CA2161125A1 - Cloning and expression of the chondroitinase i and ii genes from p. vulgaris - Google Patents

Abstract

This invention relates to the DNA sequence encoding the major protein component of chondroitinase ABC, which is referred to as "chondroitinase I", from Proteus vulgaris (P. vulgaris), which is contained in the Nsi fragment shown in the figure. This invention further relates to the DNA sequence encoding a second protein component of chondroitinase ABC, which is referred to as "chondroitinase II", from P. vulgaris, to the cloning and expression of the genes containing these DNA sequences, to the amino acid sequences of the recombinant chondroitinase I and II, and to methods for the isolation and purification of recombinant chondroitinase I or II. These methods provide significantly higher yields and purity than those obtained by adapting for the recombinant enzymes the method previously used for isolating and purifying native chondroitinase I enzyme from P. vulgaris.

Description

W0941t5567 ~ 1611~ ~ PCT~S94/0~95 CLONING AND EXPRESSION OF THE
CHONDROITINASE I AND II GENES FROM P. VULGARIS

Field of the Invention This invention relates to the DNA sequence encoding the major protein component of c~onAroitinase ABC, which is referred to as "chn~roitinase I", from Proteus vulqaris (P. vulqaris). This invention further relates to the DNA sequence encoding a second protein component of r~on~roitina8e ABC, which is referred to as "rhn~roitinase II", from P. vulqaris.
This invention also relates to the cloning and expression of the genes cont~;n;ng these DNA sequences and to the amino acid se~uences of the recombinant ch~Aroitinase I and II enzymes encoded by these DNA
sequences.
This invention additionally relates to methods for the isolation and purification of the recombinantly expressed major protein component of c~QnAroitinase ABC, which is referred to as "chon~roitinaee I", from Proteus vulqaris (P.
w lqaris). This invention further relates to methods for the isolation and purification of the recombinantly expressed second protein component of ch~Aroitinase ABC, which is referred to as ~chn~roitinase II", from P. vulqaris. These methods provide significantly higher yields and purity than those obtained by adapting for the recombinant enzymes the method previously used for isolating and purifying the native cho~roitinase I enzyme from P. vulqaris.

W094/25567 PCT~S94/0~95 6112~ - 2 -Backqround of the Invention ChQ~A--oitinases are enzyme~ of bacterial origin which have been described aR having value in di~solving the cartilage of herniated discs without disturbing the stabilizing collagen components of those discs.
Examples of ch~n~roitinase enzymes are chQn~roitinase ABC, which is produced by the bacterium P. vulqaris, and rh~nAroitinase AC, which is produced by A. aurescens. The chQnAroitinaseR function by degrading polysaccharide side ch~n~ in protein-polysaccharide complexes, without degrading the protein core.
Yamagata et al. describes the purification of the enzyme chQ~Aroitinase ABC from extracts of P.
vulqaris (Bibliography entry 1). The enzyme selectively degrades the glycosaminoglycans chQnAroitin-4-sulfate, dermatan sulfate and chQnAroitin-6-sulfate (also referred to respectively as chn~Aroitin sulfates A, B and C) at pH 8 at higher rates than ~honAroitin or hyaluronic acid. However, the enzyme did not attack keratosulfate, heparin or heparitin sulfate.
~ih~Ch; et al. describes the purification of glycosaminoglycan degrading enzymes, such as chQnAroitinase ABC, by fractionating the enzymes by adsorbing a solution conts~;n;ng the enzymes onto an insoluble sulfated polysaccharide carrier and then desorbing the individual enzyme~ from the carrier (2).
Brown describes a method for treating intervertebral disc displacement in mammals, including humans, by injecting into the intervertebral disc space effective amounts of a ~olution cont~;n;ng c~oIAroitinase ABC (3). The rhon~roitina~e ABC wa~

_ W094/25567 ~ 16112 S PCT~S94/0~95 i~olated and purified from extracts of P. vulqari~.
This native enzyme material functioned to dissolve cartilage, such as herniated spinal discs.
-Specifically, the enzyme causes the selective 5 chemonucleolysis of the nucleus pulposus which contains proteoglycans and randomly dispersed collagen fibers.
Hageman describes an ophthalmic vitrectomy method for selectively and completely disinserting the ocular vitreous body, epiretinal membranes or fibrocellular m~mhranes from the neural retina, ciliary epithelium and posterior lens surface of the mammalian eye as an adjunct to vitrectomy, by administering to the eye an effective amount of an enzyme which disrupts or degrades ch~n~roitin sulfate proteoglycan localized specifically to sites of vitreoretinal adhesion and thereby permit complete disinsertion of said vitreous body and/or epiretinal membranes (4). The enzyme can be a protease-free glycosaminoglycanase, such as chQ~roitinase ABC.
Hageman utilized rhnn~roitinase ABC obt~;ne~ from Seikagaku Kogyo Co., Ltd., Tokyo, Japan.
In isolating and purifying the chn~roitinase ABC enzyme from the Seikagaku Kogyo material, it was noted that there was a correlation between effective preparations of the ~hQ~roitinase in vitrectomy procedures and the presence of a second protein having an apparent molecular weight (by SDS-PAGE) slightly greater than that of the major protein - 30 component of ch~roitinase ABC. The second protein is now designated "chnn~roitinase II", while the major protein component of rhon~roitinase ABC iB referred to as "cho~roitinase I." The chQ~roitinase I and II
proteins are basic proteins at neutral pH, with similar isoelectric points of 8.30-8.45. Separate W094/25567 PCT~S94/04495 purification of the cho~roitinase I and II forms of the native enzyme revealed that it was the combination of the two proteins that was active in the surgical vitrectomy rather than either of the proteins individually.
Use of the cho~roitinase I and II forms of the native enzyme to date has been limited by the small amounts of enzymes obtained from native sources.
The production and purification of the native forms of the enzyme has been carried out using fermentations of P. w lqaris in which its substrate has been used as the inducer to initiate production of these forms of the enzyme. A combination of factors, including low levels of synthesis, the cost and availability of the inducer (cho~roitin sulfate), and the O~G ~u~istically pathogenic nature of P. w laaris, has resulted in the reguirement for a more efficient method of production. In addition, the native forms of the enzyme produced by conventional techniques are subject to degradation by proteases present in the bacterial extract. Therefore, there i8 a need for a reliable supply of pure material free of contaminants in order for the medical applications of the two forms of this enzyme to be evaluated properly and exploited. There is also a need for methods to isolate and purify a reliable supply of the chQn~roitinase I and II enzymes free of contaminants.

Summary of the Invention Accordingly, it is an object of this invention to produce ch~n~roitinase I and chon~roitinase II in quantities not readily achievable using present non-recombinant bacterial fermentation and extraction techniques.

W094l25567 2161 12 5 PCT~S94/0~95 It is a further object of this invention to produce cho~roitinase I and cho~roitinase II, each in a form substantially free of protease~ which would otherwise degrad,e the enzyme and cause a 1088 of its activity.
These objects are achieved through the use of an alternative approach to the problems presented by large scale bacterial fermentation of these two forms of the enzyme. Separately for chon~roitinase I
and chn~roitinase II, the gene that encodes the enzyme is cloned and the enzyme i8 expressed at high levels in a heterologous host. In a preferred embodiment, this invention is directed to the cloning of the P. vulaari~ gene for ch~roitinase I and the high level expression of that enzyme in E. coli, as well as the cloning of the P. vulq,aris gene for chon~roitinase II and the high level expression of that enzyme in E. ,coli.
This invention provides a purified isolated DNA fragment of P. vulqaris which comprises a sequence encoding for chon~roitinase I. This invention further provides a purified isolated DNA fragment of P.
vulqaris which hybridizes with a nucleic acid sequence encoding for amino acids as follows:
(a) the chQn~oitinase I enzyme with its signal peptide (SEQ ID NO: 2, amino acids 1-1021) or a biological equivalent thereof (encoded for example by: (l) nucleotides numbered 119-3181 of SEQ ID NO:l, and (2) nucleotides numbered 119-3181 of SEQ ID NO:3, where the three nucleotides immediately upstream of the initiation codon are changed (SEQ ID NO:3, nucleotide~ 116-3~ 118));

W094/25567 . PCT~S94/~95 (b) the mature chn~roitinase I enzyme (SEQ
ID N0:2, amino acids 25-1021) or a biological equivalent thereof (encoded for example by: (1) nucleotides numbered 191-3181 of SEQ ID N0:1, and

(2) nucleotides numbered 191-3181 of SEQ ID N0:3, where the three nucleotides immediately upstream of the initiation codon are changed (SEQ ID
N0:3, nucleotides 116-118)); and (c) the mature rhQ~roitinase I enzyme where the sequence encoding the signal peptide has been replaced with a seauence which adds a methionine residue to the amino terminus of the enzyme (SEQ ID N0:5, a~;no acids 24-1021) or a biological equivalent thereof (encoded for example by nucleotides numbered 188-3181 of SEQ ID
N0:4).
The recombinant cho~roitinase I is produced by transforming a host cell with a plasmid cont~;n;ng a purified isolated DNA fragment of P. vulaaris which contains one of the above-described seauences, and culturing the host cell under conditions which permit expression of the enzyme by the host cell.
This invention also provides a purified isolated DNA fragment of P. vulqaris which comprises a sequence encoding for chn~roitinase II. This invention further provides a purified isolated DNA
fragment from P. vulaaris which hybridizes with a nucleic acid sequence ~nroA; ng for amino acids as follows:
(a) the chQ~roitinase II enzyme with its signal peptide (SEQ ID N0:40, amino - W094t25567 PCT~S94/0~95 acids 1-1013) or a biological equivalent thereof (encoded for example by nucleotides numbered 3238-6276 of SEQ ID N0:39); and (b) the mature cho~roitinase II enzyme (SEQ ID N0:40, amino acids 24-1013) or a biological equivalent thereof (encoded for example by nucleotides numbered 3307-6276 of SEQ ID N0:39).
The recombinant chQ~roitinase II is produced by transforming a host cell with a plasmid cont~;n;ng a purified isolated DNA fragment of P.
vulqaris which contains one of the above-described sequences, and culturing the host cell under conditions which permit expression of the enzyme by the host cell.
It is an additional object of this invention to provide methods for the isolation and purification of the recombinantly expressed cho~roitinase I enzyme of P. vulgaris.
It is a particular object of this invention to provide methods which result in significantly higher yields and purity of the recombinant chQn~roitinase I enzyme than those obtained by adapting for the recombinant enzyme the method previously used for isolating and purifying the native chonAroitinase I enzyme from P. vulqaris.
These objects are achieved through either of two methods described and claimed herein for the - 30 ch~n~roitinase I enzyme. The first method comprises the steps of:
(a) lysing by homogenization the host cell~
which express the recombinant ch~roitinase I enzyme to release the enzyme into the supernatant;

