staphylothermus, Pyrodictium, Archaeoglobus, Aquifex, M11TL, Thermococcus, Teredinibacter and Sulfolobus organisms are disclosed. The enzymes are produced from native or recombinant host cells and can be utilized in the pharmaceutical, agricultural and other industries.

Description

WO 9713016~ PCTIUS9'J102039 ESTERASES

This invention relates to newly identified polynucleotides, polypeptides encodedby such polynucleotides. the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly~ the polynucleotides and polypeptides of the present invention have been putatively identified as esterases. Esterases are enzymes that catalyze the hydrolysis of ester groups to or~anic acids and alcohols.

Many esterases are kno~n and have been discovered in a broad variety of or~ni.cm~. including bacteria. yeasl and higher ~nim~l.c and plants. A principal example of esterases are the lipases. which are used in the hydrolysis of lipids, acidolysis(replacement of an esterified fatty acid with a free fatty acid) reactions, transesterification(exchan~e of fatt~ acids between triglycerides)reactions, and in ester synthesis. The major industrial applications for lipases include: the detelge-lL industry, where they are employed to decompose fatty materials in laundry stains into easily removable hydrophilic substances: the food and beverage industry where they are used in the m~mlf~tllre of cheese~ the ripening and flavoring of cheese, as ~nfi~t~ling agents for bakery products. and in the production of malgalille and other spreads with natural CA 02246737 l998-08-l7 W O 97/30160 PCTrUS97/02039 butter flavors; in waste systems; and in ~e pharrn~-~el1tical industry where they are used as digestive aids.

The polynucleotides and polypeptides of the present invention have been i~lentifi~A as esterases as a result of their enzymatic activity.

In accordance with one aspect of the present invention, thlere are provided novel enzymes, as well as active fr~m~nt~, analogs and derivatives thereof.

In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the enzymes of the present invention including ml~NAs, cDNAs, genomic DNAs as well as active analogs and fragments of such enzymes.

In accordance with another aspect of the present invention there are provided isolated nucleic acid molecules encoding mature polypeptides expressed by the DNA
contained in ATCC Deposit No.

In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant t~chniqlle~ comprising culturing recombinant prokaryotic and/or eukaryotic host cells, cont~ining a nucleic acid sequence of the present invention, under conditions promoting expression of saidenzymes and subsequent recovery of said enzymes.

In accordance with yet a further aspect of the present invention, there is provided a process for ~ltili7ing such enzymes. or polynucleotides encoding such enzymes for hydrolyzing ester groups to yield an organic acid and an alcohol. The esterases of the invention are stable at high temperatures and in organic solvents and, thus, are superior for use in production of optically pure chiral compounds used in pharm~.el1tical, agricultural and other chemical industries.

Wo 97130160 PCTJUS97102û39 In acco~dance with yet a fur~her aspect of ~e present invention. there are also - provided nucleic acid probes comprising nucleic acid molecules of sufficient length to hybridize to a nucleic acid sequence of the present invention.

In accordance with yet a further aspect of the present invention~ there is provided a process for nti1i7ing such enzymes, or polynucleotides encoding such enzymes. for in vitro purposes related to scientific leseal~;h, for example, to generate probes for identifying similar sequences which might encode similar enzymes from other org~ni~m~
by using certain regions, i.e., conserved sequence regions, of the nucleotide sequence.

These and other aspects of the present invention should be a~uale.ll to those skilled in the art from the te~rllin~ herein.

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

Figure 1 is an illust~ation of the full-length DNA (SEQ ID NO:23) and corresponding ded1~red amino acid sequence (SEQ ID NO:33) of Staphylothermus marinus F1-12LC of the present invention. Sequencing was performed using a 378 automated DNA sequencer (Applied 13iosystems, Inc.) for all sequences of the present invention.

Figure 2 is an illustration of the full-length DNA (SEQ ID NO:24) and corresponding (led~lce~ amino acid sequence (SEQ ID NO:34) of Pyrodictium TAGl 1-17LC.-Figure 3 is an illustration of the full-length DNA (SEQ ID NO:25) and corresponding ~çl1llrecl amino acid sequence (SEQ ID NO:35) of Archaeoglobus venificus SNP6-24LC.

W O 97/30160 PCTrUS97/02039 Figure 4 is an illustration of the full-length DNA (SEQ ID NO:26) and corresponding cle~ ced amino acid sequence (SEQ ID NO:36) of Aquffex pvrophilus-28LC.

Figure 5 is an illustration of the full-length DNA (SEQ ID NO:27) and corresponding ~1e~ ee(l amino acid sequence (SEQ ID NO:37) of MllTL-29L.

Figure 6 is an illustration of the full-length DNA ~SEQ ID NO:28) and corresponding ~leduce(l amino acid sequence (SEQ ID NO:38) of Thermococcus CL-2-30LC.

Figure 7 is an illustration of the full-length DNA (SEQ ID NO:29~ and corresponding ~le~ ced amino acid sequence (SEQ ID NO:39) of Aquifex VF5-34LC.

Figure 8 is an illustration of the full-length DNA (SEQ ID NO:3û) and corresponding ~ ce~:l amino acid sequence (SEQ ID NO:40) of Teredinibacter-42L.

Figure 9 is an illustration of the full-length DNA (SEQ ID NO:31) and corresponding de~ re(l amino acid sequence (SEQ ID NO:41) of Archaeoglobusfulgidus VC16-16MC.

Figure 10 is an illustration of the full-length DNA (SEQ ID NO:32) and corresponding f~ cefl amino acid sequence (SEQ ID NO:42~ of Sulfolo~us solfatarzcus Pl -8LC .

Figure 11 is an illustration of the full-length DNA (SEQ ID NO:23) and corresponding ~le~ ced amino acid sequence (SEQ ID NO:33) of LAll.l Esterase es2of the present invention.

W ~ 97J3~160 PCTAUS97/02039 -Fi~;ure 12 is an illustration of the full-length DNA (SEQ ID NO:24) and corresponding de~ cecl amino acid sequence (SEQ ID NO:34) of Whale Mat Sample 11.801 Esterase es9.

Figure 13 is an illustration of the full-length DNA (SEQ ID NO:25) and corresponding ~ ce(l amino acid se~uence ~SEQ ID NO:35) of Metallosphaera PrunaeRon 12/2 Esterase 23mcl .

Figure 14 is an illustration of the full-length DNA (SEQ ID NO:26) and corresponding deduced amino acid seq1len~e (SEQ ID N0:36) of Thermotoga neapolitana 5068 Esterase 56mc4.

~ igure 15 is an illustlation of the full-length DNA (SEQ ID NO:27) and corresponding ~ledl-ce(l amino acid sequence (SEQ ID NO:37) of Meliltangium lichenicola Esterase 77mcl.

Figure 16 is an illustration of the full-length DNA (S~Q ID NO:28) and corresponding A~ ced amino acid sequence (SEQ ID NO:38) of Whale Mat Sample 1 1 . 801 Esterase es2 .

Figure 17 is an illustration of the full-length DNA (SEQ ID NO:293 and corresponding dPd~ed amino acid sequence (SEQ ID NO:39) of Whale Mat Sample AD3059 Esterase es4.

Figure 18 is an illustration of the full-length DNA (SEQ ID NO:30) and corresponding dt-d~lced amino acid sequence (SEQ ID NO:40) of Microscilla furvescens Esterase 53sc2.

' CA 02246737 1998-08-17 W O 9~30160 PCT~US97/02039 Figure 19 is an illustration of the full-length DNA ~(SEQ ID NO:31) and corresponding dedllce(l amino acid sequence (SEQ ID NO:41) of Thermotoga marztima MSB8 Esterase 6sc1.

..
Figure 20 is an illustration of the full-length DNA (SEQ ID NO:32) and corresponding ~ledl~e~l amino acid sequence (SEQ ID NO:42) of Polyangium brachysporum Esterase 78mcl.

The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions prece-ling and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

A coding sequence is "operably linked to" another coding sequence when RNA
polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having amino acids derived from both codingsequences. The coding sequences need not be contiguous to one another so long as the expressed sequences ul~im~t~ly process to produce the desired protein.

"Recombinant" enzymes refer to enzymes produced by recombinant DNA
techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired enzyme. "Synthetic" enzymes are those prepared by chemical synthesis.

A DNA "coding sequence of" or a "nucleotide sequence encoding" a particular enzyme, is a DNA sequence which is transcribed and tr~n~l~te~l into an enzyme when placed under the control of approp,iate regulatory sequences.

In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which encode for the mature enzymes having the (led~lce(l amino acid sequences of Figures 1-10 (SEQ ID NOS:23-32).

In accordance with another aspect of ~e present invention. there are provided isolated polynucleotides encoding the enzymes of the present invention. The deposited material is a mixture of genomic clones comprising DNA encoding an enzyme of thepresent invention. Each genomic clone comprising the respective DNA has been inserted into a pBluescript vector (Stratagene, La Jolla. CA). The deposit has been deposited with the American Type Culture Collection, 12301 Parklawn Drive.
~ Rockville, Maryland 20852, USA, on December 13, 1995 and a~si~nPcl ATCC Deposit No.

The deposit(s) have been made under the terms of the Budapest Treaty on the International Recognition of the deposit of micro-org~ni~m.c for purposes of patent procedure. The strains will be irrevocably and without restriction or condition released to the public upon the i~s--~n~e of a patent. These deposits are provided merely as convenience to those of skill in the art and are not an ~rlmis~ion that a deposit would be required under 35 U.S.C. 112. The sequences of the polynucleotides contained in the deposited materials~ as well as the amino acid sequences of the polypeptides encoded there~y, are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

The polynucleotides of this invention were originally recovered from genomic gene libraries derived from the followin or~ni~m~:

Staphylothermus marinus F I is a thermophilic sulfur archaea which was isolated in Vulcano, ltaly. It grows optimally at 85~C (Tma" = 98~C) at pH 6.5.

Pyrodictium TAGI I is a thermophilic sulfur archaea which was isolated in the Middle Atlantic Ridge. It grows optimally at 103~C (Tma,~ = llO~C) at pH 6.~.

Archaeoglobus venificus SNP6 was isolated in the Middle Atlantic Rid~e and grows optimally at 75~C ~Tm~ = 92~C) at pH 6.9.

Aquifex pyrophilus K01 5a was isolated at Kolbeinsey Ridge, North of Iceland.
This marine organism is a gram-ne~ative, rod-shaped, strictly chemolithoautrophic, knall gas bacterium. It grows optimally at 85~C (Tm~ = 95~C) at pH 6.8.

MllTL is a new species of Desulfurococcus which was isolated from Diamond Pool (formerly Jim's Black Pool) in Yellowstone. The organism grows helelo~ hically by fel~nentation of different organic materials (sulfur is not n.ocec~ly) in grape-like aggregates optimally at 85 - 88~C in a low salt medium at pH 7Ø

The~7nococcus CL-2 was isolated in the North Cleft Segment of the Juan de ~uca Ridge from a severed alvinellid worm residing on a "black smoker" sulfide structure.
This marine archaea forms pleomorphic cocci, and grows optimally at 88~C.

Aquifex VF5 was isolated at a beach in Vulcano, Italy. This marine olXal~
is a gram-negative, rod-shaped, strictly chemolithoaulollophic, knall gas bacterium. It grows optimally at 85~C (Tm~ = 95~C) at pH 6.8.

~ eredinibacter (pure) is an endosymbiont of the shipworm Bankia gouldi. The organism has strai~ht to slightly bent 5-10 ~m rods, and forms spiral cells as stationary phase is met. The organism was described in Science (1983) 22:1401-1403. It grows optimally at 30~C at pH 8Ø

Archaeoglobus fulgidus VC16 was isolated in Vulcano, Italy. The organism grows optimally at 85~C (Tm~; = 92~C) at pH 7Ø

Sulfolobus solfataricus Pl ~rows optimally at 85~C (Tma,~ = 87~C) at pH 2Ø

. CA 02246737 l998-08-l7 W O 9~J3~160 PCTnUS971U2039 Accordingly, tne polynucleotides and enzymes encoded thereby are identified by the or~anism from which they were isolated. and are som~times hereinafter referred to as Fl/12LC (Figure 1 and SEQ ID NOS:23 and 33), TAG11/17LC (Figure 2 and SEQ
- - ID NOS:24 and 34), SNP6/24LC (Figure 3 and SEQ ID NOS:25 and 35), AqP/28LC
(Figure 4 and SEQ ID NOS:26 and 36), MllTL/29L ~Figure 5 and SEQ ID NOS:27 and 37), CL-2/30LC (Figure 6 and SEQ ID NOS:28 and 38), VF5/34LC (Figure 7 and ~ SEQ ID NOS:29 and 39), Trb/42L (Fig,ure 8 and SEQ ID NOS:30 and 40), VC16/16MC (Figure 9 and SEQ ID NOS:31 and41) and Pl/8LC (Figure 10 and SEQ
ID NOS: 32, amd 42).

The polynucleotides and polypeptides of the present invention show identity at the nucleotide and protein level to known genes and proteins encoded tnereby as shown in Table 1.

W O 97/30160 PCT~US97/02039 _ Table 1 Protein Protein D~A
~ene wlclosest .~ Identity Identity Enzyme .H~Imolugy (O~ %~ ~%) F1/12LC No significant homology TAG11/17LC No .~ignifir~.nt homology - - -carboxylesterase Pseudomones sp. (strain KWI-56) open reading frame of unknown function in E.coli.
AqP/29LC 53 31 38 MllTL/29LC No significant homology CL02/30LC No significant homology - - -VF5/34LC Identified by homology 84 71 71 to 28LC; also homologous to ORF of unknown function 5' of tgs in E. coli Trb/42L No significant homology Pl -8LC

All the clones identified in Table 1 encode polypeptides which have esterase activity.

This invention, in addition to the isolated nucleic acid molecules encoding t~e enzymes of the present invention, also provides substantially similar sequences. Isolated nucleic acid sequences are substantially similar if: (i) they are capable of hybridizing under conditions hereinafter described, to the polynucleotides of SEQ ID NOS:23-32;
(ii) or they encode DNA sequences which are degenerate to the polynucleotides of SEQ

Wo 97130160 PCTIUS97102039 ID NOS:23-32. Degenerate DNA sequences encode the amino acid sequences of SEQ
ID NOS:33-42, but have variations in the nucleotide coding sequences. As used herein, subst~nti~lly similar refers to the se~uences having similar identity to the sequences of - the instant invention. The nucleotide sequences that are substantially the same can be identified by hybridization or by sequence comparison. Enzyme sequences that aresubstantially the same can be identified by one or more of the following: proteolytic digestion, gel electrophoresis and/or microsequencing.

One rneans for isolating the nucleic acid molecules encoding the enzymes of the present invention is to probe a gene library with a natural or artificially designed probe using art recognized procedures (see, for example: Current Protocols in Molecular Biology, Ausubel F.M. et al. (EDS.) Green Publishing Company Assoc. and John Wiley lnterscience, New York, 1989, 1992). It is appreciated by one skilled in the art that the polynucleotides of SEQ ID NOS:23-32, or fragments thereof (cc,lllplisillg at least 12 contiguous nucleotides~, are particularly useful probes. Other particularly useful probes for this purpose are hybridizable fragments of the sequences of SEQ ID
NOS:1-22 {i.e., comprising at least 12 contiguous nucleotides~.
-With respect to nucleic acid sequences which hybridize to specific nucleic acidsequences disclosed herein~ hybridization may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions. As an example of oligonucleotide hybridization. a polymer membrane cont~inin~ immobilized denatured nucleic acids is first prehybridized for 30 mimlt~s at 45~C in a solution consisting of 0.9 M NaCl, 50 mM Na~,PO4~ pH 7.0, 5.0 rnM Na2EDTA, 0.5% SDS, 10X Denhardt's, and 0.5 mg/mL polyriboadenylic acid. Approximately 2 X 107 cpm (specific activity 4-9 X 108 cpm/ug) of 32p end-labeled oligonucleotide probe are then added to thesolution. After 12-16 hours of inc~ tion, the membrane is washed for 30 minl-~es at room temperature in lX SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na~EDTA) cont,qining 0.5 % SDS. followed by a 30 minute wash in fresh lX SET

W O 97/30160 CA 02246737 1998-08-17 PCTrUS97/02039 at Tm -I0~C for the oligo-nucleotide probe. The membrane is then exposed to auto-radiographic film for detection of hybridization signals.

Stringent conditions means hybridization will occur oniy if there is at least 907c identity, preferably at least 95% identity and most preferably at least 97% identity between the sequences. See J. Sambrook et al., Molecular Cloning, A L,aboralor~
Manual, 2d Ed.1 Cold Spring Harbor Laboratory (1989) which is hereby incorporated by reference in its entirety.

As used herein, a first DNA (RNA) sequence is at least 70% and preferably at least 805~o identical to another DNA (RNA) sequence if there is at least 70% andpreferably at lest a 8()% or 90% identity, respectively, between the bases of the first sequence and the bases of the another sequence~ when properly aligned with each other, for example when aligned by BLASTN.

The present invention relates to polynucleotides which differ from the referencepolynucleotide such that the changes are silent changes, for example the change do not alter the amino acid sequence encoded by the polynucleotide. The present invention also relates to nucleotide changes which result in amino acid substitùtions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference polynucleotide. In a preferred aspect of the invention these polypeptides retain the same biological action as the poiypeptide encoded by the reference polynucleotide.

The polynucleotides of this invention were recovered from genomic gene librariesfrom the org~ni.cmc listed in Table l. Gene libraries were geDerated in the Lambda ZAP II cloning vector (Strata~ene Cloning Systems). Mass excisions were pe~ro~ ed on these libraries to generate libraries in the pBluescript phagemid. ~ibraries were generated and excisions were perforrned according to the protocols/methods hereinafter described.

~ CA 02246737 1998-08-17 WO 9713û16~ PCTIUS97102039 The polynucleotides of the present invention may be in the form of RNA or DNA which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequences which encodes the matureenzymes may be identical to the coding sequences shown in Figures 1-10 (SEQ ID
NOS:23-32) or may be a different coding sequence which coding sequence, as a result of the re~ ntl~n~y or degeneracy of the genetic code, encodes the same mature enzymes as the DNA of Figures 1-10 (SEQ ID NOS:23-32).

The polynucleotide which encodes for the mature ert7.yme of Figures 1-10 (SEQ
ID NOS:33-42) may include, but is not limited to: only the coding sequence for the mature enzyme; the coding sequence for the mature enzyme and additional coding sequence su.ch as a leader sequence or a l,ro~ in sequence; the coding sequence for the mature enzyme (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature enzyme.

Thus, the term "polynucleotide encoding an enzyme (protein)" encompasses a polynucleotide which includes only coding seq~lenre for the enzyme as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates tO variants of the hereinabove described polynucleotides which encode for fragments~ analogs and derivatives of the enzymes having the df~-luced amino acid sequences of Figures 1-10 (SEQ ID NOS:33-42). The variant of the polynucleotide ma~ be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encodLing the same mature enzymes as shown in Figures 1-10 (SEQ ID NOS:23-32) as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the W O 97/30160 PCT~US97/02039 enzymes of Figures 1-10 (SEQ ID NOS:23-32). Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

- - As hereinabove indicated, the polynucleotides may have a coding seqllenre which is a naturally occurring allelic variant of the coding se~uences shown in Figures 1-10 ~SEQ ID NOS:23-32). As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a sl~hstit-ltion~ deletion or addition of one or more nucleotides, which does not subst~nti~lly alter the function of the encoded enzyme.

Fr~mt~nts of the full length gene of the present invention may be used as hybridization probes for a cDNA or a genomic library to isolate the full length DNA
and to isolate other DNAs which have a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least 10, preferably at least 15, and even more preferably at least 30 bases and may contain, for example, at least 50 or more bases. The probe may also be used to identify a DNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons and introns. An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of genomic DNA to determine which members of the library the probe hybridizes to.

It is also appreciated that such probes can be and are preferably labeled with an analytically ~let~ct~ble reagent to facilitate i~l~ntifir~tion of the probe. Useful reagents include but are not limited to radioactivity, fluorescent dyes or enzymes capable of catalyzing the formation of a detectable product. The probes are thus useful to isolate complementary copies of DNA from other sources or to screen such sources for related se~uences.

-WO g~S3~60 PCT/US9710203g The present invention further relates to polynucleotides which hybridize tO the hereinabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term "stringent conditions"
means hybridization will occur only if there is at least 95 % and preferably at least 97 7G
identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a ~l~rell~d embodirnent encode enzymes which either retain substantially the same biological function or activity as the mature enzyme encoded by the DNA of Fig~res 1-10 (SEQ ID NOS:23-32).

Alternatively, the polynucleotide may have at least 15 bases, preferably at least 30 bases, and more preferably at least 50 bases which hybridize to any part of apolynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotides of SEQ ID NOS:23-32, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.

Thus. the present invention is directed to polynucleotides having at least a 70%identity, preferably at least 90% identity and more preferably at least a 95 % identity to a polynucleotide which encodes the enzymes of SEQ l[D NOS:33~2 as well as fragments thereof, which fragments have at least 1~ bases, preferably at least 30 bases and most preferably at least 50 bases, which fragments are at least 90% identical, preferably at least 95% identical and most preferably at least 97% identical under stringent conditions to any portion of a polynucleotide of the present invention.

The present invention further relates to enzymes which have the ~ ce~l amino acid sequences of Figures 1-10 (SEQ ID NOS:23-32) as well as fragments, analogs and derivatives of such enzyme.

W O 97/30160 PCTrUS97/02039 The terms "fragment, " "derivative" and "analog" when lef~ g to the enzymes of Figures 1-10 (SEQ IO NOS:33-42) mean enzymes which retain essentially the same biological function or activity as such enzymes. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature enzyme.

The enzymes of the present invention may be a recombinant enzyme, a natural enzyme or a synthetic enzyme, preferably a rec~ll,bhlalll enzyme.

The fragment, derivative or analog of the enzymes of Figures 1-10 (SEQ ID
NOS:33-42) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature enzyme is fused with another compound, such as a compound to increase the half life of the enzyme (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature enzyme, such as a leader or secretory seql-~n~e or a sequence which is employed for purification of the mature enzyme or a plol)luL~in sequence. Such fragments, derivatives and analogs are deem~ to be within the scope of those skilled in the art from the fe~ching~ herein.

The enzymes and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term "isolated" means that the material is removed from its original environment (e.g., the natural envirorlment if it is naturally occurring). Por example, a naturally-occurring polynucleotide or enzyme present in a living animal is not isolated, but the same polynucleotide or enzyme, separated from some or all of the coexisting materials in the natural system. is isolated. Such polynucleotides could be part of a . CA 02246737 1998-08-17 WO g~l30160 PCTlUSg7102039 vector and/or such polynucleotides or enzymes could be part of a composition. and still be isolated in that such vector or composition is not part of its natural environment.

- -The enzymes of the present invention include the enzymes of SEQ ID NOS:33-42 (in particular the mature enzyme) as well as enzymes which have at least 7~% similarity (preferably at least 70% identity) to the enzymes of SEQ ID NOS:33~2 and more ~ preferably at least 90 % similarity (more preferably at least 90 % identity) to the enzymes of SEQ ID NOS:33~2 and still more preferably at least 95% similarity (still morepreferably at least 95% identi~y) to the enzymes of SEQ ID NOS:3342 and also include portions of such enzymes with such portion of the enzyme generally cont:3inin~ at least 30 arnino acids and more preferably at least 50 amino acids.

As known in the art "sirnilarity" between two enzymes is del~lll,hled by comparing lhe amino acid sequence and its conserved amino acid substitutes of one enzyme to the sequence of a second enzyme.

A variant, i.e. a "fragment", "analog" or "derivative" polypeptide, and reference ~olypeptide may differ in amino acid se~uence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination.

Among preferred variants are those that vary from a reference by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements aî~nong the aromatic residues Phe, Tyr.

' CA 02246737 1998-08-17 W O 97/30160 PCTrUS97/02039 Most highly ~rerelled are variants wh~ch retain the same biological function andactivity as the reference polypeptide from which it varies.

Fragments or portions of the enzymes of the present invention may be employed for producing the corresponding full-length enzyme by peptide synthesis; therefore, the fragments may be employed as interrn~ tPs for producing the full-length enzymes.Fragments or portions of the polynucleotides of the present invention may be used to synthesi7e full-length polynucleotides of the present invention.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of enzymes of the invention by recombinant techniques.

Host cells are genetically engineered (tr~n~ ce!1 or transforrned or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as a~ .""iate for activating promoters, selecting transformants or amplifying the genes of the present invention. The culture conditions, such as temperature, p~I and the like, are those previously used with the host cell selected for expression, and will be a~ nt to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed for producing enzymes by recombinant techniques. Thus, for examp}e, the polynucleotide may be included in any one of a variety of expression vectors for expressing an enzyme. Such vectors include chromosomal. nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast pl~mi-ls;vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus~ fowl pox virus. and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host. -~ CA 02246737 1998-08-17 W O g M 016U PCTnUS9~102U39 The a~l~ro~liate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an app~ iate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are - deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an ~ appropriate expression control sequence(s) ~promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, tltle ~. coli. Iac. or t~p, the pha~e lambda PL promoter and other promoters known to control expression of genes in prolcaryotic or eukaryotic cells or their viruses.
The expression vector also contains a ribosome binding site for translation initiation and a ~ ,s~ tion terminator. The vector may also include a~lol)liate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of ll~n~roLll~ed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

As representative examples of a~propriate hosts, there may be mentioned:
bacterial cells, such as E. coli. Streplom!ces, Bacillus subtilis; fungal cells, such as yeast; insect cells such as Drosopflila 52 and Spodoptera S~); animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an a~ iate host is deemed to be within the scope of those skilled in the art from the teal~hinp.s herein.

' CA 02246737 1998-08-17 W O 97/30160 PCTrUS97/02039 More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the - - invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embo-iiment, the construct further comprises regulatory s~equences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of exannple; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBluescript II KS, ptrc99a, pKK2~-3, pDR54(~, pRlT2T
(Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene) pSVK3, pBPV, pMSG, pSVL, SV40 (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacl, lacZ, T3, T7, gpt~ lambda PR, PL and trp. Eukaryotic promoters include CMV imlnediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the a~lo~iate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment. the present invention relates to host cells cont~ining the above-described constructs. The host cell can be a higher eukaryotic cell, such as a m~mm~ n cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mP(li~tecl transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)3.

. CA 02246737 1998-08-17 wo 97130160 PCT/US97102039 The constructs in host cells can be used in a conventional manner to produce thegene product encoded by the recombinant seqll~n~e. Alternatively, the enzymes of the invention can be synthetically produced by conventional peptide synthesizers.

Manlre proteins can be expressed in m~mm~ n cells, yeast, bacteria, or other cells under ~he control of applo~liate promoters. Cell-free translation systems can also ~ be employed to produce such ~uteins using RNAs derived from the DNA constructs of the present invention. Apl"opliate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., Molecular Cloning.-A Laboratory Mam~al, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby inco~porated by ,er~l~nce.

Transcription of the DNA encoding the enzymes of the present invention by higher eukaryotes is increased by inserting an enh~n~er sequence into the vector.
F.nh~nrers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples include the SV40 enh~nrer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyorna enhancer on the late side of the replication origin, and adenovirusenh~n~ers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of ~. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), cY-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in ap~ iate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of tr~n~!~t~d enzyme. Optionally, the heterologous sequence can encode a fusion enzyme including an N-ter~in~l identifir~tion peptide ,a~ ling desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

- - Useful ~ ssion vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include ~.
coli, RcJcill-~ subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but nonlimitin~ example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an approl.liate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strainto an appropriate cell density, the selected promoter is in~ ce~l by appl~liate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemi~means, and the resulting crude extract retained for further purification.

WO 97/30160 PCTtUS97102039 Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication~ meçh~nir~I disruption, or use of cell Iysing agents, such methods are well known tO those skilled in the art.

Various m~mm~ n cell culture systems can also be employed tO express recombinant protein. Examples of m~mm~ n expression systems include the COS-7 - lines of monkey kidney fibroblasts, described by Gll~7m~n~ Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3.
CHO, HeLa and BHK cell lines. ~mm~ n ~ ssion vectors will comprise an origin of replication, a suitable promoter and enh~nrer, and also any n~ce~ry ribosomc binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' fl~nkTng nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

The erl7yme can be recovered and purified from recombinant cell cultures by methods in~ ing ammonium sulfate or ethanol precipitation, acid extraction, anion or ~tion exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as nPces~ry, in completing configuration of the mature protein. ~inally, high performance liquidchromatography (HPLC) can be employed for final purification steps.

The enzymes of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and m~mm~ n cells in culture). Depending upon the host employed in a recombinantproduction procedure, the enzymes of the present invention may be glycosylated or may be non-glycosylated. Enzymes of the invention may or may not also include an initial methionine amino acid residue.

W O 97/30160 PCT~US97/02039 Esterases are a group of key enzymes in the metabolism of fats and are found in all Olg~ from microbes to l~ l.c. In the hydrolysis reaction, an ester group is hydrolysed to an organic acid and an alcohol.

Esterases enantiomerically dir~lenliate dicarboxylic diesters and di~ce~t-?s of diols. Using the approach disclosed in a commonly ~ n~-l, copending provisional app!ication Serial No. 60/008,316, filed on December ~, 1995 and entitled "Combinatorial Enzyme Development," the disclosure of which is incorporated herein by reference in its entirety, one could convert the enantiospecificity of the esterase.
Further, the thermostable esterases are believed to have superior stability at higher temperatures and in organic solvents. Thus, they are better suited for use in rigorous production procees which require robust catalysts.

There are a number of industrial and scientific applications for esterases, suchas those of the present invention, including:

1) Esterases are useful in the dairy industry as ripening starters for cheeses, such as the Swiss-type cheeses;

2) Esterases are useful in the pulp and paper industry for lignin removal from cellulose pulps, for lignin solubilization by cleaving the ester linkages between aromatic acids and lignin and between lignin and hemi~e~ oses~ and for disruption of cell wall structure when used in combination with xylanase and other xylan-degrading enzymes in biopulping and bioble~chin~ of pulps;

3) Esterases are useful in the synthesis of carbohydrate derivatives, such as sugar derivatives;

WO 97130160 PCTIUS97~2039 4) Esterases are useful, when combined with xylanases and cellu1ases, in the conversion of lignocellulosic wastes to fe~nentable sugars for producing a variety of chemicals and fuels;

5) Esterases are useful as research reagents in studies on plant cell wall structure, particularly the nature of covalent bonds between lignin and carbohydrate - po~ymers in the cell wall mat~ix;

6) Esterases are also useful as research reagents in studies on m~ch~ni~m.s related to disease resistance in plants and the process of organic matter decomposition;
and 7) Esterases are useful in selection of plants bred for production of highly digestible animal feeds, particularly for ~lmin~nt ~nim~

Antibodies generated a~ainst the enzymes co~ )ollding to a sequence of the present invention can be obtained by direct injection of the enzymes into an animal or by ~lmini~tering the enzymes to an animal, preferably a nonhllm~n. The antibody so obtained will then bind the enzymes itself. In this manner, even a sequence encoding only a fragment of the enzymes can be used to generate antibodies binding the whole native enzymes. Such antibodies can then be used to isolate the enzyme from cells expressin~ that enzyme.

For preparation of monoclonal antibodies, any technique which provides antibodies ]produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, Nature, 256:495-497, lg75), the triomatechnique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today4:72, 1983~, and the EBV-hybridoma technique to produce human monoclonal antibodies (~ole el al.~ in Monoclor2al Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985).

CA 02246737 l998-08-l7 W O 97/30160 PCT~US97/02039 Techniques described ~or the production of single chain antibodies (U.S. Patent - 4,946,77g) can be adapted to produce single chain antibodies to imml-nc~genic erl7yme products of this invention. Also, transgenic mice may be used to express hllm~ni7~od antibodies to immlmngenic enzyme products of this invention.

Antibodies generated against an enzyme of the present invention may be used in screening for sirnilar enzymes from other org~ni~m~ and samples. Such screening techniques are known in the art, for example, one such screening assay is described in Sambrook et ~l., Molecular Cloning: A Laboratory Manual (2d Ed.), Cold Spring Harbor Laboratory, Section 12.21-12.28 (1989) which is hereby incorporated by reference in its entirety.

The present invention will be further described with lc~relcl1ce to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.

In order to facilitate unders~n~lin~ of the following exa~ples certain frequently occurring methods and/or terms will be described.

"Plasmids" are ~lesi~n~tecl by a lower case "p" preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, e~uivalent plasmids to those described are known in the art and will be a~arel~t to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For ~ CA 02246737 1998-08-17 W O 97130~60 PCTnUS97102039 analytical purposes, typically 1 ~g of plasmid or DNA fragment is used with about 2 uI~its of enzyme in about 20 ,ul of buffer solution. For ~e purpose of isolating DNA
fragments for plasmid construction, typically S to 50 ~g of DNA are ~ligested with 20 - - to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the m~n-lf~ctllrer. lncubation times of about 1 hour at 37 C are ordinarily used, but may va~r in accordance with the~ supplier's instructions. After digestion ~e reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fr~gm~-nt~ is performed using 8 percent polyacrylamide gel described by Goeddel et al., Nucleic Acids Res., 8:4057 (1980).

"Oligonucleotides" refers to ei~er a single stranded polydeoxynucleotide or two complemen~ary polydeoxynucleotide strands which may be ch~rnic~lly synth.oci7P(lSuch synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleo~ide without adding a phosphate with an ATP in ~e presence of a kinase.
A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 ~bg of approximately equimolaramounts of the DNA fragments to be ligated.

~ Unless otherwise stated~ transformation was performed as described in Sambrooket al., Molecular Cloning: A Laboratory Manual (2d Ed.), Cold Spring Harbor Press (1989).

W O 97/30160 PCTrUS97/02039 lc 1 Bacterial Ex~ression and Puri~lcation of Esterases DNA encoding the enzymes of the present invention, SEQ ID NOS:33 through - - 42, were initially amplified from a pBluescript vector cont~inin~ the DNA by the PCR
t~chni~ using the primers noted herein. The ampli~led sequences were then inserted into the respective PQE vector listed beneath the primer sequences, and the enzyme was expressed according to the protocols set forth herein. The 5' and 3' primer sequences for the respective genes are as follows:

Staphylothermus marinus F1-12LC
5' CCGAGAATTC ATTAAAGAGG AGAAATTAAC TAT~'I'~'l''l"l'A AACAAGCACT CT
3' CGGAAGATCT CTATCGTTTA GTGTATGATT T
vector: pQET

Pyrodictium TAGl 1-17LC
5' CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGAAACTC CTTGAGCCCA CA Eco~
3' CGGAAGATCT CGCCGGTACA CCATCAGCCA C Bglll vector: pQET

Archaeoglobus venificus SNP6-24LC
S' CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGCCATAT GTTAGGAATG GT
3' CGGAGGTACC TTAGAACTGT GCTGAAGAAA TAAATTCGTC CATTGCTCT
3' CGGAGGTACC TTAGAACTGT GCTGAAGAAA TAAATTCGTC CATTGCTCTA TTA
vector: pQET

Aquifex pyrophilus - 28LC
5' CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGAGATTG AGGAAATTTG AAG
3' CGGAGGTACC CTATTCAGAA AGTACCTCTA A
vector: pQET

Ml lTL - 29LC
5' CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGTTTAAT ATCAATGTCT TT
3' CGGAAGATCT TTAAGGATTT TCCCTGGGTA G

. 28 . CA 02246737 1998-08-17 WO97J30160 PCT~S97/02039 vector: pQET

Ther nococcus CL-2 - 30LC
S' CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGGAGGTT TACA.AGGCCA AA
3' CGGAGGTACC TTATTGAGCC GAAGAGTACG A
- vector: pQET

Aquifex VF5 - 34LC
S' CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGATTGGC AATTTGAAAT TGA EcoRI
3' CGGAGGTACC TTAAAGTGCT CTCATATCCC C Kpnl vecto~: pQET

~eredinibacter 42L
5' CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGCCAGCT AATGACTCAC CC
3' CGGAAGATCT TCAACAGGCT CCAAATAATT TC (without His-~g) 3' CGGAAGATCT ACAGGCTCCA AATAATTTC (wi~ His-~g) vector: pQE12 ArchaeogZobus fulgidus VC16-16MC
5' CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGCTTGAT ATGCCAATCG AC ~coRl 3' CGGAGGTACC CTAGTCGAAG ACAAGAAGAG C Kpnl vector: pQET

Sulfolabus solfataricus P1-8LC
S' CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGCCCCAG GATCCTAGAA TT EcoRI
3' CGGAGGTACC TTAAATTTTA TCATAAAATA C Kplll vector: pQET
., .
The restriction enzyme sites indicated correspond to the restriction enzyme sites on the bacterial expression vector indicated for the respective gene (Qiagen, Inc.
~ Chatsworth, CA). The pQE vector encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites.

~ CA 02246737 1998-08-17 W O 97/30160 PCT~US97/02039 The pQE vector was digested with the restriction enzymes in-lic~tetl. The arnplified sequences were ligated into the respective pQE vector and inserted in frame with the sequence encoding for the I~BS. The ligation mixture was then used to - - transform the E. coli strain M15/pREP4 (Qiagen, Inc.) by electroporation. M15/pREP4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resi~t~n~e (Kan~). Transformants were identified by their ability to grow on LB plates and arnpicillin/kanamycin resistant colonies were selected.Plasmid DNA was isolated and con~ l by restriction analysis. Clones cons~ining the desired constructs were grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture was used to inoculate a large culture at a ratio of 1:100 to 1:250. 'Ihe cells were grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") was then added to a final concentration of 1 mM. IPTG
induces by inactivating the lacl repressor, clearing the P/O leading to increased gene expression. Cells were grown an extra 3 to 4 hours. Cells were then harvested bycentrifugation.

The primer sequences set out above may also be employed to isolate the target gene from the deposited material by hybridization technifllles described above.

Ex~mple 2 Isolation of a Selected Clone from the Deposite(l Genomic Clones The two oligonucleotide primers corresponding to the gene of interest are used to amplify ~e gene from the deposited material. A polymerase chain reactionis carried out in 25 ~l of reaction mixture with 0.1 ~g of the DNA of the gene of interest. The reaction mixture is 1.5-5 mM MgCl2, 0.01% ~w/v) gelatin, 20 ,uM
each of dATP, dCTP, dGTP, dTTP. 25 pmol of each primer and 1.25 Unit of Taq polymerase. Thirty cycles of PCR (denaturation at 94~C for 1 min; ~nn~ling at 55~C for l min; elongation at 72~C for 1 min) are performed with the Perkin-Elmer . CA 02246737 1998-08-17 W ~ 97/30160 PCTnUS97/02039 Cetus 9600 thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified tO be the gene of interest by subcloning and sequencing the DNA product.

FY~mple 3 Production of the Expression Gene Bank Colonies cont~inin~ pBluescript plasmids with random inserts from the orgzlni.~m~ M1 lTL, Thermococcus GUSL5, and Teredinibacter were obtained according to the method of Hay and Short, Stralegies, 5:16, 1992.

Example 4 Screenin~ for Li~ase/Esterase Activitv The resulting colonies were picked with sterile toothpicks and used to singly inoculate each of the wells of 96-well microtiter plates. The wells contained 2~0 ~L
of LB media with 100 ~g/mL ampicillim 80 ,ug/mL methicillin, and 10% v/v glycerol (LB Amp/Meth~ glycerol). The cells were grown overnight at 37~C without~h~king. This constituted ~eneration of the "Source GeneBank." Each well of the Source GeneBank thus contained a stocl; culture of E. coli cells, each of which contained a pBluescript with a unique DNA insert.