W094/25567 PCT~S94/04495 2 16 112~ _ 8 - -(b) subjecting the supernatant to diafiltration to remove salts and other small molecules;
(c) passing the supernatant through an anion eY~hAnge resin-contA;n;ng column;
(d) loAA;ng the eluate from step (c) to a cation eY~ch~nge resin-contA;n;ng column 80 that the enzyme in the eluate binds to the cation eYch~nge column; and (e) eluting the enzyme bound to the cation ~YchA~ge col~mn with a solvent capable of releasing the enzyme from the column.
In the second method, prior to step (b) of the first method just described, the following two steps are performed:
(1) treating the ~u~e.~atant with an acidic solution to precipitate out the enzyme;
and (2) recovering the pellet and then dissolving it in an alkali solution to again place the enzyme in a basic environment.
It is a further object of this invention to provide methods for the isolation and purification of the recombinantly eYpressed chQ~Aroitinase II enzyme of P. vulqaris.
It is an additional object of this invention to provide methods which re~ult in ~ignificantly higher yields and purity of the recombinant ch~Aroitinase II enzyme than those obtA;neA by adapting for the recombinant enzyme the method previously used for isolating and purifying the native chQ~roitinase I enzyme from P. vulqaris.
These objects are achieved through either of - W094/25567 PCT~S94/~95 two methods described and claimed herein for the rhn~roitinase II enzyme. The first method comprises the steps of:
(a) lysing by homogenization the host cells which express the recombinant rho~roitinase I enzyme to release the enzyme into the supernatant;
(b) su~jecting the supernatant to diafiltration to remove salts and other small molecules;
(c) passing the supernatant through an anion eYchan~e resin-contAin;ng column;
(d) loA~;ng the eluate from step (c) to a cation ~Y~hAn~e resin-contA;n;ng col~n 80 that the enzyme in the eluate binds to the cation eYchAnge column;
(e) obta;n;ng by affinity elution the enzyme bound to the cation ~Ych~An~e column with a solution of chon~roitin sulfate, such that the enzyme is co-eluted with the cho~roitin sulfate;
(f) loA~;ng the eluate from ~tep (e) to an anion eYrhAn~e resin-contA;n;ng column and eluting the enzyme with a sol~ent such that the ~hnn~roitin sulfate binds to the column; and (g) concentrating the eluate from step (f) and crystallizing out the enzyme from the ~upernatant which contains an - 30 approximately 37 kD contaminant.
In the second method, prior to step (b) of the first method just described, the following two steps are performed:
(1) treating the supernatant with an acidic solution to precipitate out the enzyme;

W094/25567 PCT~S94/04495 and (2) recovering the pellet and then dissolving it in an alkali solution to again place the enzyme in a basic environment.
Use of the methods of this invention results in significantly higher yields and purity of each recombinant enzyme than those obt~;ne~ by adapting for each recombinant enzyme the method previously ueed for isolating and purifying the native ch~roitinase I
enzyme from P. vulqaris.

Brief DescriPtion of the Fi res Figure 1 depict~ a preliminary restriction map for the subcloned approximately 10 kilobase Nsi fragment in pIBI24. The Nsi fragment contains the complete gene encoding chon~oitinase I and a portion of the gene encoding chon~roitinase II. The restriction site~ are shown in their approximate positions. The restriction sites are useful in the constructions described below; other restriction sites present are not shown in this Figure; some are ~et forth in Example 13 below.
Figure 2 depicts the elution of the recombinant cho~noitinase I enzyme from a cation eY~h~nge chromatography column using a sodium chloride gradient. The method used to purify the native enzyme is used here to attempt to purify the recombinant enzyme. The initial fractions at the left do not bind to the column. They contain the majority of the chon~oitinase I enzyme activity. The fractions at right cont~; n; ng the enzyme are marked "eluted activity". The gradient is from 0.O to 250 mM NaCl.
Figure 3 depicts the elution of the W094/25567 PCT~S94/0~95 2l6ll25 recombinant chon~roitinase I enzyme from a cation ge column, after first passing the supernatant through an anion eY~h~nge column, in accordance with a method of this invention. The initial fractions at the left do not bind to the column, and contain only traces of chonAroitinase I activity. The fractions at right contA; n; ng the enzyme are marked "eluted activity". The gradient is from 0.O to 250 mM NaCl.
Figure 4 depicts sodium dodecyl sulfate-polyacrylamide gel chromatography (SDS-PAGE) of the recombinant chnn~roitinase I enzyme before and after the purification methods of this invention are used.
In the SDS-PAGE gel photograph, Lane 1 i8 the enzyme purified using the method of the first ~hodiment of the invention Lane 2 is the enzyme purified using the method of the second embodiment of the in~ention;
Lane 3 represents the supernatant from the host cell prior to purification -- many other proteins are present; Lane 4 represents the following molecular weight st~n~rds: 14.4 kD - lysozyme; 21.5 kD -trypsin inhibitor; 31 kD - carbonic anhydrase;
42.7 kD - ovalbumin: 66.2 kD - bovine serum albumin;
97.4 kD - phosphorylase B; 116 kD - beta-galactosidase; 200 kD - myosin. A single sharp band is seen in Lanes 1 and 2.
Figure 5 depicts SDS-PAGE chromatography of the recombinant chQn~oitinase II enzyme during various stages of purification u~ing a method of this invention. In the SDS-PAGE gel photograph, Lane 1 i~
the crude supernatant after diafiltration; Lane 2 the eluate after passage of the supernatant through an anion eYch~nge resin-cont~;ning col~ ; Lane 3 is the enzyme after elution through a cation eY~hange resin-conta;n;ng column; ~ane 4 is the enzyme after elution through a second anion ~Yrh~nge resin-cont~;n;ng W094/25S67 - PCT~S94/0~95 ~161125 column; Lane 5 represents the same molecular weight st~n~Ards as described for Figure 4, plus 6.5 kD -aprotinin; Lane 6 is the same as Lane 4, except it iR
overloaded to show the approximately 37 kD
cont~m;nAnt; Lane 7 is the 37 kD contA~;nAnt in the supernatant after crystallization of the chon~-oitinaee II enzyme; ~ane 8 i~ first wa~h of the crystals; ~ane 9 is the second wash of the crystals;
Lane 10 is the enzyme in the washed crystals after redissolving in water.

Detailed Descri~tion of the Invention Preliminary experiments indicated that E.
coli could not use the hydrolysis products yielded by chnn~roitinase I as a sole carbon source, suggesting that this gene could not be cloned by selecting for its expression in E. coli. Another approach, followed in this application, is to use a physical method to identify DNA fragments that encode the chQ~roitinase I enzyme. This is accomplished using an appropriately labeled probe for hybridization with individual clones that, together, make up a gene bank comprising the complete genome of P. vulqaris. The probe itself is generated using Polymerase Chain Reaction (PCR) (5).
In this procedure, the genomic DNA of P. vulqaris is denatured and oligonucleotides (designed to bracket part of the rhQn~roitinase I gene) are Anne~led and DNA synthesis is carried out i vitro. This cycle of denaturation, annealing and DNA synthesis using the oligonucleotides as primers is repeated many times (e.g., 30), with the yield of the desired product (the DNA fragment that lies between the two oligonuc-leotides) increasing eYpo~entially with each cycle.
A putative nucleotide sequence of the WOg4/25567 - 216112 S PCT~S94/0~95 appropriate oligonucleotides is constructed from available amino acid sequence information derived from the protein purified from P. vulqaris bacteria. Once this is done, the DNA fragment produced by PCR is cloned and its DNA sequence determined to verify that it is part of the chnn~roitinase I gene. It is then labeled and used as a probe to indicate which members of the gene bank actually contain the chon~roitinase I
gene. Subsequent restriction mapping and Southern hybridization narrows the location to a piece of DNA
of approximately four thousand base-pairs (bp). This i8 then sequenced using the Sanger dideoxy chain termination method (6) to reveal the exact position of the gene and guide the subsequent manipulations u~ed to place the gene into a high-level expression system in E. coli. A fermentation at a 10 liter ~cale carried out with this E. coli strain contain;ng a recombinant plasmid expressing the P. vulgaris chQ~roitina~e I gene yields a maximum chQn~roitinase I titer of a~lo~imately 600 units/ml (which is the same as 1.2 mg/ml). This yield far exceeds that of the native P. vul~aris fermentation process which had not achieved a titer of more than 2 units/ml.
The process of cloning and expression of the chQ~roitina~e I gene is summarized by the following series of stages:
1) The isolation of P. vul~aris genomic DNA and the construction of a cosmid gene bank.
2) PCR experimentation designed to yield an authentic piece of the chQn~roitinase I gene for u~e as a hybridization probe.