The plates of the Source GeneBank were used to multiply inoculate a single plate (the "Con~en.ce~i Plate") containin~ in each well 200 ~L of LB Amp/Meth, glycerol. This step was performed usin~ the High Density Replicating Tool (HDRT)of the Beclcman Biomek with a I '~t bleach, water, isop~ )allol, air-dry sterilization cycle in between each inoculation. Each well of the Con~ .cecl Plate thus contained W O 97/30160 PCTrUS97/02039 10 to 12 different pBluescript clones from each of the source Iibrary plates. The Con~ n~ed Plate was grown for 16 hours at 37~C and then used to inoculate two white 96-well Polyfiltronics microtiter ~ ght~r plates cont~ining in each well 250 - - ,uL of LB Amp/Meth (no glycerol). The original condensed plate was put in storage -80~C. The two condensed ~ ghter plates were inrub~tec~ at 37~C for 18 hours.

The short chain esterase '600 ~M substrate stock solution' was prepared as follows: 25 mg of each of the following compounds was dissolved in the ap~ iate volume of DMSO to yield a 25.2 mM solution. The compounds used were 4-methylumbelliferyl proprionoate, 4-methylumbelliferyl butyrate, and 4-methylumbelliferyl heptanoate. Two hundred fifty microliters of each DMSO
solution was added to ca 9 mL of 50 mM, pH 7.5 Hepes buffer which contained 0.6% of Triton X-100 and 0.6 mg per mL of dodecyl maltoside (Anatrace). The volume was taken to 10.5 mL with the above Hepes buffer to yield a slightly cloudy suspension.

The long chain '600 ,uM substrate stock solution' was yle~aled as follows:
25 mg of each of the following compounds was dissolved in ~MSO to 25.2 mM as above. The compounds used were 4-methylumbelliferyl elaidate, 4-methylumbelliferyl p~lmi~te, 4-methylumbelliferyl oleate, and 4-methylumbelliferyl stearate. All required brief warming in a 70~C bath to achieve dissolution. Two hundred fifty microliters of each DMSO solution was added to the Hepes buffer and diluted to 10.5 mL as above. All seven umbelliferones were obtained from Sigma Chemical Co.

Fifty ~L of the long chain esterase or short chain esterase '600 ~M substrate stock solution' was added to each of the wells of a white condensed plate using the Biomek to yield a final concentration of substrate of about I00 ,uM.. The fluorescence values were recorded (excitation = 326 nm, emission = 450 nm) on a plate-reading fluorometer im mer~i~tely after addition of the substrate. The plate was wo g7130160 PCTIUS97102039 incubated at 70~C for 60 minutes in the case of the long chain substrates, and 30 minutes at RT in tne case of the short chain substrates. The fluorescence valueswere recorded again. The initial and final fluorescence values were compared to - determine if an active clone was present.

Example 5 Isolation and Puri~lcation of the Active Clone To isolate the individual clone which carried the activity, the Source GeneBank plates were thawed and the individual wells used to singly inoculate a new plate con~ining LB Amp/Meth. As above, the plate was inr~1h~tr-l at 37~C to growthe cells, 50 ,LL of 600 ,uM substrate stock solution was added using the Biomek and the fluorescence was determined. Once the active well from the source plate was identified, cells from this active well were streaked on agar with LB/Amp/Meth and grown overnight at 37~C to obtain single colonies. Eight single colonies were picked witll a sterile toothpick and used to singly inoculate the wells of a 96-well microtiter plate. The wells contained 250 ~L of LB Amp/Meth. The cells were grown overnight at 37~C without ~h~king. A 200 ~L aliquot was removed from each well and assayed with the aL,pl-op,iate long or short chain substrates as above.
The most active clone was identified and the rem~inin~ 50 ~L of culture was used to streak an agar plate with LBlAmp/Meth. Eight single colonies were picked, grown and assayed as above. The most active clone was used to inoculate 3 mL cultures of LB/Amp/Meth, which were grown overnight. The plasmid DNA was isolated from the cultures and utilized for sequencing.

Numerous modifications and variations of the present invention are possible in light of the above trarhing~ and. therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.

W O 97130160 PCTrUS97/02039 ~U~N~ LISTING
( 1 ) ~.~M~AT. INFORMATION:
(i) APPLICANTS:
ROBERTSON, Daniel E.
MURPHY, Denni 8 REID, John MAFFIA, Anthony LINK, Steven SWANSON, Ronald V.
WARREN, Patrick V.
KOSMOTKA, Anna CALLEN, Walter (ii) TITLE OF lNV~N'l'lON:
ESTERASES

(iii) N~3ER OF ~QD~N~S: 62 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI, STEWART & OLSTEIN
(B' STREET: 6 BECKER FARM ROAD
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tD STATE: NEW JERSEY
(E~ COUN'1'K~: USA
(Fj ZIP: 07068 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 INCH DISKETTE
(B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WORD PERFECT 5.1 (vi) CURRENT APPLICATION DATA:
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(viii) AllORN~Y/AGENT INFORMATION:
(A) NAME: HERRON, CHARLES J.
(B) REGISTRATION NUMBER: 28,019 (C) REFERENCE/DOCKET NUMBER: 331400-39 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 201~994-1700 . 34 (B) TELEFAX: 201-994-1744 (2) INFORMATION FOR SEQ ID NO:1:
U~'N~h' CHARACTERISTICS
~A) LENGTH: 52 NUCLEOTIDES
- - ~B) TYPE NUCLEIC ACID
:C) ST~ANn~nMEqs: S INGLE
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(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
CCGAGAATTC ATTAAAGAGG AGAAATTAAC TA'l~l~L~ A AACAAGCACT CT 52 (2) INFORMATION FOR SEQ ID NO 2:
(i) SEQUENCE CHARACTERISTICS
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(ii) MOLECULE TYPE: CDNA
(Xi ) ~UU~N - '~' DESCRIPTION SEQ ID NO:2:
CGGAAGATCT CTA1~1~11A GTGTATGATT T 31 (2) INFORMATION FOR SEQ ID NO: 3:
(1) SEQUENCE CHARACTERISTICS
(A) LENGTH: 52 NUCLEOTIDES
(B) TYPE NUCLEIC ACID
(C) STR~Nn~nN~q.~: SINGLE
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(Xi) ~EQU~N~ DESCRIPTION SEQ ID NO:3 (2) 1N~O~ ~TION FOR SEQ ID NO:4:
W~N~'~' CHARACTERISTICS
(A) LENGTH 3 I NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
(C) STR~Nn~nN~S SINGLE
(D) TOPOLOGY LINEAR
(ii) MOLECULE TYPE CDNA
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

W O 97130160 PCT~US97/02039 (2) INFORMATION FOR SEQ ID NO:5:
U~N~'~ CHARACTERISTICS
(A) LENGTH: 52 NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
(C) STRANn~N~SS: SINGLE
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(ii) Mnr.~CTrr.~ TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

.. . . .
(2) INFORMATION FOR SEQ ID NO:6:
(i) S~U~N~ CHARACTERISTICS
(A) LENGTH: 53 NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
(C) STRAN~N~SS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 49 NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

(2) lN~O~ ~TION FOR SEQ ID NO:8:
(i) ~UU~N~ CHARACTERISTICS
(A) LENGTH: 53 NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) S~UU~N~ DESCRIPTION: SEQ ID NO:8:

. 36 .. CA 02246737 l998-08-l7 WO g7130~6~ - PCTIUS97102039 (2) lN~G~L.TION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS
(A LENGTH: 31 NUCLEOTIDES
(B TYPE: NUCLEIC ACID
(C~ STR~nRnNR-~S: SINGLE
~D) TOPOLOGY: LINEAR
(ii) MOhECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS
(A) hENGTH: S2 NUChEOTIDES
~8) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
~ii) MOhECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:lO:

(2) INFORMATION FOR SEQ ID NO:ll:
(i) ~D~N~: CHARACTERISTICS
(A) ~ENGTH: 31 NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
CGGAAGATCT TTAAGGATTT lCC~lGGGTA G 31 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 52 NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: .

(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS
(A) hENGTH: 31 NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE

W O 97/30160 PCT~US97/02039 (D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) ~Qu~N~ DESCRIPTION: SEQ ID NO:13:

(2) INFORMATION FOR SEQ ID NO:14:
( i ) S~U~N~' CHARACTERISTICS
~A) LENGTH: 53 NUCL~Ull~S
B) TYPE: NUCLEIC ACID
C) STR~Nn~nN~-~S: SINGLE
~D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: CDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS
tA) LENGTH: 31 NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
(C) STRAN~N~SS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) ~Uu~N~ DESCRIPTION: SEQ ID NO:15:

(2) INFORMATION FOR SEQ ID NO:16:
(i) ~Qu~N~ CHARACTERISTICS
~A LENGTH: 31 NUCLEOTIDES
(B TYPE: NUCLEIC ACID
(C STRANDEDNESS: SINGLE
(Dl TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) ~UU~N~ DESCRIPTION: SEQ ID NO:16:

(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS
A) LENGTH: 32 NUCLEOTIDES
B) TYPE: NUCLEIC ACID
C) STRANDEDNESS: SINGLE
~D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA

W ~ 97130160 PCTnUS97/02039 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

(2) INFORMATION FOR SEQ ID NO:18:
- - (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 29 NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
(C) ST~Nr~R~NE~ SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) S~Uu~Nch~ DESCRIPTION: SEQ ID NO:18:

(2) INFORMATION FOR SEQ ID NO:l9:
(i) ~UU~NU~' CHARACTERISTICS
'A) LENGTH: 52 NUCL~u~
~B) TYPE: NUCLEIC ACID
C) STR~NI)~:I)N~:.C.~ SINGLE
D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(Xi) ~UU~N~ DESCRIPTION: SEQ ID NO:l9:
CCGAGAATTC ATTAAAGAGG AGAAATTAAC TAl~LL~T ATGCCAATCG AC 52 (2) INFORMATION FOR SEQ ID NO:20:
(i) S~u~:N~ CHARACTERISTICS
(A) LENGTH: 31 NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
(C) STRAN~N~SS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOT~Cr~ TYPE: cDNA
(xi) S~Qu~Nu~ DESCRIPTION: SEQ ID NO:20:
CGGAGGTACC CTAGTCGAAC AGAAGA~GAG C 31 (2) INFORMATION FOR SEQ ID NO:21:
~i) SEQUENCE CHARACTERISTICS
(A LENGTH: 52 NUCLEOTIDES
(Bl TYPE: NUCLEIC ACID
(C STRANDEDNESS: SINGLE
(DJ TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(xi) ~uu~u~ DESCRIPTION: SEQ ID NO:21:
CCGAGAATTC ATTAAAGAGG AGAAATTAAC TAL~CCC~l~A GATCCTAGAA TT 52 . 39 ' CA 02246737 l998-08-l7 WO 97/30160 PCT~uS97/02039 -(2) INFORMATION FOR SEQ ID NO:22:
( i ) ~yU~N~ CHARACTERISTICS
~ (A) LENGTH: 31 NUCL~Oll~:S
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
- - (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CGGAGGTACC TTA~A'l-lTIA TCATAAAATA C 31 (2) INFORMATION FOR SEQ ID NO:23:
(i) ~UU~N~ CHARACTERISTICS
(A) LENGTH: 555 NUCL~ ~S
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: GENOMIC DNA
(Xi) ~U~:N~ DESCRIPTION: SEQ ID NO:23:

Met Ser Leu Asn Lys His Ser Trp Met Agp Met Ile Ile Phe Ile Leu Ser Phe Ser Phe Pro Leu Thr Met Ile Ala Leu Ala Ile Ser Met Ser Ser Trp Phe A6n Ile Trp Asn Asn Ala Leu Ser Asp Leu Gly His Ala Val Lys Ser Ser Val Ala Pro Ile Phe Agn Leu Gly Leu Ala Ile Gly Gly Ile Leu Ile Val Ile Val Gly Leu Arg Asn Leu Tyr Ser Trp Ser Arg Val Lys Gly Ser Leu Ile Ile Ser Met Gly Val Phe Leu Asn Leu Ile Gly Val Phe Asp Glu Val Tyr Gly Trp Ile Hi6 Phe Leu Val Ser Val Leu Phe Phe Leu Ser Ile Ile Ala Tyr Phe Ile Ala Ile Ser Ile Leu Asp Lys Ser Trp Ile Ala Val Leu Leu Ile Ile Gly Hi6 Ile Ala Met Trp Tyr Leu His Phe Ala Ser Glu Ile Pro Arg Gly Ala Ala Ile WO 97/30160 PCTJUS97~02039 CCC GAG TrA TTA GCG GTA TTC TCG TTT TTA CCA TTC TAT ATA AGA CAG 528 Pro Glu Leu Leu Ala Val Phe Ser Phe Leu Pro Phe Tyr Ile Arg Asp Tyr Phe hys Ser Tyr Thr Lys Arg - (2) INFORMATION FOR SEQ ID NO:24:
(i) ~Uu~N~ CHARACTE~ISTICS
(A) LENGTH: 1041 NUCLEOTIDES
~ (B) TYPE: NUCLEIC ACI~
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: GENOMIC DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
ATG A~A CTC CTT GAG CCC ACA AAT ACC TCC TAC ACG CTG TTA CAG GAT 48 Met Lys Leu Leu Glu Pro Thr Asn Thr Ser Tyr Thr Leu Leu Gln Asp TTA GCA TTG CAT TTT GCA TTT TAC TGG TTT CTG GCC GTG TAT ACG TGG

Leu Ala Leu His Phe Ala Phe Tyr Trp Phe Leu Ala Val TYr Thr Trp Leu Pro Gly Val Leu Val Arg Gly Val Ala Val Asp Thr Gly Val Ala Arg Val Pro Gly Leu Gly Arg Arg Gly Lys Arg Leu Leu Leu Ala Ala Val Ala Val Leu Ala Leu Val Val Ser Val Val Val Pro Ala Tyr Val Ala Tyr Ser Ser Leu His Pro Glu Ser Cys Arg Pro Val Ala Pro Glu 85 90 g5 GGG CTC ACC TAC A~A GAG TTC AGC GTG ACC GCG GAG GAT GGC TTG GTG 336 Gly Leu Thr Tyr Lys Glu Phe Ser Val Thr Ala Glu Asp Gly Leu Val Val Arg Gly Trp Cal Leu Gly Pro Gly Ala Gly Gly Asn Pro Val Phe 1~.5 120 125 Val Leu Met His Gly Tyr Thr Gly Cys Arg Ser Ala Pro Tyr Met Ala Val Leu Ala Arg Glu Leu Val Glu Trp Gly Tyr Pro Val Val Val Phe GAC TTC CG& GGC CAC GGG GAG AGC &GG GGC TCG ACG ACG ATT GGG CCC 528 Asp Phe Arg &ly His Gly Glu Ser Gly Gly Ser Thr Thr Ile Gly Pro CA 02246737 l998-08-l7 WO 97t30160 PCTrus97/02039 -Arg Glu Val Leu Asp Ala Arg Ala Val Val Gly Tyr Val Ser Glu Arg Phe Pro Gly Arg Arg Ile Ile Leu Val Gly Phe Ser Met Gly Gly Ala Val Ala Ile Val Glu Gly Ala Gly Asp Pro Arg Val Tyr Ala Val Ala Ala Asp Ser Pro Tyr Tyr Arg Leu Arg A p Val Ile Pro Arg Trp Leu Glu Tyr Lys Thr Pro Leu Pro Gly Trp Val Gly Val Leu Ala Gly Phe Tyr Gly Arg Leu Met Ala Gly Val Asp Leu Gly Phe Gly Pro Ala Gly 260 265 2'70 GTG GAG CGC GTG GAT AAG CCG TTG CTG GTG GTG TAT GGG CCC CGG &AC 864 Val Gly Arg Val Asp Lys Pro Leu Leu Val Val Tyr Gly Pro Arg Asp Pro Leu Val Thr Arg Asp Glu Ala Arg Ser Leu Ala Ser Arg Ser Pro TGT GGC CGT CTC GTC GAG GTT CCT GGG GCT GGC CAC GTG GAG &CC GTG 960 Cys Gly Arg Leu Val Glu Val Pro Gly Ala Gly His Val Glu Ala Val Asp Val Leu Gly Pro Gly Arg Tyr Ala Asp Met Leu Ile Glu Leu Ala His Glu Glu Cys Pro Pro Gly Ala Gly Gly 2) INFORMATION EOR SEQ ID NO:25:
(i) S~u~ CHARACTERISTICS
(A) LENGTH: 789 NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: GENOMIC DNA

CA 02246737 l998-08-l7 Wo 97/30160 P~TIUS971û~039 (xi) ~yU~N~ DESCRIPTION: SEQ ID NO:25:

Met Pro Tyr Val Arg Asn Gly Gly Val Asn Ile Tyr Tyr Glu Leu Val - - Asp Gly Pro Glu Pro Pro Ile Val Phe Val His Gly Trp Thr Ala Asn ATG AAT TTT TGG A~A GAG CAA AGA CGT TAT TTT GCA GGC AGG AAT ATG 144 Met Asn Phe Trp Lys Glu Gln Arg Arg Tyr Phe Ala Gly Arg Asn Met ~ ATG TTG TTT GTC GAT AAC AGA GGT CAT GGC AGG TCC GAT AAG CCA CTT 192 Met Leu Phe Val Asp Asn Arg Gly HiS Gly Arg Ser Asp Lys Pro Leu GGA TAC GAT TTC TAC AGA TTT GAG AAC TTC ATT TCA GAT TTA GAT &CG 240 Gly Tyr Asp Phe Tyr Arg Phe Glu Asn Phe Ile Ser Asp Leu Asp Ala Val Val Arg Glu Thr Gly Val Glu Lys Phe Cal Leu Val Gly His Ser 85 go 95 Phe Gly Thr Met Ile Ser Met Lys Tyr Cys Ser Glu Tyr Arg ARn Arg Val Leu Ala Leu Ile Leu Ile Gly Gly Gly Ser Arg Ile Lys Leu Leu Hls Arg Ile Gly Tyr Pro Leu Ala Lys Ile ~eu Ala Ser Ile Ala Tyr Lys Lys Ser Ser Arg Leu Val Ala Asp Leu Ser Phe Gly Lys Asn Ala GGT GAA CTT AAA GAG TGG GGA TGG AAA CAG GC~ ATG GAT TAT ACA CCC 528 Gly Glu Leu Lys Glu Trp Gly Trp Lys Gln Ala Met Asp Tyr Thr Pro TCC TAC GTG GCA ATG GAC ACG TAC AGA ACT CTA ACG AAA GTG A~T CTT 576 Ser Tyr Val Ala Met Tyr Thr Tyr Arg Thr Leu Thr Lys Val Asn Leu Glu Asn Ile Leu Glu Lys Ile Asp Cys Pro Thr Leu Ile Ile Val Gly Glu Glu Asp Ala Leu Leu Pro Val Ser Lys Ser Val Glu Leu Ser Arg Arg Ile Glu Asn Ser Lys Leu Val Ile Ile Pro Asn Ser Gly His Cys GTA ATG CTT GAG AGT CCA AGT GAG GTT AAT AGA GCA ATG GAC G~AA TTC 768 Val Met Leu Clu Ser Pro Ser Glu Val Asn Arg Ala Met Asp Glu Phe CA 02246737 l998-08-l7 W O 97/30160 PCTrUS97/02039 -Ile Ser Ser Ala Gln Phe ~2~ INFORMATION FOR SEQ ID NO:26:
- - (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 756 NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
( C ) STR A N ~ N ~ S: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: GENOMIC DNA
(Xi) ~UU~N~ DESCRIPTION: SEQ ID NO:26:
TTG AGA TTG AGG A~A TTT GAA GAG ATA AAC CTC GTT CTT TCG GGA GGA 48 Leu Arg Leu Arg Lys Phe Glu Glu Ile Asn Leu Val Leu Ser Gly Gly l 5 10 15 GCT GCA AAG GGC ATA GCC CAC ATA GGT GTT TT& AAA GCT ATA AAC GAG 96 Ala Ala Lys Gly I}e Ala His Ile Gly Val Leu Lys Ala Ile Asn Glu Leu Glu Ile Arg Val Arg Ala Leu Ser Gly Val Ser Ala Gly Ala Ile Val Ser Val Phe Tyr Ala Ser Gly Tyr Ser Pro Glu Gly Met Phe Ser Leu Leu Lys Arg Val Asn Trp Leu Lys Leu Phe Lys Phe Lye Pro Pro CTG AAG GGA TTG ATA GGG TGG GAG AAG GCT ATA AGA TTC CTT GAG GA~ 288 Leu Lys Gly Leu Ile Gly Trp Glu Lys Ala Ile Arg Phe Leu Glu Glu Val Leu Pro Tyr Arg Arg Ile Glu Lys Leu GLu Ile Pro Thr Tyr Ile Cys Ala Thr Asp Leu Tyr Ser Gly Arg Ala Leu Tyr Leu SEr Glu Gly Ser Leu Ile Pro Ala Leu Leu Gly Ser Cy~ Ala Ile Pro Gly Ile Phe Glu Pro Val Glu Tyr Lys Asn Tyr Leu Leu Val Asp Gly Gly Ile Val Asn Asn Leu Pro Val Glu Pro Phe Gln Glu Ser Gly Ile Pro Thr Val Cys Val Asp Val Leu Pro Ile Glu Pro Glu Lys Asp Ile Lys Asn Ile CA 02246737 l998-08-l7 Wo 97J30160 PCTIUS97/02039 heu His Ile Leu Leu Arg Ser Phe Phe heu Ala Val Arg Ser Asn Ser ~ 195 200 205 Glu Lys Arg Lys Glu Phe Cys Asp Leu Val Ile Val Pro Glu Leu Glu GAG TTC ACA CCC CTT GAT GTT AGA A~A GCG GAC CAA ATA ATG GAG AGG 720 Glu Phe Thr Pro Leu Asp Val Arg LYB Ala Asp Gln Ile Met Glu Arg Gly Tyr Ile Lys Ala Leu Glu Val Leu Ser Glu (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS
~A) LENGTH: 894 NUCLEOTIDES
(B~ TYPE: NUChEIC ACID
tC) STRANDEDNESS: SINGLE
~D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: GENOMIC DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