3) Colony hybridization studies to identify at least a portion of the chQn~roitinase I
gene.

4) Restriction mapping, Southern hybridi-W094t25567 PCT~S94/04495 ~l6li2s ;

zation, DNA Bequencing~ and chon~roitinase I enzyme assays that, collectively, serve to place the location of the chQ~roitinase I gene more precisely within the cloned DNA.

5) DNA 8equence analysis to reveal the exact coding region and location of the cho~roitinase I gene.

6) Site-specific mutagenesis, related manipulations, and genetic engineering le~;ng to the regulated, high-level expression of the P. vulqaris gene in E. coli.
These 8ix stages are described in specific detail in Examples 1-7 below. The rationale for the stages is as follows. In the first stage, genomic DNA
is obta;n~A. DNA is separated from protein and other material contained in a P. vulqaris fermentation.
Study of the genomic DNA is facilitated by the insertion of fragments of the DNA into cosmid vectors.
The genomic DNA is digested with an a~ o~riate restriction en~n~nclease, such as Sau3A, and then ligated into a cosmid vector. The p~~ged recombinant cosmids cont~;n;ng the P. vulaaris DNA
fragments are introduced into an a~lo~riate bacterial host strain, such as an E. coli strain, and the resulting culture is grown to allow gene expression.
The gene banks are engineered to contain a marker, such as ampicillin or kanamycin resistance, to assist in the screening of the gene banks for the presence of the chQ~roitinase I gene.
Applicants have conducted some amino acid sequencing of the native cho~roitinase I enzyme.
Samples of the enzyme are generated by fermentation of P. vulqaris. Samples may also be obt~;ne~ from Seikagaku Rogyo Co., Ltd., Tokyo, Japan. The amino acid sequence information is used to design 2161~ 2~

oligonucleotides for use in screening for the chon~oitinase I gene.
In the second stage, oligonucleotides are designed for use in PCR. A first set of oligonucleotides is designed 80 as to encode a heptapeptide that has minimal degeneracy of its genetic code. Seven amino acids near the amino terminus of the chon~roitinase I enzyme (SEQ ID NO:2, amino acids 19-25) are potentially encoded by 512 different nucleotide sequences (SEQ ID NO:6; see Example 2). The number of potential sequences is reduced to 32 by selecting specific nucleotides at the 5' end, because of the observation that mismatched nucleotides in PCR primers are of less consequence at the 5' end than at the 3' end of the primer (7). The sequences of the pool of 32 primers are set out at SEQ
ID NOS:7-14.
Applicants have discovered that the approximately 110 kD rhon~oitinase I enzyme is cleaved proteolytically into an 18,000 NW (nl8 kDn) fragment and an approximately 90,000 MW (ngo kDn) fragment. Furthermore, the 18 kD fragment is further fragmented by treatment with cyanogen bromide and trypsin. The various fragments are then used to design additional sets of oligonucleotide primers for PCR.
Seven amino acids within the 18 kD fragment (SEQ ID NO:2, amino acids 114-120) are potentially encoded by 512 different nucleotide sequences (SEQ ID
NO:15; see Example 2). The complementary strand has the same number of potential sequences (SEQ ID NO:16;
see Example 2). Using the criteria described above for the first set of oligonucleotides, the number of potential sequences is reduced to 128, whose sequences are set out at SEQ ID NOS:17-24.

W094t25567 PCT~S94/0~95 Six amino acids located near the amino-terminus of the "90 kD" fragment (SEQ ID NO:2, amino acids 165-170) are potentially encoded by a large ~umber of different nucleotide sequences (SEQ ID
NOS:25 and 26; see Example 2). The complementary strand has the same number of potential sequences (SEQ
ID NOS:27 and 28; see Example 2). Using the criteria described above for the first set of oligonucleotides, the number of potential sequences is reduced to the sequences set out at SEQ ID NOS:29-36.
PCR amplifications are conducted using these 24 mixtures of oligonucleotides. The most effective ampl$fications are observed as discrete bands on electrophoretic gels. Products approxlmately 500 and 350 base pairs (bp) in size are obt~ineA. The approximately 350 bp product is a subfragment of the approximately 500 bp product. The approximately 500 bp product is isolated and, following successive cloning procedures described in Example 2, is isolated as a 455 bp PCR product.
This 455 bp fragment is sequenced and translated into an amino acid sequence which is in virtual agreement with the sequence available from the native ~ho~roitinase I enzyme. The sequences differ by one amino acid; subsequent experiments reveal that the nucleotide and amino acid sequences of the 455 bp fragment are correct, while the native amino acid sequence identification is in error.
In the third stage, the PCR amplification fragment is used as a probe to identify the cosmid gene banks prepared in the first stage which contain the chon~roitinase I gene. The PCR fragment i~
denatured and labelled with, for example, digoxigenin-labelled d~TP (Boehringer-MAnnhe;m, Tn~; ~n~poli8, IN).
The cosmid gene banks are then used to infect a W094/25567 PCT~S94/0~95 bacterial strain. The resulting colonies are lysed and their DNA subjected to colony hybridization with the labelled probe, followed by exposure to an alkaline phosphatase-conjugated antibody to the digoxigenin-labelled material. Positive clones are visualized and then picked to be grown in selective media.
In the fourth stage, Southern hybridization (8) and restriction mapping are used to localize the position of the chon~roitinase I gene within individual clones. The PCR-generated fragment described above is used as a Southern hybridization probe against P. vulaari~ genomic DNA that is first digested by restriction enzymes and fractionated. In a second PCR amplification, several of the oligonucleotides described above are used as primers.
The results indicate that the portion of the cho~roitinase I gene that hybridizes to the probe i8 carried on several large DNA fragments.
These large DNA fragments are digested to yield individual fragments which are isolated, tested for the presence of chon~roitinase I sequences by Southern hybridization, and then subcloned into appropriate vectors. Example 3 details the cloning strategy used. Restriction maps are generated to assist in the identification of the portions of the fragments carrying the desired sequences. In addition, i vitro rhQn~roitinase I assays in which the acti~ity of the enzyme based on measuring the release of unsaturated disaccharide from rhQ~roitin sulfate C at 232 nm are conducted on several samples to assist in the placement and orientation of the chQn~roitinase I gene. The results of these procedures suggest that a 4.2 kb EcoRV-EcoRI fragment of a larger 10 kb NsiI fragment could contain the W094/25567 PCT~S94/0~95 entire chon~roitinase I gene.
In the fifth stage, the above-mentioned 4.2 kb fragment iB subjected to DNA sequence analysis.
The resulting DNA sequence iB 3980 nucleotides in length (SEQ ID NO:l). Translation of the DNA sequence into the putative amino acid sequence reveals a continuous open reA~;n~ frame (SEQ ID NO:l, nucleotides 119-3181) encoding 1021 amino acids (SEQ
ID NO:2).
In turn, analysis of the amino acid sequence reveals a 24 residue signal sequence (SEQ ID NO:2, amino acids 1-24), followed by a 997 residue mature (processed) rhQ~roitinase I enzyme (SEQ ID NO:2, amino acids 25-1021).
Signal ~equences are required for a complex series of post-translational processing steps which result in secretion of a protein from a host cell.
The signal ~equence constitutes the amino-terminal end of the protein to be secreted. In most cases, the signal sequence is cleaved off by a specific protease, called a signal peptidase.
The "18 kD" and "90 kD" fragments are found to be adjacent to each other, with the "18 kD"
fragment constituting the first 157 amino acids of the mature protein (SEQ ID NO:2, amino acids 25-181), and the "90 kD" fragment constituting the remaining 840 amino acids of the mature protein (SEQ ID NO:2, amino acids 182-1021).
The chQn~roitinase I enzyme of this invention is expressed using established recombinant DNA methods. Suitable host organisms include bacteria, viruses, yeast, insect or mammalian cell lines, as well as other conventional organisms. The host cell is transformed with a plasmid cont~;n;ng a purified isolated DNA fragment encoding for W094/25567 PCT~S94/0~95 2161125 ~

chon~roitinase I enzyme. The host cell i8 then cultured under conditions which permit expression of the enzyme by the host cell.
In the sixth stage, the gene is subjected to site-directed mutagenesis to introduce unique restriction sites. These permit the gene to be moved, in the correct reading frame, into an expression system which results in expression of ~ho~roitinase I
enzyme at high levels. Such an appropriate host cell is the bacterium _. coli.
As detailed in Example 6 below, two different constructs are prepared. In the first, the three nucleotides immediately upstream of the initiation codon are changed (SEQ ID N0:3, nucleotides 116-118) through the use of a mutagenic oligonucleotide (SEQ ID N0:37). The coding region and amino acid sequence encoded by the resulting construct are not changed, and the signal sequence is preserved (SEQ ID N0:3, nucleotides 119-3181; SEQ ID N0:2).
In a preferred ~odiment of this invention, the second construct is used. In the second construct, the site-directed mutagenesis is carried out at the junction of the signal sequence and the start of the mature protein. A mutagenic oligonucleotide (SEQ ID N0:38) is used which differs at six nucleotides from those of the native sequence (SEQ ID N0:1, nucleotides 185-190). The sequence differences result in (a) the deletion of the signal sequence, and (b) the addition of a methionine residue at the amino-terminus, resulting in a 998 amino acid protein (SEQ ID N0:4, nucleotides 188-3181; SEQ ID
N0:5).
In the absence of a signal sequence, the enzyme is not secreted. Fortunately, it is not retAine~ within the cell in the form of insoluble W094/25567 PCT~S94/0~95 216112~ `

inclusion bodies. Instead, at least some of the enzyme is produced intracellularly as a soluble active enzyme. The enzyme is extracted by homogenization, which serves to lyse the cells and thereby release the enzyme into the supernatant. Even with the signal sequence present, much of the enzyme is not secreted, because it is thought that this expression system provides such high yields of enzyme that it exceeds the capacity of the host cell to secrete that much enzyme.
As described in Example 7 below, the gene lacking the signal sequence is inserted into an appropriate expression vector. One such vector is pET-9A (9; Novagen, Madison, WI), which is derived from elements of the E. coli bacteriophage T7. The resulting recombinant plasmid is designated pTM49-6.
The plasmid is then used to transform an appropriate expression host cell, such as the E. coli B strain BL21/(DE3)/pLysS (10; Novagen).
Samples of this E. coli B strain BL21(DE3)/pLysS carrying the recombinant plasmid pTM49-6 were deposited by Applicants on February 4, 1993, with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A., and have been assigned ATCC accession number 69234.
Expression of the chQn~roitinase I enzyme using the deposited host cell yields approximately 300 times the amount of the enzyme as was possible using a same size fermentation vessel with native (non-recombinant) P. vulqaris.
After expression of the ~hQn~roitinase I
enzyme, the supernatant from the host cellg i8 treated to isolate and purify the enzyme. Initial attemptg to isolate and purify the recombinant chon~roitinase I
enzyme do not result in high yield~ of purified W094/25567 ~16 11 2 5 ~ PCT~S94/04495 protein. The previous method for isolating and purifying native chon~oitinase I from fermentation cultures of P. vulqaris is found to be inappropriate for the recombinant material.
The native enzyme is produced by fermentation of a culture of P. vul~aris. The bacterial cells are first recovered from the medium and resuspended in buffer. The cell suspension is then homogenized to lyse the bacterial cells. Then a charged particulate such as Bioacryl (Toso Haas, Philadelphia, PA), is A~A~ to remove DNA, aggregates and debris from the homogenization step. Next, the solution iB brought to 40% saturation of ammonium sulfate to precipitate out undesired proteins. The ch~nA~oitinase I remains in solution.
The solution is then filtered and the retentate is wa~h~ to recover most of the enzyme.
The filtrate is concentrated and subjected to diafiltration with a phosphate to remove the salt.
The filtrate cont~in;ng the rhQn~oitinase I
is subjected to cation ~Y~hAnge chromatography using a cellulose sulfate column. At pH 7.2, 20 mM sodium phosphate, more than 98% of the chQn~oitinase I binds to the col~ . The native chQnA~oitinase I is then eluted from the column using a sodium chloride gradient.
The eluted enzyme is then subjected to additional chromatography steps, such as anion ~Ych~nge and hydrophobic interaction column chromatography. As a result of all of these procedures, rhQn~oitinase I is obt~;ne~ at a purity of 90-97%. The level of purity is measured by first performing SDS-PAGE. The proteins are st~;n~ using Coomassie blue, destained, and the lane on the gel is scanned using a laser beam of wavelength 600 nm. The W094/25567 PCT~S94/04495 6~2~ ~

purity is expressed as the percentage of the total absorbance accounted for by that band.
However, the yield of the native protein is only 25-35%. The yield is measured as the remaining activity in the final purified product, expressed a~ a percentage of the activity at the start (which is taken as 100%). In turn, the activity of the enzyme is based on measuring the release of unsaturated disaccharide from chon~roitin sulfate C at 232 nm.
This purification method also results in the extensive cleavage of the approximately 110,000 dalton (110 kD) chQ~roitinase I protein into a 90 kD and an 18 kD fragment. Nonetheless, the two fragments remain non-covalently bound and exhibit chQn~roitinase I
activity.
When this procedure is repeated with homogenate from lysed host cells carrying a recombinant plasmid encoding chn~roitinase I, significantly poorer results are obt~;neA. Less than 10% of the rhn~roitinase I binds to the cation eYch~nge column at st~n~rd stringent conditions of pH

7.2, 20 mM sodium phosphate.
Under less stringent bin~;ng conditions of pH 6.8 and 5 mM phosphate, an improvement of b; n~; ng with one batch of material to 60-90% is observed.
However, elution of the recombinant protein with the NaCl gradient gives a broad activity peak, rather than a sharp peak (see Figure 2). This indicates the product is heterogeneous. Furthermore, in subsequent fermentation batches, the recombinant enzyme binds poorly (1-40%), even using the less stringent b;n~;ng conditions. Most of these batches are not processed to the end, as there is poor b;n~;ng. Therefore, their overall recovery is not quantified.
Based on these result~, it is concluded that W094/2S567 PCT~S94/04495 ~161125 the recombinant chn~roitinase I enzyme has a reduced basicity compared to the native enzyme, and that the basicity also varies between batches, as well as within the same batch.
It is evident that the method used to isolate and purify the native enzyme is not appropriate for the recombinant enzyme. The method produces low yields of protein at high cost.
Furthermore, for large batches, large amounts of solvent waste are produced cont~in;ng large amounts of a nitrogen-cont~;n;ng compound (ammonium sulfate).
This is undesirable from an environmental point of view.
A hypothesis is then developed to explain the~e poor results and to provide a basis for developing improved isolation and purification methods. It is known that the native chQn~oitinase I
enzyme is ba~ic at neutral pH. It is therefore assumed that the surface of the enzyme has a net excess of positive charges.
Without being bound by this hypothesis, it is believed that, in recombinant expression of the enzyme, the host cell contains or produces small, negatively charged molecules. These negatively charged molecules bind to the enzyme, thereby reducing the number of positive charges on the enzyme. If these negatively charged molecules bind with high enough affinity to copurify with the enzyme, they can cause an alteration of the behavior of the enzyme on the ion ~Y~h~nge column.
Support for this hypothesi6 is provided by the data described below. In general, cation eYch~nge resins bind to proteins better at lower pH's than higher pH's. Thus, a protein which is not very basic, and hence does not bind at a high pH, can be made to W094/25567 216112 a PCT~S94/0~95 '.`

bind to the cation eYch~nger by carrying out the operation at a lower pH. At pH 7.2, the native enzyme binds completely to a cation ~Ych~nge resin. However, the recombinant-derived enzyme, due to the lowered basicity as a result of b; n~; ng of the negatively charged molecule~, does not bind very well (less than 10%). This enzyme can be made to bind up to 70% by using a pH of 6.8 and a lower phosphate concentration (5 mM rather than 20 mM), but heterogeneity and low yield remain great problems. Indeed, only one fermentation results in a 70% bin~;ng level;
typically, it is much less (less than 10%) even at pH
6.8. This level of b; n~; ng varies dramatically between different fermentation batches.
This hypothesis and a possible solution to the problem are then tested. If negatively charged molecules are att~ch; n~ non-covalently to ch~n~roitinase I, thus decreasing its basicity, it should be possible to remove these undesired molecules by using a strong, high capacity anion eYch~nge resin.
Removal of the negatively charged molecules should then restore the basicity of the enzyme. The enzyme could then be bound to a cation eY~ch~n~e resin and eluted therefrom in pure form at higher yields.
Experiments demonstrate that this approach indeed provides a solution to the problem encountered with the isolation and purification of the recombinantly expressed cho~roitinase I enzyme.
As is discussed below, chon~roitinase I is recombinantly expressed in two forms. The enzyme is expressed with a signal peptide, which is then cleaved to produce the mature enzyme. The enzyme is also expressed without a signal peptide, to produce directly the mature enzyme. The two embodiments of this invention which will now be discussed are W094l25567 216112 5 PCT~S94/0~95 suitable for use in purifying either of these forms of the enzyme.
In the first embodiment of this aspect of the invention, the host cells which express the recombinant rho~roitinase I enzyme are lysed by homogenization to release the enzyme into the supernatant. The supernatant is then subjected to diafiltration to remove salts and other small molecules. However, this step only removes the free, but not the bound form of the negatively charged molecules. The bound form of these cha~ged species is next removed by passing the supernatant through a strong, high capacity anion eY~han~e resin-conta;n;ng col-~n. An example of such a resin is the Macro-PrepTM
High Q resin (Pio-Rad, Melville, N.Y.). Other strong, high capacity anion ~Ychange columns are also suitable. Weak anion eYchan~ers conta;n;n~ a diethylaminoethyl (DEAE) ligand also are suitable, although they are not as effective. Similarly, low capacity resins are also suitable, although they too are not as effective. The negatively charged molecules bind to the column, while the enzyme passes through the column. It is also found that some unrelated, undesirable proteins also bind to the cOll--nn .
Next, the eluate from the anion ~Ychange column is directly loaded to a cation ~Y~hange resin-conta;n;ng column. Examples of such resins are the S-SepharoseTM (Pharmacia, Piscataway, N.J.) and the Macro-PrepTM High S (Bio-Rad). Each of the~e two resin-cont~;n;ng columns has S03- ligands bound thereto in order to facilitate the ~Ychan~e of cations. Other cation eY~hange columns are also suitable. The enzyme binds to the column and is then eluted with a solvent capable of releasing the enzyme from the column.