Met Phe A~n Ile Asn Val Phe Val Asn Ile Ser Trp Leu Tyr Phe Ser GGG ATA Gl'T ATG AAG ACT GTG GAA GAG TAT GCG CTA CTT GAA ACA GGC 96 Gly Ile Val Met Lys Thr Val Glu Glu Tyr Ala Leu Leu Glu Thr Gly GTA AGA GTG TTT TAT CGG TGT GTA ATC CCG GAG A~A GCT TTT AAC ACT 144 Val Arg Val Phe Tyr Arg Cys Val Ile Pro Glu Lys Ala Phe Asn Thr Leu Ile Ile Gly Ser His Gly Leu Gly Ala His Ser Gly Ile Tyr Ile Ser Val Ala Glu Glu Phe Ala Arg His Gly Phe Gly Phe Cy8 Met His Asp Gln Arg Gly His Gly Arg Thr Ala Ser Asp Arg Glu Arg Gly Tyr _ Val Glu Gly Phe Hi8 Asn Phe Ile Glu Asp Met Lys Ala Phe Ser A8p TAT GCC A~G TGG CGC GTG GGA GGT GAC GAA ATA ATA TTG CTA GGA CAC 384 Tyr Ala Lys Trp Arg Val Gly Gly Asp Glu Ile Ile Leu Leu Gly His AGT ATG GGC GGG CTG ATA GCG CTC GGA ACA GTT GCA ACT TAT AAA GA~ 432 Ser Met Gly Gly Leu Ile Ala Leu Leu Thr Val Ala Thr Tyr Lys Glu .

CA 02246737 l998-08-l7 W O 97/30160 PCT~US97/02039 Ile Ala Lys Gly Val Ile Ala heu Ala Pro Ala Leu Gln Ile Pro Leu Thr Pro A}a Arg Arg Leu Val Leu Ser Leu Ala Ser Arg Leu Ala Pro ~ - CAT TCT AAG ATC ACC TTA CAA AGG AGA TTG CCG CAG A~A CCA GAG GGT 576 His Ser Lys Ile Thr Leu Gln Arg Arg Leu Pro Gln Lys Pro Glu Gly Phe Gln Arg Ala Lys Asp Ile Glu Tyr Ser Leu Ser Glu Ile Ser Val AAG CTC GTG GAC GAA ATG ATT A~A GCA TCA TCT ATG TCT TGG ACC ATA 672 Lys Leu Val Asp Glu Met Ile Lys Ala Ser Ser Met Phe Trp Thr Ile GCA GGG GAA ATT AAT ACT CCC GTC CTG CTT ATT CAT GGG GAA A~A CAG 720 Ala Gly Glu Ile Asn Thr Pro Val Leu Leu Ile His Gly Glu Lys Asp AAT GTC ATA CCT CCG GAG GCG AGC A~A A~A GCC RTAC CAA TTA ATA CCT 768 Asn Val Ile Pro Pro Glu Ala Ser Lys Lys Als Tyr Gln Leu Ile Pro TCA TTC CCT A~A GAG TTG A~A AAA TAC CCC GAT CTT GGA CAC AAC TTG 816 Ser Phe Pro Lys Glu Leu Lys Ile Tyr Pro Asp Leu Gly His Asn Leu Phe Phe Glu Pro Gly Ala Val Lys Ile Val Thr Asp Ile Val Glu Trp Val Ly~ Asn Leu Pro Arg Glu Asn Pro (2) INFORMATION FOR SEQ ID NO:28:
(i) ~yu~N~ CHARACTERISTICS
~A) LENGTH: 789 NUCL~Oll~S
~B) TYPE: NUCLEIC ACID
~C) STRPNn~n~qS: SINGLE
~D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: GENOMIC DNA
(Xi) S~QU~N~ DESCRIPTION: SEQ ID NO:28:
ATG GAG GTT TAC AAG GCC A~A TTC GGC GAA GCA AAG CTC GGC TGG GTC 48 Met Glu Val Tyr Lys Ala Lys Phe Gly Glu Ala Lys Leu Gly Trp Val Val Leu Val His Gly Leu Gly Glu His Ser Gly Arg Tyr Gly Arg Leu Ile Lys Glu Leu Asn Tyr Ala Gly Phe Gly Val Tyr Thr Phe Asp Trp 35 ~0 45 ~ CA 02246737 1998-08-17 W 097J30160 PcTnusg7f02039 pro Gly His Gly Lys Ser Pro Gly Lys Arg Gly His Thr Ser Val Glu Glu Ala Met Glu Ile Ile Asp Ser Ile Ile Glu Glu Ile Arg Glu Lys - - CCC TTC CTC TTC GGC CAC AGC CTC GGT GGT CTA ACT GTC ATC AGG TAC 288 Pro Phe Leu Phe Gly His Ser Leu Gly Gly Leu Thr Val Ile Arg Tyr GCT GAG ACG. CGG CCC GAT A~A ATA CGG GGA TTA ATA GCT TCC TCG CCT 336 Ala Glu Thr Arg Pro Asp Lys Ile Arg Gly Leu Ile Ala Ser Ser Pro 100 105 .110 Ala Leu Ala Lys Ser Pro Glu Thr Pro Gly Phe Met Val Ala Leu Ala Lys Phe Leu Gly Lys Ile Ala Pro Gly Val Val Leu Ser Asn Gly Ile Lys Pro Glu Leu Leu Ser Arg Asn Arg Asp Ala Val Arg Arg Tyr Val Glu A~p Pro Leu Val HiS Asp Arg Ile Ser Ala Lys Leu Gly Arg Ser ATC TTC GTG AAC ATG GAG CTG GCC CAC AGG GAG GCG GAC AAG ATA A~A 5'76 Ile Phe Val Asn Met Glu Leu Ala His Arg Glu Ala Asp Lys Ile Lys Val Pro Ile Leu Leu Leu Ile Gly Thr Gly Asp Val Ile Thr Pro Pro Glu Gly Ser ARg Arg Leu Phe Glu Glu Leu Ala Val Glu Asn Lys Thr Leu Arg Glu Phe Glu Gly Ala Tyr His Glu Ile Phe Glu Asp Pro Glu TGG GCC GAG GAG TTC CAC GAA ACA ATT GTT AAG TGG CTG GTT GAA A~A 768 Trp Ala Glu Glu Phe His Glu Thr Ile Val Lys Trp Leu Val Glu Lys TCG TAC TCT TCG GCT CAA TAA 7'75 ~- Ser Tyr Ser Ser Ala Gln (2) INFORMATION FOR SEQ ID NO:29:
ti) SEQUENCE CHARACTERISTICS
(A) LENGTH: 750 NUCLEOTIDES
tB) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) M~T.~Cr~ TYPE: GENOMIC DNA

WO 97/30160 PCT~us97/02039 (xi) ~uu~h DESCRIPTION: SEQ I~ NO:29:

Leu Ile Gly Asn Leu Lys Ley Lys Arg Phe Glu Glu Val Asn Leu Val - ~ Leu Ser Gly Gly Ala Ala Lys Gly Ile Ala ~i8 Ile Gly Val Leu Lys Ala Leu Glu Glu Leu Gly Ile Lys Val Lys Arg Leu Ser Gly Val Ser Ala Gly Ala Ile Val Ser Val Phe Tyr Ala Ser G y Tyr Thr Pro Asp GAG ATG TTA AAA CTC CTG AaA GAG GTA AAC TGG CTC AAA CTT TTT AAG 240 Glu Met Leu Lys Leu Leu Lys Glu Val Asn Trp Leu Lys Leu Phe Lys TTC AAA ACA CCG AAA ATG GGC TTA ATG GGG TGG GAG A~G GCT GCA GAG 288 Phe Lys Thr Pro Lys Met Gly Leu Met Gly Trp Glu Lys Ala Ala Glu Phe Leu Glu Lys Glu Leu Gly Val Lys Arg Leu Glu Asp Leu Asn Ile Pro Thr Tyr Leu Cys Ser Ala Asp Ley Tyr Thr Gly Lys Ala Leu Tyr Phe Gly Arg Gly Asp Leu Ile Pro Val Leu Leu Gly Ser Ly~ Ser Ile Pro Gly Ile Phe Glu Pro Val Glu Tyr Glu Asn Phe Leu Leu Val Asp Gly Gly Ile Val Asn Asn Leu Pro Val Glu Pro Leu Glu Ly~s Phe Lys Glu Pro Ile Ile Gly Val Asp Val Leu Pro Ile Thr Gln Glu Arg Lys Ile Lye Asn Ile Leu His Ile Leu Ile Arg Ser Phe Phe Leu Ala Val Arg SEr Asn Ser Glu Lys Arg Lys Glu Phe Cys Asn Val Val Ile Glu Pro Pro Leu Glu Glu Phe Ser Pro Leu Asp Val Asn Lys Ala Asp Glu ~ CA 02246737 1998-08-17 97/30160 PCTn~S97J0~03g Ile Phe cy6 Gly Asp Met Arg Ala Leu (2) INFORMATION FOR SEQ ID NO:30:
?U~;N~J~: CHARACTERISTICS
(A) LENGTH: 1017 NUCLEOTIDES
~B) TYPE: NUCLEIC ACID
~C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii ) M~T.~CT~.~ TYPE: GENOMIC DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

Met Pro Ala Asn Asp Ser Pro Thr Ile Asp Phe Asn Pro Arg Gly Ile CTT CGC AAC GCT CAC GCA CAG GTT ATT TTA GCG ACT TCC GGC TTG CGC g6 Leu Arg Asn Ala His Ala Gln Val Ile Leu Ala Thr Ser Gly ~eu Arg Lys Ala Phe Leu Lys Arg Thr His Lys Ser Tyr Leu Ser Thr Ala Gln Trp Leu Glu Leu Asp Ala Gly Asn Gly Val Thr Leu Ala Gly Glu Leu Asn Thr Ala Pro Ala Thr Ala Ser Ser Ser His Pro Ala His Lys Asn Thr Leu Val Ile Val Leu His Gly Trp Glu Gly Ser Ser Gln Ser Ala Tyr Ala Thr Ser Ala Gly Ser Thr Leu Phe Asp Asn Gly Phe Asp Thr Phe Arg Leu Asn Phe Arg Asp His Gly Asp Thr Tyr His Leu Asn Arg GGC ATA TIT AAC TCA TCG CTG ATT GAC GAA GT~ GTG GGC GCA GTC AAA 432 Gly Ile Phe Asn Ser Ser Leu Ile Asp Glu Val Val Gly Ala Val Lys Ala Ile Gln Gln Gln Thr Asp Tyr Asp Lys Tyr Cys Leu Met Gly Phe Ser Leu Gly Gly Asn Phe Ala Leu Arg Val Ala Val Arg Glu Gln His Leu Ala Lys Pro Leu Ala Gly Val Leu Ala Val Cys Pro Val Leu Asp . 49 CA 02246737 l998-08-l7 W O 97/30160 PCTrUS97/02039 Pro Ala His Thr Met Met Ala Leu Asn Arg Gly Ala Phe P~e Tyr Gly Arg Tyr Phe Ala His Lys Trp Lys Arg Ser Leu Thr Ala Lys Leu Ala - - GCT TTC CCA GAC TAC AAA TAC GGC A~A GAT TTA AAA TCG ATA CAC ACG 720Ala Phe Pro Asp Tyr Lys Tyr Gly Lys Asp Leu Lys Ser Ile His Thr Leu Asp Glu Leu Asn Asn Tyr Phe Ile Pro Arg Tyr Thr Gly Phe Asn Ser Val Ser Glu Tyr Phe Lys Ser Tyr Thr Leu Thr Gly Gln Lys Leu Ala Phe Leu Asn Cys Pro Ser Tyr Ile Leu Ala Ala Gly Asp Asp Pro ATA ATT CCA GCA TCC GAC TTT CAG A~A ATA GCC AAG CCT GCG AAT CTG 912 Ile Ile Pro Ala Ser Asp Phe Gln Lys Ile Ala Lys Pro Ala Asn Leu His Ile Thr Val Thr Gln Gln Gly Ser His Cys Ala Tyr Leu Glu Asn CTG CAT AAA CCT AGT GCT GCC GAC A~A TAT GCG GTG A~A TTA TTT GGA 1,008 Leu His Lys Pro Ser Ala Ala Asp Lys Tyr Ala Val Lys Leu Phe Gly GCC TGT TGA 1,111 Ala Cys ~2) INFORMATION FOR SEQ ID NO:31:
( i ) ~U~N~ CHARACTERISTICS
(A) LENGTH: 936 NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: GENOMIC DNA
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:31:

Met Leu Asp Met Pro Ile Asp Pro Val Tyr Tyr Gln Leu Ala Glu Tyr Phe Asp Ser Leu Pro Lys Phe Asp GLn Phe Ser Ser Ala Arg Glu Tyr Arg Glu Ala Ile Asn Arg Ile Tyr Glu Glu Arg Asn Arg Gln Leu Ser Gln His Glu Arg Val Glu Arg Val Glu Asp Arg Thr Ile Lys Gly Arg 5(~

W ~ ~7/30160 PCTnJS97/02039 Asn Gly Asp Ile Arg Val Arg Val Tyr Gln Gln Lys Pro ASp Ser Pro Val Leu Val Tyr Tyr His Gly Gly Gly Phe Val Ile Cys Ser Ile Glu 85 ~ 9O

Ser HIs Asp Ala Leu Cys Arg ARg Ile Ala Arg Leu Ser Asn Ser Thr Val Val Ser Val Asp Tyr Arg Leu Ala Pro Glu His Lys Phe Pro Ala Ala Val Tyr Asp Cys Tyr Aso Ala Thr Lys Trp Val Ala Glu Asn Ala Glu Glu Leu Arg Ile Asp Pro Ser Lys Ile Phe Val Gly Gly Asp Ser Ala Gly Gly Asn Leu Ala Ala Ala Val Ser Ile Met Ala Arg Asp Ser Gly Glu Asp Phe Ile Lys His Gln Ile Leu Ile Tyr Pro Val Val Asn Phe Val A].a Pro Thr Pro Ser Leu Leu Glu Phe GLy Glu Gly Leu Trp Ile Leu Asp Gln Lys Ile Met Ser Trp Phe Ser Glu Gln Tyr Phe Ser Arg Glu Glu Aso Lys Phe Asn Pro Leu Ala Ser Val Ile Phe Ala Asp Leu Glu Asn Leu Pro Pro Ala Leu Ile Ile Thr Ala Glu Tyr Asp Pro Leu Arg Asp Glu Gly Glu Val Phe Gly Gln Met Leu Arg Arg Ala Gly Val Glu Ala Ser Ile Val Arg Tyr Arg Gly Val Leu His Gly Phe Ile Asn Tyr Tyr Pro Val Leu Lys Ala Ala Arg Asp Ala Ile Asn Gln Ile Ala Ala Leu leu Val Phe Asp 97/30160 PCTrus97/02039 (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 918 NUCLEOTIDES
(B) TYPE: NUCLEIC ACID
(C) STR~n~nM~S: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: GENOMIC DNA
(Xi ) S~YU~N~ DESCRIPTION: SEQ ID NO:32:

Met Pro Leu Asp Pro Arg Ile Lys Ly6 Leu Leu Glu Ser Ala Leu Thr ATA CCA ATT GGT AAA GCC CCA GTA GAA GAG GTA AGA AAG A~.A TTT AGG 96 Ile Pro Ile Gly Lys Ala Pro Val Glu Glu Val Arg Lys Ile Phe Arg CAA TTA GCG TCG GCA GCT CCC A~A GTC GAA GTT GGA AAA GTA GAA GAT 144 Gln Leu Ala Ser Ala Ala Pro Lys Val Glu Val Gly Lys Val Glu Asp Ile Lys Ile Pro Gly Ser Glu Thr Val Ile Asn Ala Arg Val Tyr Phe Pro Lys Ser Ser Gly Pro Tyr Gly Val Leu Val Tyr Leu His Gly Gly Gly Phe Val Ile Gly Asp Val Glu Ser Tyr Asp Pro Leu Cys Arg Ala Ile Thr Asn Ala Cys Asn Cys Val Val Val Ser Val Asp Tyr Arg Leu Ala Pro Glu Tyr Lys Phe Pro Ser Ala Val Ile Asp Ser Phe Asp Ala Thr Asn Trp Val Tyr Asn Asn Leu Asp Lyg Phe Asp Gly Lys Met Gly Val Ala Ile Ala Gly Asp Ser Ale Gly Gly Asn Leu Ala Ala Val Val Ala Leu Leu Ser Lys Gly Lys Ile Asn Leu Lys Tyr Gln Ile Leu Val Tyr Pro Ala Val Ser Leu Asp Asn Val Ser Arg Ser Met Ile Glu Tyr Ser ARP Gly Phe Phe Leu Thr Arg Glu Hls Ile Glu Trp Phe Gly Ser WO 97130160 PCTIUS97/~2039 Gln Tyr Leu Arg Ser Pro Ala Asp Leu Leu Asp Phe Arg Phe Ser Pro Ile Leu Ala Gln Asp Phe Asn Gly Leu Pro Pro Ala Leu Ile Ile Thr Ala Glu Tyr Asp Pro Leu Arg Asp Gln Gly Glu Ala Tyr Ala Asn Lys Leu Leu Gln Ala Gly Val Ser Val Thr Ser Val Arg Phe.Asn Asn Val Ile His Gly Phe Leu Ser Phe Phe Pro Leu Met Glu Gln Gly Arg Asp Ala Ile Gly Leu Ile Gly Ser Val Leu Arg Arg Val Phe Tyr Asp Lys Ile (2) INFORMATION FOR SEQ ID NO:33:
(i) ~u~ CHARACT~RISTICS
(A) LENGTH: 184 AMINO ACIDS
(B) TYPE: AMINo ACID
~D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIM
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Met Ser Leu Asn Lys His Ser Trp Met Asp Met Ile Ile Phe Ile Leu Ser Phe Ser Phe Pro Leu Thr Met Ile Ala Leu Ala Ile Ser Met Ser Ser Trp Phe Asn Ile Trp Asn Asn Ala Leu Ser Asp Leu Gly His Ala Val Lys Ser Ser Val Ala Pro Ile Phe Asn Leu Gly Leu Ala Ile Gly Gly Ile Leu Ile Val Ile Val Gly Leu Arg Asn Leu Tyr Ser Trp Ser Arg Val Lys Gly Ser Leu Ile Ile Ser Met Gly Val Phe Leu Asn Leu Ile Gly Val Phe Asp Glu Val Tyr Gly Trp Ile ~is Phe Leu Val Ser Val Leu Phe Phe Leu Ser Ile Ile Ala Tyr Phe Ile Ala Ile Ser Ile Leu Asp Lys Ser Trp Ile Ala Val Leu Leu Ile Ile Gly His Ile Ala ~ CA 02246737 1998-08-17 W O 97/30160 PC~nUS97/02039 Met Trp Tyr Leu His Phe Ala Ser Glu Ile Pro Arg Gly Ala Ala Ile Pro Glu Leu Leu Ala Val Phe Ser Phe Leu Pro Phe Tyr Ile Arg Asp Tyr Phe Lys Ser Tyr Thr Lys Arg (2) INFORMATION FOR SEQ ID NO:34:
(i) ~QU~N~ CHARACTERISTICS
(A) LENGTH: 346 AMINO ACIDS
- (B) TYPE: AMINO ACID
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:34:
Met Lys Leu Leu Glu Pro Thr Asn Thr Ser Tyr Thr Leu Leu Gln Asp Leu Ala Leu His Phe Ala Phe Tyr Trp Phe Leu Ala Val TYr Thr Trp Leu Pro Gly Val Leu Val Arg Gly Val Ala Val Asp Thr Gly Val Ala Arg Val Pro Gly Leu Gly Arg Arg Gly Lys Arg Leu Leu Leu Ala Ala Val Ala Val Leu Ala Leu Val Val Ser Val Val Val Pro Ala Tyr Val Ala Tyr Ser Ser Leu His Pro Glu Ser Cys Arg Pro Val Ala Pro Glu Gly Leu Thr Tyr Lys Glu Phe Ser Val Thr Ala Glu Asp Gly Leu Val Val Arg Gly Trp Cal Leu Gly Pro Gly Ala Gly Gly Asn Pro Val Phe Val Leu Met His Gly Tyr Thr Gly Cys Arg Ser Ala Pro Tyr Met Ala Val Leu Ala Arg Glu Leu Val Glu Trp Gly Tyr Pro Val Val Val Phe Asp Phe Arg Gly His Gly Glu Ser Gly Gly Ser Thr Thr Ile Gly Pro Arg Glu Val Leu Asp Ala Arg Ala Val Val Gly Tyr Val Ser Glu Arg Phe Pro Gly Arg Arg Ile Ile Leu Val Gly Phe Ser Met Gly Gly Ala Val Ala Ile Val Glu Gly Ala Gly Agp Pro Arg Val Tyr Ala Val Ala Ala Asp Ser Pro Tyr Tyr Arg Leu Arg Asp Val Ile Pro Arg Trp Leu CA 02246737 l998-08-l7 WO 97/30160 PCTJUS9~/02039 Glu Tyr Lys Thr Pro Leu Pro Gly Trp Val Gly Val Leu Ala Gly Phe Tyr Gly Arg Leu Met Ala Gly Val Asp Leu Gly Phe Gly Pro Ala Gly Val Gly Arg Val Asp Ly8 Pro Leu Leu Val Val Tyr Gly Pro Arg Asp Pro Leu Val Thr Arg Asp Glu Ala Arg Ser Leu Ala Ser Arg Ser Pro Cys Gly Arg Leu Val Glu Val Pro Gly Ala Gly His Val Glu Ala Val Asp Val Leu Gly Pro Gly Arg Tyr Ala Asp Met Leu Ile Glu Leu Ala His Glu Glu cy8 Pro Pro Gly Ala Gly Gly 3g0 345 (2) INFORMATION FOR SEQ ID NO:35:
~i) S~U~N~'~: CHARACTERISTICS
~A) LENGTH: 262 AMINO ACIDS
(B) TYPE: AMINO ACID
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Met Pro Tyr Val Arg Asu Gly Gly Val Asn Ile Tyr Tyr Glu Leu Val Asp Gly Pro Glu Pro Pro Ile Val Phe Val His Gly Trp Thr Ala A8n Met Asn Phe Trp Lys Glu Gln Arg Arg Tyr Phe Ala Gly Arg Asn Met Met Leu Phe Val Asp Asn Arg Gly His Gly Arg Ser Asp Lys Pro Leu Gly Tyr Asp Phe Tyr Arg Phe Glu Asn Phe Ile Ser Asp Leu Asp Ala Val Val Arg Glu Thr Gly Val Glu Lys Phe Cal Leu Val Gly His Ser ~ 85 g0 95 Phe Gly Thr Met Ile Ser Met Lys Tyr cy8 Ser Glu Tyr Arg A8n Arg 100 105 1~0 Val Leu Ala Leu Ile Leu Ile Gly Gly Gly Ser Arg Ile Lys Leu Leu His Arg Ile Gly Tyr Pro Leu Ala Lys Ile ~eu Ala Ser Ile Ala Tyr Lys Lys Ser Ser Arg Leu Val Ala Asp Leu Ser Phe Gly Lys Asn Ala Gly Glu Leu Lys Glu Trp Gly Trp Lys Gln Ala Me~ Asp Tyr Thr Pro WO g7/30160 P~TrUS97/02039 Ser Tyr Val Ala Met Tyr Thr Tyr Arg Thr Leu Thr Lys Val Asn Leu Glu Asn Ile Leu Glu Lys Ile Asp Cys Pro Thr Leu Ile Ile Val Gly Glu Glu Asp Ala Leu Leu Pro Val Ser Lys Ser Val Glu Leu Ser Arg Arg Ile Glu A6n Ser Lys Leu Val Ile Ile Pro Agn Ser Gly His Cys Val Met Leu Glu Ser Pro Ser Glu Val Asn Arg Ala Met Asp Glu Phe Ile Ser Ser Ala Gln Phe (2) INFORMATION FOR SEQ ID NO:36:
u~ CHARACTERISTICS
(A) LENGTH: 251 AMINO ACIDS
(B) TYPE: AMINO ACID
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Leu Arg Leu Arg Lys Phe Glu Glu Ile Asn Leu Val Leu Ser Gly Gly Ala Ala Lys Gly Ile Ala His Ile Gly Val Leu Lys Ala Ile Asn Glu 3~
Leu Glu Ile Arg Val Arg Ala Leu Ser Gly Val Ser Ala Gly Ala Ile Val Ser Val Phe Tyr Ala Ser Gly Tyr Ser Pro Glu Gly Met Phe Ser Leu Leu Lys Arg Val Asn Trp Leu Lys Leu Phe Lys Phe Lye Pro Pro Leu Lys Gly Leu Ile Gly Trp Glu Ly8 Ala Ile Arg Phe Leu Glu Glu Val Leu Pro Tyr Arg Arg Ile Glu Lys Leu GLu Ile Pro Thr Tyr Ile 100 105 11~
Cys Ala Thr Asp Leu Tyr Ser &ly Arg Ala Leu Tyr Leu SEr Glu Gly Ser Leu Ile Pro Ala Leu Leu Gly Ser Cys Ala Ile Pro Gly Ile Phe Glu Pro Val Glu Tyr Lys Asn Tyr Leu Leu Val Asp Gly Gly Ile Val Asn Asn Leu Pro Val Glu Pro Phe Gln Glu Ser Gly Ile Pro Thr Val Cys Val Asp Val Leu Pro Ile Glu Pro Glu Lys Asp Ile Lys Asn Ile .

WO 97130160 PCTnJs97l02039 -Leu His Ile Leu Leu Arg Ser Phe Phe Leu Ala Val Arg Ser Asn Ser Glu Lys Arg Lys Glu Phe Cy8 Asp Leu Val Ile Val Pro Glu Leu Glu Glu Phe Thr Pro Leu Asp Val Arg Lys Ala Asp Gln Ile Met Glu Arg ~ly Tyr Ile Lys Ala Leu Glu Val Leu Ser Glu (2) INFO~MATION FOR SEQ ID NO:37:
(i) ~;~U~;N~ rT~RT.~TICS
(A) LENGTH: 297 AMINO ACIDS
(B) TYPE: AMINO ACID
(D) TOPOLOGY: LIN~AR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Met Phe Asn Ile Asn Val Phe Val Asn Ile Ser Trp Leu Tyr Phe Ser 1 5 10 lS
~ly Ile Val Met Lys Thr Val Glu Glu Tyr Ala Leu Leu Glu Thr Gly Val Arg Val Phe Tyr Arg ~ys Val Ile Pro Glu Lys Ala Phe Asn Thr Leu Ile Ile Gly Ser His Gly Leu Gly Ala His Ser Gly Ile Tyr Ile Ser Val Ala Glu Glu Phe Ala Arg His Gly Phe Gly Phe Cys Met His ~sp Gln Arg Gly His Gly Arg Thr Ala Ser Asp Arg Glu Arg Gly Tyr ~al Glu Gly Phe His Asn Phe Ile Glu Asp Met Lys Ala Phe Ser Asp Tyr Ala Lys Trp Arg Val Gly Gly Asp Glu Ile Ile Leu Leu Gly His Ser Met Gly Gly Leu Ile Ala Leu Leu Thr Val Ala Thr Tyr Lys Glu Ile Ala Lys Gly Val Ile Ala Leu Ala Pro Ala Leu Gln Ile Pro Leu 145 . 150 155 160 ~hr Pro Ala Arg Arg Leu Val Leu Ser Leu Ala Ser Arg Leu Ala Pro ~is Ser Lys Ile Thr Leu Gln Arg Arg Leu Pro Gln Ly~ Pro Glu Gly Phe Gln Arg Ala Lys Asp Ile Glu Tyr Ser Leu Ser Glu Ile Ser Val Lys Leu Val Asp Glu Met Ile Lys Ala Ser Ser Met Phe Trp Thr Ile W O 97/30160 PCTrUS97/02039 Ala Gly Glu Ile Asn Thr Pro Val Leu Leu Ile His Gly Glu Lys Asp Asn Val Ile Pro Pro Glu Ala Ser Lys Lys Als Tyr Gln Leu Ile Pro Ser Phe Pro Lys Glu Leu Lys Ile Tyr Pro Asp Leu Gly His Asn Leu Phe Phe Glu Pro Gly Ala Val Lys Ile Val Thr Agp Ile Val Glu Trp Val Ly6 Asn Leu Pro Arg Glu Asn Pro (2) INFORMATION FOR SEQ ID NO:38:
(i) ~u~ CHARACTERISTICS
tA) LENGTH: 262 AMINO ACIDS
~B) TYPE: AMINO ACID
(D) TOPOLOGY: LINEAR
tii) MOLECULE TYPE: PROTEIN
(xi) ~Qu~N~ DESCRIPTION: SEQ ID NO:38:
Met Glu Val Tyr Lys Ala Lys Phe Gly Glu Ala Lys Leu Gly Trp Val Val Leu Val His Gly Leu Gly Glu His Ser Gly Arg Tyr Gly Arg Leu Ile Lys Glu Leu Asn Tyr Ala Gly Phe Gly Val Tyr Thr Phe Asp Trp Pro Gly His Gly Lys Ser Pro Gly Lys Arg Gly Hig Thr Ser Val Glu Glu Ala Met Glu Ile Ile Asp Ser Ile Ile Glu Glu Ile Arg Glu Lys Pro Phe Leu Phe Gly His Ser Leu Gly Gly Leu Thr Val Ile Arg Tyr 9o 95 Ala Glu Thr Arg Pro Asp Lys Ile Arg Gly Leu Ile Ala Ser Ser Pro Ala Leu Ala Lys Ser Pro Glu Thr Pro Gly Phe Met Val Ala Leu Ala Lys Phe Leu Gly Lys Ile Ala Pro Gly Val Val Leu Ser Asn Gly Ile Lys Pro Glu Leu Leu Ser Arg Asn Arg Agp Ala Val Arg Arg Tyr Val Glu Asp Pro Leu Val His Asp Arg Ile Ser Ala Lys Leu Gly Arg Ser Ile Phe Val Asn Met Glu Leu Ala His Arg Glu Ala Asp Ly8 Ile Lys Val Pro Ile Leu Leu Leu Ile Gly Thr Gly Agp Val Ile Thr Pr.o Pro WO 97/30160 PCTnuS97r02039 Glu Gly Ser ARg Arg Leu Phe Glu Glu Leu Ala Val Glu Asn Lys Thr Leu Arg Glu Phe Glu Gly Ala Tyr His Glu Ile Phe Glu Asp Pro Glu Trp Ala Glu Glu Phe His Glu Thr Ile Val Lys Trp Leu Val Glu Lys Ser Tyr Ser Ser Ala Gln (2) INFORMATION FOR SEQ ID NO:39:
U~'N~ CHARACTERI STICS
(A) LENGTH: 249 AMINO ACIDS
(B) TYPE: AMINO ACID
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) S~Qu~ DESCRIPTION: SEQ ID NO:39:
Leu Ile Gly Asn Leu Lys Ley Ly8 Arg Phe Glu Glu Val Asn Leu Val Leu Ser Gly Gly Ala Ala Lys Gly Ile Ala Hi8 Ile Gly Val Leu Lys Ala Leu Glu Glu Leu Gly Ile ~ys Val Lys Arg Leu Ser Gly Val Ser Ala Gly Ala Ile Val Ser Val Phe Tyr Ala Ser Gly Tyr Thr Pro Asp Glu Met Leu Lys Leu Leu Lys Glu Val Asn Trp Leu Lys Leu Phe Lys -~5 70 75 80 Phe Lys Thr Pro Lys Met Gly Leu Met Gly Trp Glu Lys Ala Ala Glu Phe Leu Glu Lys Glu Leu Gly Val Lys Arg Leu G1U Asp Leu Asn Ile Pro Thr Tyr Leu Cys Ser Ala Asp Ley Tyr Thr ~ly Lys Ala Leu Tyr Phe Gly Arg Gly Asp Leu Ile Pro Val Leu Leu Gly Ser Lys Ser Ile 130 135 1~0 Pro Gly Ile Phe Glu Pro Val Glu Tyr Glu Asn Phe Leu Leu ~al Asp Gly Gly Ile Val Asn Asn Leu Pro Val Glu Pro Leu Glu Ly8 Phe Lys Glu Pro Ile Ile Gly Val Asp Val Leu Pro Ile Thr Gln Glu Arg Lys Ile Lye Asn Ile Leu His Ile Leu Ile Arg Ser Phe Phe Leu Ala Val lg5 200 205 Arg SEr Asn Ser Glu Lys Arg Lys Glu Phe Cys Asn Val Val Ile Glu W O 97/30160 PCTrUS97/02039 Pro Pro Leu Glu Glu Phe Ser Pro Leu Asp Val Asn Lys Ala Asp Glu Ile Phe Cys Gly Asp Met Arg Ala Leu ~2) INFORMATION FOR SEQ ID NO:40:
(i) ~UhN~ CHARACTERISTICS
(A) LENGTH: 339 AMINO ACIDS
(B) TYPE: AMINO ACID
(D) TOPOLOGY: LINEAR
tii) MOLECULE TYPE: PROTEIN
(xi) ~Qu~N~ DESCRIPTION: SEQ ID NO:40:
Met Pro Ala Asn Asp Ser Pro Thr Ile Asp Phe Asn Pro Arg Gly Ile l 5 10 15 Leu Arg Asn Ala His Ala Gln Val Ile Leu Ala Thr Ser Gly Leu Arg Lys Ala Phe Leu Lys Arg Thr His Lys Ser Tyr Leu Ser Thr Ala Gln Trp Leu Glu Leu Asp Ala Gly Asn Gly Val Thr Leu Ala Gly Glu Leu A8n Thr Ala Pro Ala Thr Ala Ser Ser Ser His Pro Ala His Lys Asn Thr Leu Val Ile Val Leu His Gly Trp Glu Gly Ser Ser Gln Ser Ala Tyr Ala Thr Ser Ala Gly Ser Thr Leu Phe Asp Asn Gly Phe Asp Thr Phe Arg Leu Asn Phe Arg Asp His Gly Asp Thr Tyr His Leu Asn Arg Gly Ile Phe Asn Ser Ser Leu Ile Asp Glu Val Val Gly Ala Val Lys Ala Ile Gln Gln Gln Thr Asp Tyr Asp Lys Tyr Cys Leu Met Gly Phe Ser Leu Gly Gly Asn Phe Ala Leu Arg Val Ala Val Arg Glu Gln His Leu Ala Lys Pro Leu Ala Gly Val Leu Ala Val Cy8 Pro Val Leu Asp Pro Ala His Thr Met Met Ala Leu Asn Arg Gly Ala Phe Phe Tyr Gly Arg Tyr Phe Ala His Lys Trp Lys Arg Ser Leu Thr Ala Lys Leu Ala Ala Phe Pro Asp Tyr Lys Tyr Gly Lys Asp Leu Lys Ser Ile His Thr Leu Asp Glu Leu Asn Asn Tyr Phe Ile Pro Arg Tyr Thr Gly Phe Asn 97/30160 PCT~us97/020~9 Ser Val Ser Glu Tyr Phe Lys Ser Tyr Thr Leu Thr Gly Gln Lys ~eu Ala Phe Leu Asn Cys Pro Ser Tyr Ile heu Ala Ala Gly Asp Asp Pro Ile Ile Pro Ala Ser Asp Phe G}n Lys Ile Ala Lys Pro Ala Asn Leu His Ile Thr Val Thr Gln Gln Gly Ser His CyS Ala Tyr Leu Glu Asn Leu His Lys Pro Ser Ala Ala Asp Lys Tyr Ala Val Lys Leu Phe Gly Ala Cys (2) INFO~MATION FOR SEQ ID NO:41:
(i) S~UU~:N~ CHARACTERISTICS
~A) LENGTH: 311 AMINO ACIDS
~B) TYPE: AMINO ACID
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) ~u~ DESCRIPTION: SEQ ID NO:41:
Met Leu Asp Met Pro Ile Asp Pro Val Tyr Tyr Gln Leu Ala Glu Tyr Phe Asp Ser Leu Pro Lys Phe Asp GLn Phe Ser Ser Ala Arg Glu Tyr Arg Glu Ala Ile Asn Ary Ile Tyr Glu Glu Arg Asn Arg Gln Leu Ser Gln His Glu Arg Val Glu Arg Val Glu Asp Arg Thr Ile Lys Gly Arg Asn Gly Asp Ile Arg Val Arg Val Tyr Gln Gln Lys Pro Asp Ser Pro Val Leu Val Tyr Tyr His Gly Gly Gly Phe Val Ile Cys Ser Ile Glu Ser HIs Asp Ala Leu Cys Arg ARg Ile Ala Arg Leu Ser Asn Ser Thr Val Val Ser Val Asp Tyr Arg Leu Ala Pro Glu His Lys Phe Pro Ala Ala Val Tyr Asp Cys Tyr Aso Ala Thr ~ys Trp Val Ala Glu Asn Ala Glu Glu Leu Arg Ile Asp Pro Ser Lys Ile Phe Val Gly Gly Asp Ser Ala Gly Gly Asn Leu Ala Ala Ala Val Ser Ile Met Ala Arg Asp Ser Gly Glu Asp Phe Ile Lys ~is Gln Ile Leu Ile Tyr Pro Val Val Asn Phe Val Ala Pro Thr Pro Ser Leu Leu Glu Phe GLy Glu Gly Leu Trp .