W094/25567 PCT~S94/04495 Any salt which increases the conductivity of the solution is suitable for elution. Examples of such salts include sodium salts, as well as potassium salts and ammonium salts. An aqueous sodium chloride solution of appropriate concentration is suitable. A
gradient, such as 0 to 250 mM sodium chloride is acceptable, as is a step elution using 200 mM sodium chloride.
A sharp peak is seen in the sodium chloride gradient elution (Figure 3). The improvement in enzyme yield over the prior method is striking. The recombinant rh~n~noitinase I enzyme is recovered at a purity of 99% at a yield of 80-90%.
The purity of the protein is measured by 8CAnn; ng the bands in SDS-PAGE gels. A 4-20% gradient of acrylamide is used in the development of the gels.
The band(s) in each lane of the gel is scAnne~ using the procedure described above.
These improvements are related directly to the increase in b;n~;ng of the enzyme to the cation eYrhAnge column which results from first using the anion ~YchAnge column. In comparative experiments, when only the cation ~YchAnge column is used, only 1%
of the enzyme binds to the col~"~. However, when the anion eYrhAnge column is used first, over 95% of the enzyme binds to the column.
The high purity and yield obtA; ne~ with the first embodiment of this invention make it more feasible to manufacture the recombinant chQn~oitinase I enzyme on a large scale.
In a second embodiment of this aspect of the invention, two additional steps are inserted in the method before the diafiltration step of the first embodiment. The supernatant is treated with an acidic solution to precipitate out the desired enzyme. The W094/25567 216112 ~ PCT~S94/04495 pellet i8 recovered and then dissolved in an alkali solution to again place the enzyme in a basic environment. The solution is then subjected to the diafiltration and subsequent steps of the first embodiment of this invention.
In comparative experiments with the second embodiment of this invention, when only the cation eYchange column is used, only 5% of the enzyme binds to the coln~n. However, when the anion exchange column is used first, essentially 100% of the enzyme binds to the column. The second embodiment provides comparable enzyme purity and yield to the first emho~iment of the invention.
Acid precipitation removes proteins that remain soluble; however, these proteins are re~moved anyway by the cation and anion eYrhange steps that follow (although smaller columns may be used). An advantage of the acid precipitation step is that the sample volume is decreased to about 20% of the original volume after dissolution, and hence can be handled more easily on a large scale. However, the additional acid precipitation and alkali dissolution steps of the second ~bodiment mean that the second embodiment is more time consuming than the first embodiment. On a manufacturing scale, the marginal improvements in purity and yield provided by the second embodiment may be outweighed by the simpler procedure of the first embodiment, which still provides highly pure cho~oitinase I enzyme at high yields. An additional benefit of the two embodiments of the invention is that cleavage of the enzyme into - 90 kD and 18 kD fragments is avoided.
The high purity of the enzyme produced by the two ~hodiments of this invention is depicted in Figure 4. A single sharp band is seen in the SDS-PAGE

W094t25567 - PCT~S94/04495 2161125 ` ~

gel photograph: Lane 1 is the enzyme using the method of the first embodiment; Lane 2 is the enzyme using the method of the second embodiment (Lane 3 represents the supernatant from the host cell prior to purification -- many other proteins are present;
Lane 4 represents molecular weight st~n~rds).
The material deposited with the ATCC can also be used in conjunction with the sequences disclosed herein to regenerate the native cho~roitinase I gene sequence (SEQ ID NO:1) or the modified ch~roitinase I gene sequence which includes the signal sequence (SEQ ID NO:3) using conventional genetic engineering technology.
Production of native chon~roitinaee I enzyme in P. vulqaris after induction with rh~n~roitin sulfate does not provide a high yield of enzyme; the enzyme represents approximately 0.1% of total protein present. When the recombinant construct with the signal sequence deleted is used in E. coli, approximately 15% of the total protein is the ch~roitinase I enzyme.
In addition to the three DNA seguences just described for the chon~roitinase I gene (SEQ ID NOS:1, 3 and 4), the present invention further comprises DNA
sequences which, by virtue of the re~n~ncy of the genetic code, are biologically equivalent to the sequences which encode for the enzyme, that is, these other DNA sequences are characterized by nucleotide sequences which differ from those set forth herein, but which encode an enzyme having the same amino acid sequences as those encoded by the DNA sequences set forth herein.
In particular, the invention contemplates those DNA sequences which are sufficiently duplicative of the sequences of SEQ ID NOS:1, 3 or 4 80 as to W094/2~567 216112 5 permit hybridization therewith under stAn~rd high stringency Southern hybridization conditions, such as those described in Sambrook et al. (11), as well as the biologically active enzymes produced thereby.
This invention also comprises DNA sequences which encode amino acid sequences which differ from those of the chon~oitinase I enzyme, but which are the biological equivalent to those described for the enzyme (SEQ ID NOS:2 and 5). Such amino acid sequences may be said to be biologically equivalent to those of the enzyme if their sequences differ only by minor deletions from or con~ervative substitutions to the enzyme sequence, such that the tertiary configurations of the sequences are essentially ~nch~nged from those of the enzyme.
For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon ~nco~; ng another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, as well as changes based on similarities of residues in their hydropathic index, can also be expected to produce a biologically equivalent product. Nucleotide changes which result in alteration of the N-terminal or C-terminal portionQ of the protein molecule would also not be expected to alter the activity of the protein.
Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded - products. Therefore, where the terms "chon~oitinase I gene" or "chnn~oitinase I enzyme" are used in W094/25567 PCT~S94/04495 3l6ll25 either the specification or the claims, each will be understood to encompass all such modifications and variations which result in the production of a biologically equivalent protein.
The starting point for the cloning and expression of the cho~noitinase II gene is partial amino acid sequencing of the mature native chnn~oitinase II protein obtained from P. vulqaris.
The N-terminal sequence of the mature native ~hQ~oitinase II protein is found to include the following 22 amino acids:

Leu-Pro-Thr-Leu-Ser-His-Glu-Ala-Phe-Gly-Asp-Ile-Tyr-Leu-Phe-Glu-Gly-Glu-~eu-Pro-Asn-Thr (SEQ ID NO: 40, amino acids 1-22) The nucleotide sequence determined above for the region encoding the cho~oitinase I gene includes an additional approximately 800 base pairs beyond the translation termination codon (SEQ ID NOS:1 and 39, nucleotides 3185-3980). An inspection of this region reveals that the sequence between nucleotides 3307 and 3372 (SEQ ID NOS:1 and 39) encodes the identical 22 amino acids in the same order as the first 22 amino acids of native chQ~oitinase II.
Furthermore, an ATG initiation codon (SEQ ID
NOS:l and 39, nucleotides 3238-3240) is found upstream of this region and in-frame, indicating that this gene is expressed with a 23 amino acid signal peptide sequence for the export of cho~oitinase II (SEQ ID
NO:40, amino acids 1-23). Although a Shine-Dalgarno sequence (AGGA; SEQ ID NOS:l and 39, nucleotides 3225-3228) is found upstream of the initiation codon, there is no apparent promoter sequence, suggesting that both the 110 kD and 112 kD forms of the P. vul~aris WO 94/25567 - PCT~S94/0~95 chQn~roitinase enzyme are expressed as part of a single messenger RNA.
The coding sequence that starts with this ATG was originally not found to be continuous in SEQ
ID NO:1, since a termination codon (TAA) was thought to be present in-frame at base-pairs identified as 3607-3609. Re-examination of the sequencing data, however, revealed that a residue was overlooked and that a T should be inserted between nucleotides originally identified as 3593 and 3594. This change restores the open re~; ng frame which then extends through the end of SEQ ID NO: 39 (SEQ ID NOS:l and 39 include the inserted T as nucleotide 3594). (Thus, the three bases TAA at base-pairs 3608-3610, properly nu~bered, do not constitute a termination codon.) With this information available, the cloning and expression of the P. vulqaris rh~n~roitinase II
gene is performed $n three stages. In the first stage, because the N-terminal sequences are known, a site-specific mutagenesis is carried out. This is necessary in order for this gene to be placed, eventually, directly into the desired T7-based expression vector pET9A that is used (as described above) for the chQn~roitinase I gene. The mutagenized bases are upstream of the coding region (an AT
sequence (SEQ ID NOS:1 and 39, base pairs 3235 and 3236) is replaced by a CA sequence).
The second stage, which can be carried out in parallel with the first, involves the identification, isolation and DNA sequencing of an appropriate DNA fragment which will include the C-terminal coding region of the rhon~roitinase II gene.
The available DNA 8equence information is adequate to account for approximately 220 amino acids of an 3~ estimated 1000 for the entire cho~roitinase II

W094/25567 PCT~S94/0~95 ~16 1125 protein. The missing coding sequences, therefore, would extend for another 2400 base pairs beyond the end of SEQ ID NO: 1.
The third stage involves the assembly of an intact gene for cho~roitinase II that has been modified to include the initiation codon as part of an NdeI site and to be followed by a BamHI site downstream of the coding region. This allows a directed insertion of this gene into the pET9A
expression vector (Novagen, Madison, WI) without further modification.
Seguencing of the entire assembled gene confirms the presence of the initiation codon at nucleotides 3238-3240, where this codon represents the start of the region coding for the signal peptides at nucleotides 3238-3306, the region coding for the mature protein at nucleotides 3307-6276, and a termination codon at nucleotides 6277-6279 (SEQ ID
NO:39). The translation of this sequence results in 1013 amino acids, of which the first 23 amino acids are the signal peptide and 990 amino acids constitute the mature chQn~roitinase II protein at residues numbered 24-1013 (SEQ ID NO:40). In this construction, the signal peptide i8 ret~; ne~, such that the expressed gene is processed and secreted to yield the mature native enzyme structure that has a leucine residue at the N-terminus.
As described in Example 13 below, the gene encoding the chon~roitinase II protein is in~erted into pET9A and the resulting recombinant plasmid is designated LP21359. The plasmid is then used to transform an a~ o~riate expression host cell, such as the E. coli B strain BL21(DE3)/pLysS (which is also used for the expression of the ~hon~roitinase I gene.
Samples of this E. coli B strain designated W094/~567 PCT~S~ 5 ~161125 TD112, which is BL21(DE3)/pLysS carrying the recombinant plasmid LP21359, were deposited by Applicants on April 6, 1994, with the American Type Çulture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A., and have been assigned ATCC
accession number 69598.
Expression of the rhQn~roitinase II enzyme using the deposited host cell yields approximately 25 times the amount of the enzyme as was possible using a same size fermentation vessel with native (non-recombinant) P. w l~aris.
After expre~sion of the enzyme, the supernatant from the host cells is treated to isolate and purify the enzyme. Because of the virtually identical isoelectric points and similar molecular weights for the two proteins, the first method described above for isolating and purifying t_e recombinant rhnn~ oitinase I protein is adapted for isolating and purifying the recombinant rhQn~oitinase II protein, and then modified as will now be described.
The need for the modification of the method is based on the fact that the recombinant rh~n~oitinase II protein is expressed at levels approximately several-fold lower than the recombinant rhon~roitinase I protein; therefore, a more powerful and selective solution is necessary in order to obtain a final rhon~roitinase II product of a purity eguivalent to that obt~; ne~ for the chQn~oitinase I
protein.
The first several steps of the method for the rhnn~roitinase II protein are the same as those used to isolate and purify the chsn~roitinase I
protein. Initially, the host cells which express the recombinant ~hQ~noitinase II enzyme are lysed by WOg4/25567 2 ~ 6 l 12 5 ~ PCT~S94/04495 homogenization to release the enzyme into the supernatant. The supernatant iB then subjected to diafiltration to remove salts and other small molecule~. However, this step only removes the free, but not the bound form of the negatively charged molecules. The bound form of these charged specie~ i~
next removed by passing the supernatant through a strong, high capacity anion ~Ych~nge resin-conta;n;ng column. An example of such a resin is the Macro-PrepTM
High Q resin (Bio-Rad, Melville, N.Y.). Other strong, high capacity anion eYchange columns are also suitable. Weak anion eYchangers contA;n;ng a diethylaminoethyl (DEAE) ligand also are suitable, although they are not as effective. Similarly, low capacity resins are also ~uitable, although they too are not as effective. The negatively charged molecules bind to the column, while the enzyme passes through the column. It is also found that some unrelated, undesirable proteins also bind to the column.
Next, the eluate from the anion ~Y~hange column is directly loaded to a cation eY~h~nge resin-contA;n;ng column. Examples of such resins are the S-SepharoseTM (Pharmacia, Piscataway, N.J.) and the Macro-Prep'~ High S (Bio-Rad). Each of these two resin-conta;n;ng columns has S03- ligands bound thereto in order to facilitate the ~Yrhange of cations. Other cation eY~hange col~ns are also suitable. The enzyme binds to the column, while a significant portion of contaminating proteins elute unbound.
At this point, the method diverges from that used for the chQn~roitinase I protein. Instead of eluting the protein with a a non-specific salt solution capable of releasing the enzyme from the cation ~Ychange column, a specific elution using a W094l25567 2 1 6 1 1 2 ~ PCT~S94/04495 solution contA;n;ng cho~Aroitin sulfate is used.
This procedure utilizes the affinity the positively charged rhQ~Anoitinase II protein has for the negatively charged rho~Aroitin sulfate. The affinity is larger than that accounted for by a simple positive and negative interaction alone. It is an enzyme-substrate interaction, which is similar to other specific biological interactions of high affinity, such as antigen-antibody, ligand-receptor, co-factor-protein and inhibitor/activator-protein.
Hence, the ch~nAroitin sulfate is able to elute the enzyme from the negatively charged resin. In contrast, the resin-enzyme interaction is a simple positive and negative interaction.
Although affinity elution chromatography is as easy to practice as ion-~YrhAnge chromatography, the elution is specific, unlike salt elution. Thus, it has the advantages of both affinity chromatography (specificity), as well as ion-eYchAnge chromatography (low cost, ease of operation, reusability).
Another advantage is the low conductivity of the eluent (approximately 5% of that of the salt eluent), which allows for further ion-eYchAnge chromatography without a diafiltration/dialysis step, which is required when a salt is used. Note, that this is not a consideration in the method for the chQ~Aroitinase I protein, because no further ion-ge chromatography is needed in order to obtain the purified c~nAroitinase I protein.
There is another reason for not using the method for purifying recombinant chs~Aroitinase I.
ChQ~Aroitinase II obtained using the rhQnAroitinase I
salt elution purification method has poor stability;
there is extensive degradation at 4C within one week.
In contra~t, rhonAroitinase II obtA;n~A by affinity W094/25567 PCT~S94/04495 ;~161125 - 36 -elution is stable. The reason for this difference in stability is not known. It is to be noted that chQn~roitinase I obtained by salt elution is stable.
The cation eYrhAnge column is next washed with a phosphate buffer to elute unbound proteins, followed by w~Rh;ng with borate buffer to elute loosely bound cont~mi n~ ting proteins and to increase the pH of the resin to that required for the optimal elution of the rhQ~roitinase II protein using the substrate, chnn~roitin sulfate.
Next, a solution of cho~roitin sulfate in water, adjusted to pH 9.0, is used to elute the rhon~roitinase II protein, as a sharp peak (recovery 65%) and at a high purity of approximately 95%. A 1%
concentration of chQn~roitin sulfate i8 used. A
gradient of this sol~ent is also acceptable.
BerAllRe the rhnn~roitin sulfate has an affinity for the ch~n~roitinase II protein which i8 stronger than its affinity for the resin of the column, the chnn~roitin sulfate co-elutes with the protein. This ensures that only protein which recognizes chon~roitin sulfate is eluted, which is desirable, but also means that an additional process step is necessary to separate the ch~n~roitin sulfate from the chon~roitinase II protein.
In this separation step, the eluate is adjusted to a neutral pH and is loaded as is onto an anion ~Y~h~nge resin-cont~;n;ng column, such as the Macro-Prep~ High Q resin. The column is ~ ~he~ with a phosphate buffer. The ch~n~roitin sulfate binds to the column, while the chQn~roitinase II protein flows through in the unbound pool with greater than 95%
recovery. At this point, the protein is pure, except for the presence of a single minor contaminant of approximately 37 kD. The cont~;n~nt may be a W094/25567 2161 12 ~ PCT~S94/0449S

breakdown product of the chQ~roitinase II protein.
This contaminant is effectively removed by a crytallization step. The eluate from the anion eYch~nge coll~n is concentrated and the solution is maintained at a reduced temperature, such as 4C, for several days to crystallize out the pure chon~roitinase II protein. The supernatant contains the 37 kD contaminant. Centrifugation causes the crystals to form a pellet, while the supernatant with the 37 kD contaminant is removed by pipetting. The crystals are then w-~he~ with water. The washed crystals are compo~ed of the ~h~n~roitinase II protein at a purity of greater than 99%.
In a second embodiment of this aspect of the invention for the chQn~roitinase II protein, two additional steps are inserted in the method before the diafiltration step of the first embodiment. The supernatant i8 treated with an acidic solution to precipitate out the desired enzyme. The pellet is recovered and then dissolved in an alkali solution to again place the enzyme in a basic environment. The solution is then subjected to the diafiltration and subsequent steps of the first embodiment of this invention.
Acid precipitation removes proteins that remain soluble; however, these proteins are removed a..y~y by the cation and anion eY~h~nge steps that follow (although Qmaller columns may be used). An advantage of the acid precipitation step is that the sample volume is decreased compared to the original volume after dissolution, and hence can be handled more easily on a large scale. However, the additional acid precipitation and alkali dissolution steps of the second embodiment mean that the second embodiment is more time consuming than the first ~hodiment. On a W094t25567 - PCTtUS94/04495 216112S:

manufacturing scale, the marginal improvements in purity and yield provided by the second embodiment may be outweighed by the simpler procedure of the first embodiment, which still provides highly pure chon~roitinase II enzyme at high yields.
Production of native rhon~roitinase II
enzyme in P. vulqaris after induction with rhsn~roitin sulfate does not provide a high yield of enzyme; the enzyme represents approximately 0.1% of total protein present. When the recombinant construct is used in E.
coli, approximately 2.5% of the total protein is the chon~oitinase II enzyme.
In addition to the DNA sequence just described for the chon~roitinase II gene (SEQ ID
NO:39), the present invention further comprises DNA
sequences which, by virtue of the re~nn~ncy of the genetic code, are biologically equivalent to the sequences which encode for the enzyme, that is, these other DNA Bequence8 are characterized by nucleotide sequences which differ from those set forth herein, but which encode an enzyme having the same amino acid sequences as those encoded by the DNA sequences set forth herein.
In particular, the invention contemplates those DNA sequences which are sufficiently duplicative of the sequence of SEQ ID NO:39 80 as to permit hybridization therewith under st~n~rd high stringency Southern hybridization conditions, such as those described in Sambrook et al. (11), as well as the biologically active enzymes produced thereby.
This invention also comprises DNA sequences which encode amino acid sequences which differ from those of the rh~n~roitinase II enzyme, but which are the biological equivalent to those described for the enzyme (SEQ ID NO:40). Such amino acid sequences may W094/25567 PCT~S94/0~95 be said to be biologically equivalent to those of the enzyme if their sequences differ only by minor deletions from or conservative substitutions to the enzyme sequence, such that the tertiary configurations of the sequences are essentially ~nrh~nged from those of the enzyme.
For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, as well as changes based on similarities of residues in their hydropathic index, can also be expected to produce a biologically equivalent product. Nucleotide changes which result in alteration of the N-terminal or C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.
Each of the ~lo~osed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Therefore, where the terms "c-hon~roitinase II gene" or "ch~n~roitinase II enzyme" are used in either the specification or the claims, each will be understood to encompass all such modifications and variations which result in the production of a biologically equivalent protein.
If desired, one of ordinary skill in the art can ligate together the two pieces of DNA from the two deposits, for example, at the HindIII site at nucleotide 3326, ~o as to express both the rhQ~roitinase I and cho~roitinase II proteins under W094/25567 PCT~S94/0~95 216 1125 _ 40 _ the control of the T7 promoter upstream of the coding sequence for chQnAroitinase I.
In order that this invention may be better understood, the following exampleR are set forth. The examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention.

Examples St~n~Ard molecular biology techniques are utilized according to the protocols described in Sambrook et al. (11).