CA 02246737 l998-08-l7 W O 97/30160 PCTrUS97/02039 Ile Leu Asp Gln Lys Ile Met Ser Trp Phe Ser Glu Gln Tyr Phe Ser Arg Glu Glu Aso LYB Phe Asn Pro Leu Ala Ser Val Ile Phe Ala Asp Leu Glu Asn Leu Pro Pro Ala Leu Ile Ile Thr Ala Glu Tyr Asp Pro Leu Arg Asp Glu Gly Glu Val Phe Gly Gln Met Leu Arg Arg Ala Gly Val Glu Ala Ser Ile Val Arg Tyr Arg Gly Val Leu His Gly Phe Ile Asn Tyr Tyr Pro Val Leu Lys Ala Ala Arg Asp Ala Ile Asn Gln Ile Ala Ala Leu leu Val Phe Asp (2) INFORMATION FOR SEQ ID NO:42:
(i) ~U~N~'~ CHARACTERISTICS
(A) LENGTH: 305 AMINO ACIDS
(B) TYPE: AMINO ACID
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Met Pro Leu Asp Pro Arg Ile Lys Lys Leu Leu Glu Ser Ala Leu Thr Ile Pro Ile Gly Lys Ala Pro Val Glu Glu Val Arg Lys Ile Phe Arg Gln Leu Ala Ser Ala Ala Pro Lys Val Glu Val Gly Lys Val Glu Asp Ile Lys Ile Pro Gly Ser Glu Thr Val Ile Asn Ala Arg Val Tyr Phe Pro Lys Ser Ser Gly Pro Tyr Gly Val Leu Val Tyr Leu His Gly Gly Gly Phe Val Ile Gly Asp Val Glu Ser Tyr Asp Pro Leu Cys Arg Ala Ile Thr Asn Ala Cys Asn Cys Val Val Val Ser Val Asp Tyr Arg Leu Ala Pro Glu Tyr Lys Phe Pro Ser Ala Val Ile Asp Ser Phe Asp Ala Thr Asn Trp Val Tyr Asn Asn Leu Asp Lys Phe Asp Gly Ly~ Met Gly Val Ala Ile Ala Gly Asp Ser Ale Gly Gly Asn Leu Ala Ala Val Val CA 02246737 l998-08-l7 WO 97J30160 PCTIUS971~2039 Ala Leu Leu Ser Lys Gly Lys Ile Asn Leu Lys T~r Gln Ile Leu Val Tyr Pro Ala Val Ser Leu Asp Asn Val Ser ~rg Ser Met Ile Glu Tyr Ser Asp Gly Phe Phe Leu Thr Ary Glu His Ile Glu Trp Phe Gly Ser - -lg5 200 205 ~ln Tyr Leu Arg Ser Pro Ala Asp Leu Leu Asp Phe Arg Phe Ser Pro Ile Leu Ala Gln Asp Phe Asn Gly Leu Pro Pro Ala Leu Ile Ile Thr Ala Glu Tyr Asp Pro Leu Arg Asp Gln Gly Glu Ala Tyr Ala Asn Lys Leu Leu Gln Ala Gly Val Ser Val Thr Ser Val Arg Phe Asn Asn Val Ile His Gly Phe Leu Ser Phe Phe Pr~ ~eu Met Glu Gln Gly Arg Asp Ala Ile Gly ~eu Ile Gly Ser Val Leu Arg Arg Val Phe Tyr Asp Lys Ile (2) INFORMATION FOR SEQ ID NO:43:
~i) SEQUENCE CHARACTERISTICS
~A) LENGTH: 605 NUCLEOTIDES
~B) TYPE: NUCLEIC ACID
~C) STR~NDEDNESS: SINGLE
~D) TOPOLOGY: LINEAR
~ii) MOLECULE TYPE: GENOMIC DNA
~Xi) ~U~N~'~ DESCRIPTION: SEQ ID NO:43:

Met Lys Val Lys His Val Ile Val Leu His Gly Leu Tyr Met Ser Gly l 5 lO 15 Leu Val Met Arg Pro Leu Cy3 Ser Arg Leu Glu Glu Ser Gly Val Lys Val Leu Asn Leu Thr Tyr Asn Thr Arg Asp Pro Asn Arg Asp Ala Ile Phe Thr Gln Ile Asp Glu Phe Ile Ser Asn Glu Pro Ser Ala Leu Val Cy9 His Ser Met Gly Gly Leu Val Ala Arg Ala Tyr Leu Glu Ala Asn Ser Ala Pro Ser His His Val Glu Lys Val Ile Thr Leu Gly Thr Pro CAT ACT GGC AGC CAT ATT GCT GAA AAA ATG CAG CAA A~A GGG TTC GAG 336 His Thr Gly Ser His Ile Ala Glu Ly~ Met Gln Gln Lys Gly Phe Glu lO0 105 llO

-CTA TTA TTA A~A AAT AGC GTT GAG TTT TTA CTC TCT AAG AAT GGT GAT 3 8 4 Leu Leu Leu Ly~ A3n Ser Val Glu Phe Leu Leu Ser Lyg Asn Gly A~p Trp Pro Phe Ly6 Ala Ly3 Leu Tyr Ser Ile 31a Gly A3p Leu Pro Ile Gly Leu Met Pro Leu Ile Val Ly6 Gly Ser Arg Ser Asp Gly Thr Val Leu Leu A3p Glu Thr Lys Leu Ly8 Gly Met Ala Glu Hi8 Ly6 Val Phe His Leu Ser His Thr Ser Met Ile Tyr Ser Arg Gln Val Val A3n Tyr Ile Leu Glu Arg Leu Asn Glu A6p Ile (2) INFORMATION FOR SEQ ID NO:44:
(i) ~:~U~N~ ~R~CT~T.~TICS
~A~ LENGTH: 77g NUCLEOTIDES
BI TYPE: NUCLEIC ACID
~CI sTRANn~nN~.c- SINGLE
~D~ TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: GENOMIC DNA
~Xi) ~b:~V~:N( ~ DESCRIPTION: SEQ ID NO:44:

Met Ile Lys A3n Phe A3p Arg Glu A~n Ser Ser Leu Val Leu Ser Gly Gly Gly Ala Leu Gly Ile Ala Hi6 Leu Gly Val Leu His A6p Leu Glu A~A CAA AAT ATT GTA C Q AAT GAA ATT GTT GGT ACA AGT ATG GGT GGT 144 Ly6 Gln A3n Ile Val Pro A3n Glu Ile Val Gly Thr Ser Met Gly Gly ATC ATT GGT G Q TCT ATG GCT ATC GGG ATG A~A GAG AAA GAA ATA CTC 192 Ile Ile Gly Ala Ser Met Ala Ile Gly Met Lys Glu Ly6 Glu Ile Leu GAA GAA ATC AAA AAC TTT TCC AAT GTC TTC AAC TGG ATA A~A TTC TCT 240 Glu Glu Ile Ly3 Asn Phe Ser Asn Val Phe A8n Trp Ile Ly8 Phe Ser Phe Ser Gly A3n Ser Val Val A3p A3n Glu Ly3 Ile Ala Lys Ile Phe Asp Thr Leu Phe Lys Asp Arg Lys Met Thr A3p Thr Val Ile Pro Leu AAA CTC ATC GCT ACA AAC TTA QT AAT GGA QT A~A A~A GTA TTT ACT 384 Ly3 Leu Ile Ala Thr A3n Leu Hi3 A3n Gly His Lys Lys Val Phe Thr GCT TCG GAT GAT GTA CTG ATC A~A GAT GCA ATA CTC TCA A Q ATG G Q 432 Ala Ser Asp Asp Val Leu Ile Ly6 Asp Ala Ile Leu Ser Thr Met Ala Ile Pro Gly Val Phe Glu Glu His Ile Ile A8p Gly Glu Thr Tyr Gly WO 97~30160 PCT/US97/û2039 Asp Gly Phe Leu ~ys Glu Asn Leu Gly Val Asn GlU Ala Thr Phe Asn GAT GTT TTA GCT GTA GAT GTC ATG GGT GAG AAC TCT TTT GAA A~A GCA 576 Asp Val Leu Ala Val Asp Val Met Gly Glu Asn Ser Phe Glu Ly6 Ala Met Pro Asp Asn Phe Phe Lys Thr Ser Asn Val ~eu Glu Met Phe Glu AAA TCA ATG CGA CTT TTT ATT TAC AAC CAG ACA CAG ACA CAT ATT A~A 672 Lys Ser Met: Arg Leu Phe Ile Tyr Asn Gln Thr Gln Thr His Ile Lys AAT GCA AAT A~A AAT ATT TAT CTT ATT GAA CCC GTT ACC AAA GAG TAT 720 Asn Ala Asn Lys Asn Ile Tyr Leu Ile Glu Pro Val Thr LyG Glu Tyr AAA A Q TTT CAA TTT CAT A~A CAT A~A GAG ATA CGT GCT TTA GGC TTG 768 Lys Thr Phe Gln Phe His Lys His Lys Glu Ile Arg Ala Leu Gly Leu Gly Leu Leu (2) INFORMATION FOR SEQ ID NO:45:
(i) ~yu~N~ r~rT~DT.~TICS
A.~ LENGTH: 905 NUCLEOTIDES
~'Bl TYPE: NUCLEIC ACID
IC~ 5TpA~ NK~x: SINGLE
,D TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: GENOMIC DNA
~xi) 5~:yu~N~ DESCRIPTION: SEQ ID NO:45:

Met Pro Leu His Pro Lys Val Lys Lys Leu Leu Ser Gln Leu Pro Pro C~G GAC TTC TCC AGA AAC GTG CAG GAC CTG AGG AAG GCC TGG GAT TTA 96 Gln ASp Phe Ser Arg Asn Val Gln Asp Leu Arg Lys Ala Trp Asp Leu Pro Phe Ser Gly Arg Arg Glu Thr Leu Lys Arg Val Glu A8p Leu Glu Ile Pro Thr Arg Asp Ala Arg Ile Arg Ala Arg Val Tyr Thr Pro Ser AGT AAG GAA AAC TTA CCC GTC CTT GTT TAC TAT CAC GGC ~T ~C TTC 240 Ser Lys Glu Asn Leu Pro Val Leu Val Tyr Tyr His Gly Gly Gly Phe GTG TTC GGT AGC GTT GAC AGC TAC GAC &GC CTC GCA TCC CTT ATT GCC 288 Val Phe Gly Ser Val Asp Ser Tyr Asp Gly Leu Ala Ser Leu Ile Ala Lys Glu Ser Gly Ile Ala Val Ile Ser Val Glu Tyr Arg Leu Ala Pro GAG CAC AAG TTC CCC ACC GCA GTC AAC GAC TCG T ~ GAT GCG CTT CTC 384 Glu His Lys Phe Pro Thr Ala Val Asn Asp Ser Trp Asp Ala Leu Leu Trp Ile Ala Glu Asn Gly Gly Lys Leu Gly Leu Asp Thr Ser Arg Leu Ala Val Ala Gly Asp Ser Ala Gly Gly Asn Leu Ser Ala Val Val Ser ~ . ~

W O 97/30160 PCT~US97/02039 Leu Leu Asp Arg Asp Gln Gly Ly8 Gly Leu Val Ser Tyr Gln Val Leu Ile Tyr Pro Ala Val Asn Met Val Asp Asn Ser Pro Ser Val Arg Glu lB0 185 190 Tyr Gly Glu Gly Tyr Phe Leu Thr Arg Ser Met Met Asn Trp Phe Gly Thr Met Tyr Phe Ser Ser Gly Arg Glu Ala Val Ser Pro Tyr Ala Ser Pro Ala Leu Ala Asp Leu His Asn Leu Pro Pro Ser Leu Val Ile Thr Ala Glu Tyr Asp Pro Leu Arg Asp Gln Gly Glu Thr Tyr Ser Eis Ser Leu Asn Glu Ala Gly Asn Val Ser Thr Leu Val Arg Tyr Gln Gly Met Ile Eis Gly Phe Leu Ser Phe Tyr Glu Trp Ile Thr Ala Gly Ly~ Leu 275 280 2a5 Ala Ile His His Ile Ala Gly Val Lue Arg Ser Val Leu Arg Ser Val Leu 301 (2) INFORMATION FOR SEQ ID NO:46:
;UU~iN~ '~A~'T~T ~TICS
A LENGTH: 978 NUCLEOTIDES
~B TYPE: NUCLEIC ACID
ST~NT~ : SINGLE
Dl TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: GENOMIC DNA
~xi) ~:UuL-~ DESCRIPTION: SEQ ID NO:46:

Val Ala Phe Phe Asp Met Pro Leu Glu Glu Leu Ly6 Lys Tyr Arg Pro 1 5 lo 15 Glu Arg Tyr Glu Glu LYB Asp Phe Asp Glu Phe Trp Arg Glu Thr Leu Lys Glu Ser Glu Gly Phe Pro Leu Asp Pro Val Phe Glu LYB Val Asp Phe His Leu LYB Thr Val Glu Thr Tyr ABP Val Thr Phe Ser Gly Tyr Arg Gly Gln Arg Ile Lys Gly Trp Leu Leu Val Pro LYB Leu Ala Glu Glu LYB Leu Pro CYB Val Val Gln Tyr Ile Gly Tyr Asn Gly Gly Arg 85 9o 95 Gly Phe Pro His ABP Trp Leu Phe Trp Pro Ser Met Gly Tyr Ile Cys WO 97/30160 PCTIUS9~102039 ~ TTT GTC ATG GAC ACC AGG GGG CAG GGA AGC GGC TGG ATG AAG GGA GAC 384 Phe Val Met Asp Thr Arg Gly Gln Gly Ser Gly Trp Met Lys Gly A8p Thr Pro A8p Tyr Pro Glu Gly Pro Val Asp Pro Gln Tyr Pro Gly Phe 130 135 14~
_ ATG ACG AGG GGC ATT CTG GAT CCG GGA ACC TAT TAC TAC AGG CGA GTC 480 Met Thr Arg Gly Ile Leu Asp Pro Gly Thr Tyr Tyr Tyr Arg Arg Val Phe Val Asp Ala Val Arg Ala Val Glu Ala Ala Ile Ser Phe Pro Arg Val A8p Ser Arg Lys Val Val Val Ala Gly Gly Ser Gln Gly Gly Gly lB0 185 190 Ile Pro Leu Ala Val Ser Ala Leu Ser Asn Arg Val Lyn Ala Leu Leu Cy~ Asp Val Pro Phe Leu Cy~ Hi~ Phe Arg Arg Ala Val Gln Leu Val A6p Thr His Pro Tyr Val Glu Ile Thr Asn Phe Leu Ly~ Thr Hi~ Arg 2~5 230 235 240 GAC A~A GAG GAG ATT GTT TTC AGA ACA CTT TCC TAC TTC GAT GGT GTG 768 Asp Ly6 Glu Glu Ile Val Phe Arg Thr Leu Ser Tyr Phe Asp Gly Val Asn Phe Ala Ala Arg Ala Lys Val Pro Ala Leu Phe Ser Val Gly Leu Met Asp Thr Ile Cys Pro Pro Ser Thr Val Phe Ala Ala Tyr Asn His Tyr Ala Gly Pro Lys Glu Ile Arg Ile Tyr Pro Tyr A8n Asn Hi8 Glu GGT GGA GGT TCT TTC CAG GCA ATT GAG CAG GTG A~A TTC TTG AAG AGA 960 Gly Gly Gly Ser Phe Gln Ala Ile Glu Gln Val Lys Phe Leu Lys Arg Leu Phe Glu Glu Gly (2) INFORMATION FOR SEQ }D NO:47:
(i) SIS~UISN~IS CH'ARACTERISTICS
(A LENGTH: 879 NUCLEOTIDES
(B TYPE: NUCLEIC ACID
( C S T~ ~ Nr~ N~ C .':: SINGLE
(Dl TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: GENOMIC DNA
(xi~ slsQu~sN-~s DESCRIPTION: SEQ ID NO:47:

Met Arg Thr Leu Ser Phe Gly Pro Met Thr Thr Gly Gly Ser Ile His Met Ala Thr Met Asp Val Met Arg Gly Pro Gly Met Gln Arg Leu Ser ' CA 02246737 1998-08-17 W O 97/30160 PCT~US97/02039 Gln Gly Ala Arg Glu Ala A1R Asn ~is Pro Trp Ala Lys Arg Leu Gly Arg Met Gly Tyr Ala Ala Lys Gly Ala Val Tyr Ala Ile Ile Gly Val Leu Ala Leu Lys Leu Ala Ala Gly Glu Gly Gly Arg Thr Thr Asp Ser His Gly Ala Val Asn Thr Val Ala His Gly Pro Phe Gly Val Ala Leu Leu Ala Val Leu Val Val Gly Leu Leu Gly Tyr Val Val Trp Arg Phe GCC CAG GCC TTC GTG GAC ACG GAG GAC AAG GGC TCC GAC GCG AaG GGA 384 Ala Gln Ala Phe Val Asp Thr Glu Asp Ly3 Gly Ser Asp Ala Lys Gly Ile Ala Thr Arg Ala Met Tyr Phe Leu Ser Gly Cys Ile Tyr Ala Ser Leu Ala Phe Phe Ala Ala Gln Ser Leu Val Gly Ala Ala His Gly Arg Ser Lys Gly Thr Gln Gly Trp Thr Ala Thr Leu Met Glu Gln Pro Phe Gly Arg Val Leu Val Ala Leu Val Gly Leu Gly Ile Val Gly Phe Ala Leu Lys Gln Phe ~is Thr Ala Trp Lys Ala Lys Phe Arg Glu Lys Leu ACC CTC ACC GGA CTG GCT GCC CGG A~G CAG CAC CAC ATC GAG CGC ATG 672 Thr Leu Thr Gly Leu Ala Ala Arg Lys Gln His His Ile Glu Arg Met Cys Gln Phe Gly Ile Ala Ala Arg Gly Val Val Phe Ala Val Ile Gly Gly Phe Leu Val Arg Ser Ala Val Asp Ala Asn Pro Gly Glu Ala Lys Gly Leu Gly Glu Ala Leu Ala Val Val Ala Arg Gln Pro Ser Gly Asp 260 265 z70 Val Leu Leu Gly Val Val Ala Ala Gly Leu Val Ala Tyr Ala Ala Tyr Leu Phe Leu Gln Ala Arg Tyr Arg Glu Leu (2) INFORMATION FOR SEQ ID NO:48:
(i) ~U~N~ CHARACTERISTICS
~A LENGT~: 914 NUCLEOTIDES
Bl TYPE: N~CLEIC ACID
~CI STRA~nRn~ SINGLE
~DI TOPOLOGY: LINEA~
(ii~ MOLEC~LE TYPE: GENOMIC DNA

(xi) .~:y~ : DESCRIPTION: SEQ ID NO:48:

Met Ser Lys Phe Ala Ile Leu Trp Ala Leu Ile Thr Ala Tyr Leu Pro Glu Pro Val Met Lys Leu Val Tyr Leu Gly Arg Arg Glu Thr Leu Gly GCA CGG ACG CTT GAC GTT A~A GCC CAA GCT GTC GGG CGG CTG GCC AAT 144 Ala Arg Thr Leu Asp Val Lys Ala Gln Ala Val Gly Arg Leu Ala Asn Ala Thr Arg Pro Val Gly Val Ile Pro Thr val Glu Glu Ser Arg Lys Met Thr Asp Ly5 Ala Val Ser Leu Phe Asp Gln Pro Ala Pro Glu Leu Phe Arg Ly8 Lya Asp Ile Gln Ile Asp Gly Ala Glu Gly Pro Ile Asp Ala Arg Ile Tyr Ser Gly Pro Ala Lys His Arg Pro Arg Pro Ile Leu Val Tyr Phe His Gly Gly Gly Trp Val Gln Gly Asn Leu Asp Ser His Asp Gly Val Cys Gly Lys Leu Ala Lys Trp Ala Asn Cys Ile Val Ile Ser Val Asp Tyr Arg Leu Ala Pro Glu His Lys Phe Pro Cys Ala Pro Leu Aap Ala Ile Ala Ala Tyr Lys Trp Val Arg Ala Asn Ala Thr Asn 16~ 170 175 Leu Gly Gly Asp Pro Glu Arg Ile Gly Val Gly Gly Asp Ser Ala Gly Gly Asn Leu Ala Ala Val Val Cys Gln Gln Thr Ala Met Asn Gly Glu Arg Thr Pro Asp Leu Gln Val Leu Ile Tyr Pro Ala ~eu Asp Ala Arg ATG ATC TCG ACC TCG ATG GAG GAA TTG CGT GAT GCC ~AC ATC TTG CCG 720 Met Ile Ser Thr Ser Met Glu Glu Leu Arg Agp Ala Tyr Ile Leu Pro Lys Ser-Arg Met Glu Tyr Phe Leu Gly Leu Tyr Thr Arg Gly Pro Asp Asp Ile Glu Asp Leu Arg Met Ser Pro Ile Leu Arg Asp Thr Val Ala Asp Gln Pro Gln Ala Cy~ Ile Val Thr cy3 Gly Phe Asp Pro Ala Arg Arg Arg Glu His Leu Arg Arg Thr Leu Asn Cyg Arg Gly Asp Arg Arg 2~0 295 300 h9 W O97/30160 PCTrUS97/02039 12) INFORMATION FOR SEQ ID NO:49:
(i) .. :t.:yU,~;N(~ TT~R2~rTpnT~cTIcs A LENGTH: 926 NUCLEOTIDES
B~ TYPE: NUCLEIC ACID
) ST~ N~ SINGLE
Dl TOPOLOGY: LINEAR
MOLECULE TYPE: GENOMIC DNA
(xi~ ~yU~:N~ DE5CRIPTION: SEQ ID NO:49:

Val Ser Ile Arg Leu Arg Leu Leu Asn Trp Phe Leu Asn Thr Phe Glu A~A CCA AAA CTG GCC GCG GCC AAA ACG CCG GAT GAT TTG CGA AAA TCG 96 Lys Pro Lys Leu Ala Ala Ala Lys Thr Pro ABP Asp Leu Arg Lys Ser Phe Glu Leu Ly6 Ala Arg Phe Leu Phe Pro Ala Pro Arg Lys Thr Arg 35 40 4~

Phe Ser His Asp Val Leu Gln Ser Gly Ile Gly Ser Val Agn Ala Gln TGG GCG A~A TCC A~A TCT GCA TCT GAT GAC AGG GTA ATC CTG TAT TTT 240 Trp Ala Lys Ser Lys Ser Ala Ser Asp Asp Arg Val Ile Leu Tyr Phe CAT GGG GGA GGG TAT GTT TTT GGG TCA CCA A~A ACG CAC CGT GCA ATG 288 His Gly Gly Gly Tyr Val Phe Gly Ser Pro Lys Thr His Arg Ala Met 85 go 95 Leu Ala Arg Leu Ser Ala Met Thr Gly Leu Ser Ala Cys Leu Pro Asp Tyr Arg Leu Ala Pro Glu His Pro Phe Pro Ala Ala Ile Glu Asp Ala GTT TTA TCG TAT AAA TGT TTA CTA GAG CGA GC~ ATC GAG CCC CAA AAT 432 Val Leu Ser Tyr Lys Cys Leu Leu Glu Arg Ala Ile Glu Pro Gln Asn Ile Ile Leu Gly Gly Asp Ser Ala Gly Gly Gly Leu Val Leu Ala Leu Leu Ala Glu I le Lys Ala Gln Ser Leu Pro Ly8 Pro Ala Gly Val Phe Ala Leu Ser Pro Leu Val Asp Leu Ser Phe Ser Gly Leu Ser Phe Ser Lys Asn Ala Gln Thr Asp Val Met Leu Pro Ala Ser Arg Ala Ala Asp Met Ala Thr Leu Tyr Leu Asp Gly Ala Asp Ala Asp Asp Pro Arg Ala Ser Pro Leu Gln Ala Asp Phe Ser Gly Met Pro Pro Val Phe Leu Thr Ala Ser ABP Ser Glu Ile Leu Leu ABP Asp Cys Leu Arg Met Ala ABP

His Leu Arg Ala Gln Gly Val Val Val Thr Aap Arg Ile val Glu Asn Wo 97/30160 PCT/US97/02039 His Pro His Val Trp His Ile Phe Gln Ary Leu LeU Pro Glu Ala Asp Gln Gly Leu Arg Ala Ile Ala Ala Trp Ile Lys Pro Leu Leu Ser Gly Ser Asn Glu Ser ~2) INFORMATION ~OR SEQ ID NO:50:
~i) ~yu~ CHARACTERISTICS
(A ~ENGTH: 713 NUCLEOTIDES
(B~ TYPE: NUCLEIC ACID
~C ST~ N~ : SINGLE
~D~ TOPOLOGY: LINEAR
~ii) MOLECULE TYPE: GENOMIC DNA
(xi) S~YU~N~ DESCRIPTION: SEQ ID NO:50: - ~

Met Leu Thr Phe Asn Val Leu Tyr Gly Met Met Lys Gln Ly~ Leu Ala Ala Ile Leu Met Phe Leu Gly Leu Ser Ala Ala Glu Ala Gln A8p Trp CCT GAC CTA CAG AAA TAT CGT AGT GCT AAT AAA GAA GCC A~A TTA CTT 144 Pro Asp Leu Gln Lys Tyr Arg Ser Ala Asn Lys Glu Ala Lys Leu Leu Pro Lys Glu Asn Arg Lys Val Val Phe Met Gly Asn Ser Ile Thr Glu 50 55 ~0 Ala Trp Ile Ser Gln Arg Pro Glu Phe Phe Ser Glu Asn Gly Phe Ile 65 ~ 70 75 80 Gly Arg Gly Ile.Ser Gly Gln Thr Thr Pro Gln Met Leu Leu Arg Phe Arg Gln Asp Val Ile Asp Leu Gln Pro Lys Ala Val Val Ile Leu Ala Gly Thr A~n Asp Val Ala Gln Asn Thr Gly Pro Met Thr Ile Glu Glu Ser Leu Ala Asn Ile Ly~ Ser Met Val Glu Leu Ala Gln Ala Asn Gly Ile Thr Pro Val Leu Cys Thr Val Leu Pro Ala Asp Arg Phe Ser Trp Arg Pro Glu Leu Thr Pro Ala Glu Thr Ile Ile Ala Leu Asn Gln Leu ATT AAG CAA. TAT GCC GAG GCA CAG GGC CTG GCC CTG GTG GAT TAT CAT 576 Ile Lys Gln Tyr Ala Glu Ala Gln Gly Leu Ala Leu Val Asp Tyr His Ala Ala Leu Thr Asn Lys Gly Gly Gly Leu Pro Val Lys Tyr Gly Glu W O 97/30160 PCTrUS97/02039 -Asp Gly Val Hi8 Pro Asn Val Ala Gly Tyr Gln Val Met Glu Asn Ile Val Leu Pro Val Ile Ser Ser Glu Leu Ala Lya Leu Lys _ (2) INFORMATION FOR SEQ ID NO:51:
iltiUU~N~.~ rT~A~ 'T~T.c:TIcs IA~ LENGT~: 978 h-UCLEOTIDES
IBI TYPE: NUCLEIC ACID
rCI ST~Nl)~ N~ : SINGLE
D~ TOPOLOGY: LINEAR
MOLECULE TYPE: GENOMIC DNA
~xi) ~uu~N~ DBSCRIPTION: SEQ ID NO:51:
ATG GCC TTC TTC GAT TTA CCA CTC GAA G~A CTG AAG AAA TAT CGT CCA 48 Uet Ala Phe Phe Asp Leu Pro Leu Glu Glu Leu Lyg Lys Tyr Arg Pro GAG CGG TAC GAA GAG A~A GAC TTC GAT GAG TTC TGG GAA GAG ACA CTC 96 Glu Arg Tyr Glu Glu Lys Asp Phe Asp Glu Phe Trp Glu Glu Thr Leu ao 25 30 Ala Glu Ser Glu Lys Phe Pro Leu Asp Pro Val Phe Glu Arg Met Glu Ser His Leu Lys Thr Val Glu Ala Tyr Asp Val Thr Phe Ser Gly Tyr Arg Gly Gln Arg Ile Lys Gly Trp Leu Leu Val Pro Lys Leu Glu Glu Glu Ly~ Leu Pro CyY Val Val Gln Tyr Ile Gly Tyr Asn Gly Gly Arg 85 go 95 Gly Phe Pro Hi9 Asp Trp Leu Phe Trp Pro Ser Met Gly Tyr Ile Cys TTC GTC ATG GAT ACT CGA GGT CAG GGA AGC GGC TGG CTG A~A GGA GAC 384 Phe Val Met Asp Thr Arg Gly Gln Gly Ser Gly Trp Leu Lys Gly Asp Thr Pro Asp Tyr Pro Glu Gly Pro Val Asp Pro Gln Tyr Pro Gly Phe Met Thr Arg Gly Ile Leu Asp Pro Arg Thr Tyr Tyr Tyr Arg Arg Val Phe Thr Asp Ala Val Arg Ala Val Glu Ala Ala Ala Ser Phe Pro Gln Val Asp Gln Glu Arg Ile Val Ile Ala Gly Gly Ser Gln Gly Gly Gly ATA GCC CTT GCG GTG AGC GCT CTC TCA AAG A~A GCA AAG GCT CTT CTG 624 Ile Ala Leu Ala Val Ser Ala Leu Ser Lyq Lys Ala Lys Ala Leu Leu Cys Asp Val Pro Phe Leu Cys His Phe Arg Arg Ala Val Gln Leu Val ~0 97/30160 PCT~US97/~2039 Asp Thr His Pro Tyr Ala Glu Ile Thr Asn Phe Leu Lys Thr His Arg Asp Lys Glu Glu Ile Val Phe Arg Thr Leu Ser Tyr Phe Asp Gly Val AAC TTC GCA GCC AGA GCG A.2~G ATC CCT GCG CTG TTT TCT GTG GGT CTC 816 ~ ~ Asn Phe Ala Ala Arg Ala Lys Ile Pro Ala Leu Phe Ser Val Gly Leu Met Asp Asn Ile Cys Pro Pro 5er Thr Val Phe Ala Ala Tyr Asn Tyr Tyr Ala Gly Pro Lys Glu Ile Arg lle Tyr Pro Tyr Asn Asn Eis Glu GGA GGA GGC TCT TTC CAA GCG GTT GAA CAG GTG AAA TTC TTG A~A AAA 912 Gly Gly Gly Ser Phe Gln Ala Val Glu Gln Val Lys Phe Leu Lys Lys Leu Phe GlU Lys Gly (2) INFORMATION FOR SEQ ID NO:52:
( i ) ~ihQUh'NC:h' r~l~p.2~r~'~n T.5TICS
A hLNGTH: 660 NUCLEOTIDES
~S~ TYPE: NUCLEIC ACID
(C. ST~2~NnRr'~S: SINGLE
( D I TOPCLOGY: LINEAR
(ii) MOLECULE TYPE: GENOMIC DNA
(xi) ~:yUhN~ DESCRIPTION: SE0 ID NO:52:

Leu Lys Tyr Phe Lys Ala Arg Leu Ala Gly Ile Thr Leu Leu Gly Leu Leu Ala Cy6 Thr Ser Ala Ser Ala Gln Thr Glu Pro Ile Val Phe Val His Gly Tyr Ser Gly Ser Ala Ser Asn Trp Asp Thr Met Leu Gly Arg Phe Ars Ser Asn Gly Tyr Ala Ser Gly Ser Leu Tyr Thr Phe Asn Tyr Asn Ser Leu Val Ser Ser Asn Ary Th2- Ser Ala Ser Glu Leu Arg Ser Phe Val Asn Thr Val Arg Ser Arg ~ Glv Asn Ala Arg Ile Ala Leu 85 go 95 Val Ala His Ser Asn Gly Gly Leu Val Ser Ary Trp Tyr Arg Ala Glu Leu Gly Gly Glu Thr Ala Thr Arg Arg Phe Val Thr Leu Gly Thr Pro His Arg Gly Thr Thr Trp Ala Tyr Ala Cys Tyr Ser Pro Ala Cys Phe , ' CA 02246737 l998-08-l7 W O 97/30160 PCTrUS97/02039 Glu Met Arg Pro Gly Ser Ser Leu Leu Thr Thr Leu Gly Ser Arg Ala TGC GAC CGC TCG CTG TGG TCG A~C ACC GAC GGC ATC ATC CTG CCG GCG 528 Cys Asp Arg Ser Leu Trp Ser Asn Thr Asp Gly Ile Ile Leu Pro Ala _ TCC AGC GCG QG TGT GGT GTC AGC ACG CGC ACT GCC GAC GTC AGC CAT 576 9er Ser Ala Gln Cys Gly Val Ser Thr Arg Thr Ala Asp Val Ser His Leu Asp Leu Leu Thr Asp Ser Arg Val Tyr Thr Gln Leu Arg Thr Gln Leu Gln End Gly End Arg Cys Thr Glu Arg Ala Pro (2) INFORMATION FOR SEQ ID NO:53:
(i) ~L~UL._L CHA~ACTERISTICS
~A) LENGTH: 201 AMINO ACIDS
(B) TYPE: AMINO ACID
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(Xi) .~r:ull~:N~ ~: DESCRIPTION: SEQ ID NO:53:
Met Lys Val Lys His Val Ile Val Leu His Gly Leu Tyr Met Ser Gly Leu Val Met Arg Pro Leu Cys Ser Arg Leu Glu Glu Ser Gly Val Lys Val Leu Asn Leu Thr Tyr Asn Thr Arg Asp Pro Asn Arg Asp Ala Ile Phe Thr Gln Ile Asp Glu Phe Ile Ser Asn Glu Pro Ser Ala Leu Val Cys His Ser Met Gly Gly Leu Val Ala Arg Ala Tyr Leu Glu Ala Asn Ser Ala Pro Ser His His Val Glu Lys Val Ile Thr Leu Gly Thr Pro His Thr Gly Ser His Ile Ala Glu Lys Met Gln Gln Lys Gly Phe Glu Leu Leu Leu Lys Asn Ser Val Glu Phe Leu Leu Ser Lys Asn Gly Asp Trp Pro Phe Lys Ala Lys Leu Tyr Ser Ile Ala Gly Asp Leu Pro Ile Gly Leu Met Pro Leu Ile Val Lys Gly Ser Arg Ser Asp Gly Thr Val Leu Leu Asp Glu Thr Lys Leu Lys Gly Met Ala Glu His Lys Val Phe His Leu Ser His Thr Ser Met Ile Tyr Ser Arg Gln Val Val Asn Tyr Ile Leu Glu Arg Leu Asn Glu Asp Ile (2) INFORMATION FOR SEQ ID NO:54:
(i) ~U~N~L CHARACTERISTICS
(A) LENGTH: 255 AMINO ACIDS
(B) TYPE: AMINO ACID
(D) TOPOLOGY: LINEAR
.

WO g7/30160 :PCTJUS97/(12039 (ii) MOLECULE TYPE: PROTEIN
(Xi) ~SUU~N~ DESCRIPTION: SEQ ID NO:54:
Met Ile Lys Asn Phe Asp Arg Glu Asn Ser Ser LeU Val Eeu Ser Gly l 5 10 15 Gly Gly Ala Leu Gly Ile Ala His Leu Gly Val Leu His Asp heu Glu Lys Gln Asn Ile Val Pro Asn Glu Ile Val Gly Thr Ser Met Gly Gly Ile Ile Gly Ala Ser Met Ala Ile Gly Met Lys Glu Lys Glu Ile Leu Glu Glu Ile Lys Asn Phe Ser Asn Val Phe Asn Trp Ile Lys Phe Ser Phe Ser Gly Asn Ser Val Val Asp Asn Glu Ly6 Ile ~la Lys Ile Phe Asp Thr heu Phe Ly8 Asp Arg Lys Mee Thr Asp Thr Val Ile Pro Leu lOo 105 110 Lys Leu Ile Ala Thr Asn ~eu His Asn Gly His Lys Lys Val Phe Thr Ala Ser Asp Asp Val Leu Ile Lys Asp Ala Ile Leu Ser Thr Met Ala Ile Pro Gly Val Phe Glu Glu His Ile Ile Asp Gly Glu Thr Tyr Gly Asp Gly Phe Leu Cys Glu Asn Leu Gly Val Asn GlU Ala Thr Phe Asn Asp Val Leu Ala Val Asp Val Met Gly Glu Asn Ser Phe Glu Lys Ala Met Pro Asp Asn Phe Phe Lys Thr Ser Asn Val Leu Glu Met Phe Glu Lys Ser Met Arg Leu Phe Ile Tyr Asn Gln Thr Gln Thr His Ile Lys Asn Ala Asn Lys Asn Ile Tyr Leu Ile Glu Pro Val Thr Ly3 Glu Tyr Ly~ Thr Phe Gln Phe Eis Lys His Lys Glu Ile Arg Ala Leu Gly Leu Gly Leu Leu ~2) INFORMATION FOR SEQ ID NO:s5:
(i) ~il:;~Ut!iN~'E CHA~ T~TCTICS
(A) LENGTH: 301 AMINO ACIDS
~B) TYPE: AMINO ACIn (D) TOPOLOGY: LINEAR
(i~) MOLECULE TYPE: PROTEIN