Exam~le 1 Isolation Of P. vulqaris Genomic DNA
And Construction Of A Cosmid Bank In E. coli Two 35 ml aliquots (designated A and B) of a P. vulqaris large-scale (1000 liter) fermentation are obta i n~A and centrifuged. Both pellets are resuspended with 7 ml of 0.05M glucose-0.025M Tri~-HCl-O.OlM EDTA (pH 8) contA;n;ng 4 mg/ml of egg-white lysozyme. After 30 minutes of incubation at 37C, 7 ml of 1% SDS-0.16M EDTA-0.02M NaCl (pH 8) are added to sample "A" and incubation is continued at 37C for another hour.
After the initial lysozyme treatment, sample "B" is centrifuged and the cell pellet taken up with 7 ml of 0.05M glucose-0.025M Tris-HCl-O.OlM EDTA (pH 8) cont~i n i ng 40 ~g/ml of DNAase-free RNAase and then 7 ml of 1% SDS-0.16M EDTA-0.02M NaCl (pH 8) are added to this resuspended material. Finally, proteinase K
(Boehringer M~nnheim, Indianapolis, IN) is added to both samples to a final concentration of 100 ~g/ml and W094l25567 PCT~S94/0~95 ~161~2~ ~

incubation is continued o~ernight at 37C.
The next day, the samples are extracted once with an equal volume (14 ml) of equilibrated phenol followed by two further extractions in which the samples are extracted with 7 ml of phenol followed by the addition of 7 ml of chloroform, continued ~hAki ng and finally, centrifugation to separate the two phases. The DNA is precipitated by ~; ng one-quarter volume of 5M ammonium acetate and 0.6 volumes of isopropanol followed by centrifugation. The pelleted DNA is rinsed once with 70% (v/v) ethanol, dried under vacuum and then resuspended with 1 ml of TE (O.OlM
Tris-HCl-O.OOlM EDTA, pH 7.4). The nucleic acid concentration for sample "A" i~ 1.2 mg/ml while that for sample "B" is 1.3 mg/ml, as determined by their ultraviolet absorption at 260 nm.
Fragmentation of the genomic DNA to yield pieces of a size suitable for insertion into cosmid vectors (approximately 25-35 kilobases (kb)) is accom-plished by partial digestion with the restriction ~n~Qn~clease Sau3A. Duplicate 0.2 ml reactions are set up (one with preparation "A" and the other with DNA from preparation "B"), each containing 100 ~g of the P. vulqaris genomic DNA, O.lM NaCl, O.OlM MgCl2, O.OlM Tris-HCl (pH 7.5) and 80 units of the enzyme Sau3A.
Incubation i8 carried out at 37C and 25 ~l aliquots are remo~ed at a~v~riate time points (5,6,7,8,9,10,11 and 20 minutes) and added to 25 ~1 of 0.2M EDTA (pH 8). The individual ~amples are heated to 70C and then 10 ~l are removed for a size-distribution analysis on an agarose gel. The sample obtained after five minutes of Sau3A digestion of preparation "A" and that obtained after 6 minutes with 3~ preparation "B" are chosen for further use.

W094t25567 PCTtUS94/04495 , 216112~ 42 -In each case, an aliquot (4 ~1, which i8 approximately equal to 2 ~g) of the chosen partial digest i8 ligated to the appropriate "left" and "right" arms of the cosmid vector DNA using approximately 1 ~g and 2 ~g of each, respectively, in 10 ~1 reactions cont~;n;ng 0.066M Tris-HCl (pH 7.4), O.OlM MgC12, O.OOlM ATP, and 400 units (as defined by the manufacturer (New England Biolabs, Beverly, MA)) of T4 DNA ligase. Incubation is carried out at 11C
overnight. The "left" and "right" arms of the cosmids are DNA fragments which, when ligated to an appropriately sized piece of P. vulqaris DNA, comprise a recombinant molecule of approximately 35-50 kb.
Both arms contain "cos" sites which are recognized by the packaging enzymes in the next step. In addition, these arms carry the origin of replication and ampicillin-resistance functions of pIBI24 (International Biochemical Inc., New Ha~en, CT).
Each of the above ligase reactions is added to one tube of a A packaging extract (Packagene~, a trademark of Promega Corp., Madison, WI) and the reaction is allowed to proceed at room temperature for two hours, at which point 0.5 ml of PDB (O.lM NaCl-O.OlM Tris-HCl (pH 7.9)-O.OlM MgS0~) is added followed by a~,G~imately 0.05 ml of chloroform. Each tube of packaged DNA is, therefore, a gene bank of the P.
vulqaris genome.
Because this method of construction creates a pool of infectious particles (i.e., ~ phage heads filled with the cosmid vector joined to approximately 25 to 35 kb of P. vulqaris DNA), the number of potential clones is guantitated by adsorbing an aliquot of the packaged material to an appropriate, sensitive E. coli host strain, and then after outgrowth, plating the mixture on selective media.

- W094/25567 PCT~$94/04495 2l6ll2~

For example, an overnight culture of the E.
coli strain ER1562 (New England Biolabs, Beverly, MA) grown in 20-10-5 medium is diluted 1:20 into fresh media (20-10-5 supplemented with 1% maltose) and grown for three hours at 37C. The cells (1 ml) are then centrifuged, resuspended with PDB (0.2 ml) and 0.02 ml of the appropriate gene bank ~AeA. After adsorption for twenty minutes at 37C, the samples are diluted to 2 ml with 20-10-5 medium and grown at 37C for 30 minutes. The culture is then spread on 20-10-5 plates cont~in;ng 100 ~g/ml of ampicillin and colonies scored after overnight incubation at 37C. The results indicate that there are approximately 68,000 and 95,000 infectious particles (potential cosmid clones) present in the two samples, designated PVl-GB and PV2-GB, corresrQ~;ng to the "A" and "B" preparation of P.
vulqaris genomic DNA, respectively.
In addition, four other P. vulqaris gene banks are prepared, as above, using two different cosmid vectors. These two cosmids differ from the above-mentioned vectors in that a kanamycin resistance determinant is used in one case rather than the ampicillin resistance, while in the other, the replication functions of pBR322 (New England Biolabs, Beverly, MA) are used instead of those of pIBI24.
These four "libraries, n designated L1974, L1975, L1976, and L1977, contain, respectively, approximately 18,000 (ampr), 34,000 (am~r), 13,000 (kanr) and 15,000 (kanr) members. Aliquots of each of these six gene banks are screened for the presence of the P. vulqaris chQn~roitinase I gene (see below).

W094l25567 PCT~S94/0~95 2l6~12S

ExamPle 2 PCR Experimentation Designed To Yield An Authentic Piece Of The ~ho~roitinase I Gene For Use As A HYbridization Probe The Polymerase Chain Reaction (PCR) (5) allows the geometric amplification of a DNA sequence that lies between oligonucleotide primers that can be extended by a DNA polymerase i vitro. The enzyme used in these experiments is the Taq DNA polymerase (isolated originally from Thermus a~uaticus), which is preferred because of its thermotolerance which allows it to ~urvive the repeated DNA denaturation steps that are carried out at 94C.
In order for this method to be employed successfully, the oligonucleotides used must have sequences that are as close as possible to those of the target sequence -- the P. vulqaris c~nn~roitinase I gene. An ~ o~imation of that sequence can be derived from the limited a~ailable amino acid sequence data. To minimize uncertainty in the sequence presented by the degeneracy of the genetic code (a given amino acid can be encoded by up to six co~on~), the first approximation involves choosing an amino acid sequence that has the least degeneracy. For example, in the amino-terminal sequence of the P.
vul~aris chn~roitinase I gene, there are the following consecutive amino acids: His-Phe-Ala-Gln-Asn-Asn-Pro (SEQ ID N0:2, amino acids 43-49).
This amino acid seguence could be encoded by any one of 512 different nucleotide sequences, repre-sented as 5'-CAY-TTY-GCN-CAR-AAY-AAY-CCN-3' (SEQ ID
N0:6), where R stands for purine (A or G), Y for pyrimidine (C or T), and N indicates that any one of the four nucleotides (A T, G, or C) at this position W094/25567 216 1 12 ~ PCT~S94/04495 will constitute a nucleotide sequence that could encode the indicated amino acid sequence. One possible approach would be to synthesize an oligonucleotide mixture contA;n;ng a total of 512 different olignucleotide~, represented as:

5'-CA(TC)-TT(TC)-GC(GATC)-CA(GA)-AA(TC)-AA(TC)-CC-(GATC)-3' (SEQ ID NO:6).