(xi) .~ DESCRIPTION: SEQ ID NO:55:
Met Pro Leu His Pro Lyg Val Lys ~ys Leu Leu Ser Gln Leu Pro Pro Gln Asp Phe Ser Arg Asn Val Gln Asp Leu Arg Lys Ala Trp Asp Leu Pro Phe Ser Gly Arg Arg Glu Thr Leu Lys Arg Val Glu Asp Leu Glu .35 40 45 Ile Pro Thr Arg Asp Ala Arg Ile Arg Ala Arg Val Tyr Thr Pro Ser ' CA 02246737 1998-08-17 W O 97/30160 PCTrUS97/02039 Ser Lys Glu Asn Leu Pro Val Leu Val Tyr Tyr His Gly Gly Gly Phe Val Phe Gly Ser Val Asp Ser Tyr Asp Gly Leu Ala Ser Leu Ile Ala Ly3 Glu Ser Gly Ile Ala Val Ile Ser Val Glu Tyr Arg Leu Ala Pro Glu His Lys Phe Pro Thr Ala Val Asn Asp Ser Trp Asp Ala Leu Leu Trp Ile Ala Glu Asn Gly Gly Lys Leu Gly Leu Asp Thr Ser Arg Leu Ala Val Ala Gly Asp Ser Al~ Gly Gly Asn Leu Ser Ala Val Val Ser Leu Leu Asp Arg Asp Gln Gly Lys Gly Leu Val Ser Tyr Gln Val Leu Ile Tyr Pro Ala Val Asn Met Val Asp Asn Ser Pro Ser Val Arg Glu Tyr Gly Glu Gly Tyr Phe Leu Thr Arg Ser Met Met Asn Trp Phe Gly 195 200 20s Thr MetO Tyr Phe Ser Ser Gly Arg Glu Ala Val Ser Pro Tyr Ala Ser Pro Ala Leu Ala Asp Leu His Asn Leu Pro Pro Ser Leu Val Ile Thr Ala Glu Tyr Asp Pro Leu Arg Asp Gln Gly Glu Thr Tyr Ser His Ser Leu Asn Glu Ala Gly Asn Val Ser Thr Leu Val Arg Tyr Gln Gly Met Ile His Gly Phe Leu Ser Phe Tyr Glu Trp Ile Thr Ala Gly Lys Leu Ala Ile His Hifi Ile Ala Gly Val Leu Arg Ser Val Leu (2) INFORMATION FOR SEQ ID NO:56:
( i ) ~'~U~N~ CHARACTERISTICS
(A) LENGTH: 326 AMINO ACIDS
(B) TYPE: AMINO ACID
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
~Xi ) ~UU~N~ DESCRIPTION: SEQ ID NO:56:
Val Ala Phe Phe Agp Met Pro Leu Glu Glu heu Lys Lys Tyr Arg Pro Glu Arg Tyr Glu Glu Lys Asp Phe Asp Glu Phe Trp Arg Glu Thr Leu Lys Glu Ser Glu Gly Phe Pro Leu Asp Pro Val Phe Glu Lys Val Asp Phe His Leu Lys Thr Val Glu Thr Tyr A6p Val Thr Phe Ser Gly Tyr Arg Gly Gln Arg Ile Lys Gly Trp Leu Leu Val Pro Lys Leu Ala Glu Glu Lys Leu Pro Cys Val Val Gln Tyr Ile Gly Tyr Asn Gly Gly Arg Gly Phe Pro His Asp Trp Leu Phe Trp Pro Ser Met Gly Tyr Ile Cys ~ CA 02246737 1998-08-17 WQ 97/30160 PCT/US97~û2039 Phe Val Met Asp Thr Arg Gly Gln Gly Ser Gly Trp Met hys Gly Asp Thr Pro Asp Tyr Pro Glu Gly Pro Val Asp Pro Gln Tyr Pro Gly Phe Met Thr Arg Gly Ile Leu Asp Pro Gly Thr Tyr Tyr Tyr Arg Arg Val Phe Val Asp Ala Val Arg Ala Val Glu Ala Ala Ile Ser Phe Pro Arg Val A3p Ser Arg Lys Val Val Val Ala Gly Gly Ser Gln Gly Gly Gly 180 185 lg0 Ile Pro Leu Ala Val Ser Ala Leu Ser Asn Arg Val Lys Ala ~eu Leu Cys Asp Val Pro Phe Leu Cys His Phe Arg Arg Ala Val Gln Leu Val Asp Thr Hi~ Pro Tyr Val Glu Ile Thr Asn Phe Leu Lys Thr His Arg Asp Lys Glu Glu Ile Val Phe Arg Thr Leu Ser Tyr Phe Agp Gly Val A3n Phe Ala Ala Arg Ala Lys Val Pro Ala Leu Phe Ser Val Gly Leu Met Asp Thr Ile Cys Pro Pro Ser Thr Val Phe Ala Ala Tyr Asn His Tyr Ala Gly Pro Lys Glu Ile Arg Ile Tyr Pro Tyr Agn Asn His Glu Gly Gly Gly Ser Phe Gln Ala Ile Glu Gln Val Lys Phe ~eu hys Arg Leu Phe Glu Glu Gly (2) INFORMATION FOR SEQ ID NO:s7:
(i) ~Uu~N~ CHARACTERISTICS
~A) LENGTH: 298 AMINO ACIDS
(B) TYPE: AMINO ACID
(D) TOPOLOGY: LINEAR
(ii) MO~ECULE TYPE: PROTEIN
(Xi ) ~U~N~'~ DESCRIPTION: SEQ ID NO:57:
Met Arg Thr Leu Ser Phe Gly Pro Met Thr Thr Gly Gly Ser Ile Hi~
l 5 10 15 Met Ala Thr Met Asp Val Met Arg Gly Pro Gly Met Gln Arg Leu Ser Gln Gly Ala Arg Glu Ala Ala Asn His Pro Trp Ala Lys Arg Leu Gly Arg Met Gly Tyr Ala Ala Lys Gly Ala Val Tyr Ala Ile Ile Gly Val Leu Ala Leu Lys Leu Ala Ala Gly Glu Gly Gly Arg Thr Thr Asp Ser His Gly Ala Val Asn Thr Val Ala Hi~ Gly Pro Phe Gly Val Ala Leu Leu Ala Val Leu Val Val Gly Leu Leu Gly Tyr Val Val Trp Arg Phe Ala Gln Ala Phe Val A3p Thr Glu A~p Lys Gly Ser Asp Ala Lys Gly . CA 0224673i 1998-08-17 Ile Ala Thr Arg Ala Met Tyr Phe Leu Ser Gly Cys Ile Tyr Ala Ser Leu Ala Phe Phe Ala Ala Gln Ser Leu Val Gly Ala Ala His Gly Arg Ser Lys Gly Thr Gln Gly Trp Thr Ala Thr Leu Met Glu Gln Pro Phe ~ Gly Arg Val Leu Val Ala Leu Val Gly Leu Gly Ile Val Gly Phe Ala Leu Lys Gln Phe His Thr Ala Trp Lys Ala Lys Phe Arg Glu Lys Leu Thr Leu Thr Gly Leu Ala Ala Arg Lys Gln His Hifi Ile Glu Arg Met Cys Gln Phe Gly Ile Ala Ala Arg Gly Val Val Phe Ala Val Ile Gly Gly Phe Leu val Arg Ser Ala Val Asp Ala Asn Pro Gly Glu Ala Lys Gly Leu Gly Glu Ala Leu Ala Val Val Ala Arg Gln Pro Ser Gly Asp Val Leu Leu Gly Val Val Ala Ala Gly Leu Val Ala Tyr Ala Ala Tyr Leu Phe Leu Gln Ala Arg Tyr Arg Glu Leu (2) INFORMATION FOR SEQ ID NO:58:
(i) ~Uu~N~ CH~RACTERISTICS
(A~ LENGTH: 304 AMINO ACIDS
(B) TYPE: AMINO ACID
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(Xi) 8~U~:N~: DESCRIPTION: SEQ ID NO:58:
Met Ser Lys Phe Ala Ile Leu Trp Ala Leu Ile Thr Ala Tyr Leu Pro Glu Pro Val Met Lys Leu Val Tyr Leu Gly Arg Arg Glu Thr Leu Gly Ala Arg Thr Leu Asp Val Lys Ala Gln Ala Val Gly Arg Leu Ala Asn Ala Thr Arg Pro Val Gly Val Ile Pro Thr Val Glu Glu Ser Arg Lys Met Thr Asp Lys Ala Val Ser Leu Phe Asp Gln Pro Ala Pro Glu Leu Phe Arg Lys Lys Asp Ile Gln Ile Asp Gly Ala Glu Gly Pro Ile Asp Ala Arg Ile Tyr Ser Gly Pro Ala Lys Hi~ Arg Pro Arg Pro Ile Leu Val Tyr Phe His Gly Gly Gly Trp Val Gln Gly Asn Leu Asp Ser His ..

Asp Gly Val Cys Gly Lys Leu Ala Lys Trp Ala Asn Cys Ile Val Ile Ser Val Asp Tyr Arg Leu Ala Pro Glu His Lys Phe Pro Cys Ala Pro Leu Asp Ala Ile Ala Ala Tyr Lys Trp Val Arg Ala Asn Ala Thr Asn Wo 97130160 PCTIUS~7/02039 ~ Leu Gly Gly A5p Pro Glu Arg Ile Gly Val Gly Gly Asp Ser Ala Gly Gly Asn Leu Ala Ala Val Val Cy8 Gln Gln Thr Ala Met Asn Gly Glu Arg Thr Pro ABP Leu Gln Val Leu Ile Tyr Pro Ala Leu A8p Ala Arg Met Ile Ser Thr Ser Met Glu Glu Leu Arg A8p Ala Tyr Ile ~eu Pro - Lys Ser Arg Met Glu Tyr Phe Leu Gly Leu Tyr Thr Arg Gly Pro Asp Asp Ile Glu Asp Leu Arg Met Ser Pro Ile Leu Arg A8p Thr Val Ala A~p Gln Pro Gln Ala Cys Ile Val Thr Cys Gly Phe Asp Pro Ala Arg Arg Arg Glu Hi~ Leu Arg Arg Thr Leu Asn Cyfi Arg Gly Asp Arg Arg ~2) INPORMATION FOR SEQ ID NO:59:
(i) ~'~'yU~N~'~' CHARACTERISTICS
~A) LENGT~: 308 AMINO ACIDS
- ~B) TYPE: AMINO ACID
(D) TOPOLOGY: ~INEAR
(ii) MOLECU~E TYPE: PROTEIN
(Xi) ~'~U~ N('~; DESCRIPTION: SEQ ID N0:59:
Val Ser Ile Arg Leu Arg Leu Leu Asn Trp Phe Leu Asn Thr Phe Glu Lys Pro Lys Leu Ala Ala Ala Lys Thr Pro Asp A8p Leu Arg Lys Ser Phe Glu Leu Lys Ala Arg Phe Leu Phe Pro Ala Pro Arg Lys Thr Arg Phe Ser His Asp Val Leu Gln Ser Gly Ile Gly Ser Val Asn Ala Gln Trp Ala Lys Ser Lys Ser Ala Ser Asp Asp Arg Val Ile Leu Tyr Phe His Gly Gly Gly Tyr Val Phe Gly Ser Pro Ly~ Thr His Arg Ala Met g5 Leu Ala Arg Leu Ser Ala Met Thr Gly Leu Ser Ala Cys Leu Pro Asp Tyr Arg Leu Ala Pro Glu His Pro Phe Pro Ala Ala Ile Glu Asp Ala Val Leu Ser Tyr Lys Cys Leu Leu Glu Arg Ala Ile Glu Pro Gln Asn Ile Ile Leu Gly Gly Asp Ser Ala Gly Gly Gly Leu Val Leu Ala Leu 1~5 150 155 160 Leu Ala Glu Ile Ly~ Ala Gln Ser Leu Pro Lys Pro Ala Gly Val Phe Ala Leu Ser Pro Leu Val Asp Leu Ser Phe Ser Gly Leu Ser Phe Ser Lys Asn Ala Gln Thr Asp Val Met Leu Pro Ala Ser Arg Ala Ala A5p Met Ala Thr Leu Tyr Leu Asp Gly Ala Asp Ala A~p Asp Pro Arg Ala . 79 W 0 ~7/ CA 02246737 1998-08-17 30160 PCT~US97/02039 Ser Pro Leu Gln Ala Asp Phe Ser Gly Met Pro Pro Val Phe Leu Thr Ala Ser Asp Ser Glu Ile Leu Leu Asp Asp Cys Leu Arg Met Ala Asp His Leu Arg Ala Gln Gly Val Val Val Thr Asp Arg Ile Val Glu Asn His Pro His Val Trp His Ile Phe Gln Arg Leu Leu Pro Glu Ala Asp Gln Gly Leu Arg Ala Ile Ala Ala ~rp Ile LYB Pro Leu Leu Ser Gly 6er Asn Glu Ser (2) INFORMATION FOR SEQ ID NO:60:
U~;N~ CHARP~CTT;~UT~:TICS
(A) LENGTH: 237 AMINO ACIDS
(~3) TYPE: AMINO ACID
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(Xi) ~ U~N~ DESCRIPTION: SEQ ID NO:60:
Met Leu Thr Phe Asn Val Leu Tyr Gly Met Met Lys Gln Lys Leu Ala Ala Ile Leu Met Phe Leu Gly Leu Ser Ala Ala Glu Ala Gln Asp Trp Pro Asp Leu Gln Lys Tyr Arg Ser Ala Asn Lys Glu Ala Lys Leu Leu Pro Lys Glu Asn Arg Lys Val Val Phe Met Gly Asn Ser Ile Thr Glu Ala Trp Ile Ser Gln Arg Pro Glu Phe Phe Ser Glu Asn Gly Phe Ile Gly Arg Gly Ile Ser Gly Gln Thr Thr Pro Gln Met Leu Leu Arg Phe 9o g5 Arg Gln Asp Val Ile Asp Leu Gln Pro Lys Ala Val Val Ile Leu Ala Gly Thr Asn Asp Val Ala Gln Asn Thr Gly Pro Met Thr Ile Glu Glu Ser Leu Ala Asn Ile Lys Ser Met Val Glu Leu Ala Gln Ala Asn Gly Ile Thr Pro Val Leu Cys Thr Val Leu Pro Ala Asp Arg Phe Ser Trp Arg Pro Glu Leu Thr Pro Ala Glu Thr Ile Ile Ala Leu A3n Gln Leu Ile Lys Gln Tyr Ala Glu Ala Gln Gly Leu Ala Leu Val Asp Tyr His Ala Ala Leu Thr Asn Ly3 Gly Gly Gly Leu Pro Val Ly6 Tyr Gly Glu Asp Gly Val His Pro Asn Val Ala Gly Tyr Gln Val Met Glu Asn Ile Val Leu Pro Val Ile Ser Ser Glu Leu Ala Lys Leu Lys 225 23~ Z35 (2) INFORMATION FOR SEQ ID NO:61:

W O 97/30160 PCT~US97102039 ( i ) ::; tS5~ U ~iN ~ ~ ~A'17 AC'r~ D r .q TI CS
(A) LENGTH: 326 AMINO ACIDS
(B) TYPE: AMINO ACID
~D~ TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
Ixi) ~Kyu~N~ DESCRIPTION: SEQ ID NO:61:
Met Ala Phe Phe Asp Leu Pro Leu Glu Glu Leu Lys Lys Tyr Arg Pro Glu Arg Tyr Glu Glu Lys ABP Phe Asp Glu Phe Trp Glu Glu Thr Leu Ala Glu Ser Glu Lys Phe Pro Leu Asp Pro Val Phe Glu Arg Met Glu Ser His Leu Lys Thr Val Glu Ala Tyr Asp Val Thr Phe Ser Gly Tyr Arg Gly Gln Arg Ile Lys Gly Trp Leu heu Val Pro Lys Leu Glu Glu Glu Lys Leu Pro CYB Val Val Gln Tyr Ile Gly Tyr A9n Gly Gly Arg go 95 Gly Phe Pro His Asp Trp Leu Phe Trp Pro Ser Met Gly Tyr Ile Cys Phe Val Met Asp Thr Arg Gly Gln Gly 5er Gly Trp Leu Lys Gly Asp Thr Pro Asp Tyr Pro Glu Gly Pro Val Asp Pro Gln Tyr Pro Gly Phe Met Thr Arg Gly Ile Leu Asp Pro Arg Thr Tyr Tyr Tyr Arg Arg Val Phe Thr Asp Ala Val Arg Ala Val Glu Ala Ala Ala Ser Phe Pro Gln Val Asp Gln Glu Arg Ile Val Ile Ala Gly Gly Ser Gln Gly Gly Gly Ile Ala Leu Ala Val Ser Ala Leu Ser Lys Lys Ala Lys Ala Leu Leu Cys Asp Val Pro Phe Leu Cy9 His Phe Arg Arg Ala Val Gln Leu Val Asp Thr His Pro Tyr Ala Glu Ile Thr Asn Phe Leu Lys Thr His Arg Asp Lys Glu Glu Ile Val Phe Arg Thr Leu Ser Tyr Phe Asp Gly Val Asn Phe Ala Ala Arg Ala Lys Ile Pro Ala Leu Phe Ser Val Gly Leu Met Asp Asn Ile Cys Pro Pro Ser Thr Val Phe Ala Ala Tyr Asn Tyr Tyr Ala Gly Pro Lys Glu Ile Arg Ile Tyr Pro Tyr Asn Asn His Glu Gly Gly Gly Ser Phe Gln Ala Val Glu Gln Val Lys Phe Leu Lys Lys Leu Phe Glu Lys Gly (2) INFORMATION POR SEQ ID NO:62:
li) ~:uu~.._~ CHARACTERISTICS
(A) LENGTH: 220 AMINO ACIDS
(B~ TYPE: AMINO ACID
~D~ TOPOLOGY: LINEAR
o W O 97/30160 PCT~US97/02039 R~ 7-R TYPE: PROTEIN
(xi ) ~iE~Iu~;N~:~ DESCRIPTION: SEQ ID NO: 62:
Leu Lys Tyr Phe Lys Ala Arg Leu Ala Gly Ile Thr Leu Leu Gly Leu Leu Ala Cys Thr Ser Ala Ser Ala Gln Thr Glu Pro Ile Val Phe Val His Gly Tyr Ser Gly Ser Ala Ser Asn Trp ABP Thr Met Leu Gly Arg Phe Arg Ser Asn Gly Tyr Ala Ser Gly Ser Leu Tyr Thr Phe Asn Tyr A~;n Ser Leu Val Ser Ser Asn Arg Thr Ser Ala Ser Glu Leu Arg Ser Phe Val Asn Thr Val Arg Ser Arg His Gly Asn Ala Arg Ile Ala Leu go 95 Val Ala His Ser Asn Gly Gly Leu Val Ser Arg Trp Tyr Arg Ala Glu Leu Gly Gly Glu Thr Ala Thr Arg Arg Phe Val Thr Leu Gly Thr Pro His Arg Gly Thr Thr Trp Ala Tyr Ala Cy~ Tyr Ser Pro Ala Cy~; Phe Glu Me'c Arg Pro Gly Ser Ser Leu Leu Thr Thr Leu Gly Ser Arg Ala Cys Asp Arg Ser Leu Trp Ser Asn Thr ABP Gly Ile Ile Leu Pro Ala Ser Ser Ala Gln Cy8 Gly Val Ser Thr Arg Thr Ala ABP Val Ser His Leu A~p Leu Leu Thr ABP Ser Arg Val Tyr Thr Gln Leu Arg Thr Gln Leu Gln End Gly End Arg CYB Thr Glu Arg Ala Pro G

Claims (20)

What Is Claimed Is:

1. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide having at least a 70% identity to a polynucleotide encoding an enzyme comprising amino acid sequences set forth in SEQ ID NOS:33-42;
(b) a polynucleotide which is complementary to the polynucleotide of (a); and (c) a polynucleotide comprising at least 15 consecutive bases of the polynucleotide of (a) or (b).

2. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.

3. The polynucleotide of Claim l wherein the polynucleotide is RNA.

4. The polynucleotide of Claim 2 which encodes an enzyme comprising amino acids 1 to 414 of SEQ ID NO:33.

5. The polynucleotide of Claim 2 which encodes an enzyme comprising amino acids 1 to 373 of SEQ ID NO:34.

6. The polynucleotide of Claim 2 which encodes an enzyme comprising amino acids 1 to 453 of SEQ ID NO:35.

7. The polynucleotide of Claim 2 which encodes an enzyme comprising amino acids 1 to 343 of SEQ ID NO:36.

8. The polynucleotide of Claim 2 which encodes an enzyme comprising amino acids 1 to 398 of SEQ ID NO:37.

9. The polynucleotide of Claim 2 which encodes an enzyme comprising amino acids 1 to 592 of SEQ ID NO:38.

10. The polynucleotide of Claim 2 which encodes an enzyme comprising amino acids 1 to 354 of SEQ ID NO:39.

11. The polynucleotide of Claim 2 which encodes an enzyme comprising amino acids 1 to 303 of SEQ ID NO:40.

12. The polynucleotide of Claim 2 which encodes an enzyme comprising amino acids 1 to 311 of SEQ ID NO:41.

13. The polynucleotide of Claim 2 which encodes an enzyme comprising amino acids 1 to 305 of SEQ ID NO:42.

14. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide having at least a 70% identity to a polynucleotide encoding an enzyme expressed by the DNA contained in ATCC Deposit No.
~ ;
(b) a polynucleotide complementary to the polynucleotide of (a); and (c) a polynucleotide comprising at least 15 consecutive bases of the polynucleotide of (a) and (b).

15. A vector comprising the DNA of Claim 2.

16. A host cell comprising the vector of Claim 15.

17. A process for producing a polypeptide comprising: expressing from the host cell of Claim 16 a polypeptide encoded by said DNA.

18. A process for producing a cell comprising: transforming or transfecting the cell with the vector of Claim 15 such that the cell expresses the polypeptide encoded by the DNA contained in the vector.

19. An enzyme comprising a member selected from the group consisting of an enzyme comprising an amino acid sequence which is at least 70% identical to the amino acid sequence set forth in SEQ ID NOS:33-42.

20. A method for transferring an amino group from an amino acid to an .alpha.-keto acid comprising:
contacting an amino acid in the presence of an .alpha.-keto acid with an enzyme selected from the group consisting of an enzyme having the amino acid sequence set forth in SEQ ID NOS:33-42.