Although use of such mixtures in PCR has been successful, another approach i8 to u~e a number of oligonucleotide mixtures, each of which is made up of a relatively smaller set of nucleotide sequences.
In order to simplify this further, advantage is taken of the observation (7) that mismatched nucleotides in PCR primers are of less consequence at the 5'-end of the primer than they are at the 3'-end. Using these criteria, a set of eight oligonucleotides (each made up of four unique sequences) is designed, where the individual sets of oligonucleotides have the following sequences:

1. 5'-CAC-TTC-GC(GATC)-CAA-AAT-AAT-CC-3' (SEQ ID NO:7) 2. 5'-CAC- TTC -GC(GATC)-CAA-AAC-AAC-CC-3' (SEQ ID NO:8) 3. 5'-CAC-TTC-GC(GATC)-CAA-AAC-AAT-CC-3' (SEQ ID NO:9) 4. 5'-CAC-TTC-GC(GATC)-CAA-AAT-AAC-CC-3' (SEQ ID NO:10) 5. 5'-CAC-TTC-GC(GATC)-CAG-AAT-AAT-CC-3' (SEQ ID NO:ll) 6. 5'-CAC-TTC-GC(GATC)-CAG-AAC-AAC-CC-3' (SEQ ID NO:12) 7. 5'-CAC-TTC-GC(GATC)-CAG-AAC-AAT-CC-3' (SEQ ID NO:13)

8. 5'-CAC-TTC-GC(GATC)-CAG-AAT-AAC-CC-3' (SEQ ID NO:14) One of these pools i8 perfectly matched for the fir6t eleven nucleotides (counting from the 3-end), and, furthermore, within this pool of four oligonucleotides, one is a perfect match for the first W094l25567 PCT~S94/04495 21611~5 - 46 -fourteen nucleotides. This is important because it permits stringent annealing conditions to be used that discriminate against imperfect matches that give rise to PCR products that are unrelated to the ~ho~roitinase I gene.
A further aid in the design of oligonucleo-tides to be used in these PCR experiments is derived from the observation that the P. vulqaris 110 kD
chodroitinase enzyme appears to have a structure that leaves one particular region hypersensitive to proteolytic cleavage. The result of this hydrolysis is that the normally a~.G~imately 110 kD protein is split into two predominant ~pecies of 18 kD and approximately 90 kD. The amino-terminal sequences of the "110 kD" protein and the "18 kD" fragment are the same, while that for the "90 kD" has been found to be different.
The "18 kD" peptide is further fragmented by treatment with cyanogen bromide and trypsin and the resulting oligopeptides sequenced, affording still more information with which to design oligonucleotides for PCR. This information from the "18 kD" and "90 kD" regions is also valuable because the locations of these amino acid sequences relative to each other and the N-terminal sequences of the intact protein are well defined. In fact, the nucleotide distance between the regions encoding the N-termini of the n 110 kD" and ~90 kD" entities can be predicted to be approximately 400-500 bp.
Two further sets of oligonucleotide pools are then designed with one further consideration: The first eight oligonucleotides hybridize to one strand of the DNA and, during the i vitro DNA syntheRis, they are extended toward the "90 kD" N-terminal coding sequences. Consequently, the oligonucleotides WOg4/25567 216112 ~ PCT~S94/0~95 corresp~n~;ng to amino acid se~uence~ from within the "18 kD" peptide and at the N-terminu~ of the "90 kD"
peptide must be designed 80 that they Anne~1 to the complementary DNA strand of the P. vulqaris genome, 80 that they extend, ln vitro, toward the region encoding the N-terminus of the intact protein.
In this way, the oligonucleotides effectively "bracket" the region of the P. w lqaris chromosome that encodes the N-termi n~ 1 region of the chon~roitinase I gene. It is worth noting that the PCR methodology offers an extremely large potential amplification of this bracketed region. Thirty PCR
cycles, in theory, increase the number of copies of this DNA segment by a factor of one billion. This allows the use of very small quantities of P. vulaaris genomic DNA as a template which will yield, potentially, miclG~I~m amounts of synthesized product which can be readily visualized, isolated and cloned.
Using the above logic, oligonucleotide mixtures are designed based on the following amino acid sequence that is found within the "18 kD"
peptide: Glu-Ala-Gln-Ala-Gly-Phe-Lys (SEQ ID N0:2, amino acids 138-144). This heptapeptide is encoded by the following nucleotide sequences:
5'-GAR-GCN-CAR-GCN-GGN-TTY-AAR-3' (SEQ ID N0:15).

The complementary strand, therefore, has the following sequences:
5'-YTT-RAA-NCC-NGC-YTG-NGC-YTC-3' which is the same as 5'-(CT)TT-(AG)AA-(GATC)CC-(GATC)GC-(CT)TG-(GATC)GC-- (CT)TC-3' (SEQ ID N0:16).

Using the same criteria as described above W094l25567 PCT~S94/~95 216~ 48 -for the first set of eight oligonucleotides, a further set of eight oligonucleotides (each made up of 16 unique sequences) is designed, where the individual sets of oligonucleotides have the following sequences: ~

9. 5'-TT-GAA-(AG)CC-(GATC)GC-(CT)TG-GGC-TTC-3' (SEQ ID NO:17)

10. 5'-TT-GAA-(AG)CC-(GATC)GC-(CT)TG-AGC-TTC-3' (SEQ ID NO:18)

11. 5'-TT-GAA-(AG)CC-(GATC)GC-(CT)TG-TGC-TTC-3' (SEQ ID NO:19)

12. 5'-TT-GAA-(AG)CC-(GATC)GC-(CT)TG-CGC-TTC-3' (SEQ ID NO:20)

13. 5'-TT-GAA-(AG)CC-(GATC)GC-(CT)TG-GGC-CTC-3' (SEQ ID NO:21)

14. 5'-TT-GAA-(AG)CC-(GATC)GC-(CT)TG-AGC-CTC-3' (SEQ ID NO:22)

15. 5'-TT-GAA-(AG)CC-(GATC)GC-(CT)TG-TGC-CTC-3' (SEQ ID NO:23)

16. 5'-TT-GAA-(AG)CC-(GATC)GC-(CT)TG-CGC-CTC-3' (SEQ ID NO:24) Unlike oligonucleotides 1-8 abo~e, one base is deleted from the 5' end of oligonucleotides 9-16 in order to reduce the number of sequence permutations.
In this case, one pool has a perfect match for the first eight nucleotides at the 3'-end, while 50% of this same pool has an eleven-nucleotide perfect match with the genomic DNA of P. vulqaris encoding c~nn~oitinase I.
For a third set of oligonucleotide mixtures, the following amino acid sequence, obt~;ne~ as part of the N-terminal amino acid sequence of the "90 kD"
peptide, is used: Gly-Ala-~ys-Val-Asp-Ser (SEQ ID
NO:2, amino acids 189-194). This hexapeptide can be _ W094/25567 PCT~S94/04495 ~161125 encoded by the following nucleotide sequences:

5'-GGN-GCN-AAR-GTN-GAY-TCN-3' (SEQ ID NO:25) or 5'-GGN-GCN-AAR-GTN-GAY-AGY-3' (SEQ ID NO:26) The complement of this sequence is:

5'-NGA-RTC-NAC-YTT-NGC-NCC-3' (SEQ ID NO:27) or 5'-RCT-RTC-NAC-YTT-NGC-NCC-3' (SEQ ID NO:28) These possible sequences are represented using the following oligonucleotide mixtures:

17. 5'-GA-GTC-(GATC)AC-(TC)TT-(AG)GC-GCC-3' (SEQ
ID NO:29)

18. 5'-GA-GTC-(GATC)AC-(TC)TT-(AG)GC-ACC-3' (SEQ
ID NO:30)

19. 5'-GA-GTC-(GATC)AC-(TC)TT-(AG)GC-TCC-3' (SEQ
ID NO:31)

20. 5'-GA-GTC-(GATC)AC-(TC)TT-(AG)GC-CCC-3' (SEQ
ID NO:32)

21. 5'-GA-GTC-(GATC)AC-(TC)TT-(TC)GC-GCC-3' (SEQ
ID NO:33)

22. 5'-GA-GTC-(GATC)AC-(TC)TT-(TC)GC-ACC-3' (SEQ
ID NO:34)

23. 5'-GA-GTC-(GATC)AC-(TC)TT-(TC)GC-TCC-3' (SEQ
ID NO:35)

24. 5'-GA-GTC-(GATC)AC-(TC)TT-(TC)GC-CCC-3' (SEQ
ID NO:36) Unlike oligonucleotides 1-8 above, one base is deleted from the 5' end of oligonucleotides 17-24 in order to reduce the number of sequence W094/25567 PCT~S94/0~95 2l6ll2s permutations.
In this case, one oligonucleotide mixture has half of its m~hers perfectly matched for the first eight nucleotides at the 3~-end, and one quarter of the oligonucleotides in the pool are perfectly matched for eleven nucleotides at the 3'-end.
The~e twenty-four oligonucleotide mixtures are purchased from Biosynthesis, Inc. (Denton, TX), and are provided as fully deprotected, purified and lyophilized samples. In each case (except oligonucleotide #20), 5 O.D. units of synthetic DNA
are obtA; ne~. This is resuspended in 0.5 ml of water to yield a ~olution that contains approximately 50-60 pmoles of oligonucleotide per microliter. The remaining sample (oligonucleotide #20) contains 15 O.D. and is resuspended with one ml of water to give a solution with approximately 90 pmole/~l.
A typical 50 ~1 PCR reaction contains approximately 20 ng of P. vulqaris genomic DNA as template; 200 ~M each of dATP, dGTP, dCTP, dTTP; 50mN
RCl; lOmM Tris-HCl (pH 8.4); 1.5 mM MgCl2; 0.01%
gelatin; 2.5 units of Ampli-TaqTM DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT); and 50 pmoles of each oligonucleotide pool to be tested. The reactions are overlaid with mineral oil (Plough) and incubated in a Perkin-Elmer/Cetus ThermalcyclerT~.
For each cycle, the instrument is programmed to denature the template DNA at 94C for 1.25 minutes, AnneAl the oligonucleotide primers to the denatured template at 60C or 62C for one minute, and to extend these primers via DNA synthesis at 72C for 2.25 minutes. Thirty such cycles are carried out in an experimental amplification. The products are analyzed by rl~nning an aliquot on a 4% NuSieveTM (FMC
Biochemicals, Rockland, ME) GTG gel contA;n~ng W094l25567 ~16 11~ PCT~S94/0~95 approximately 0.5 ~g/ml ethidium bromide u~ing either Tris-borate or Tris-acetate buffers at either full or half strength. These gels are usually run overnight at approximately lV/cm and photographed on a long wavelength W transilluminator using a red filter and Polaroid Type 57 film.
PCR experiments are run testing the pairwise combinations between oligonucleotide pools #1-8 (derived from the "110 kD" amino-terminal sequence of chQn~oitinase I), pools #9-16 (derived from a peptide sequence containeA within the "18 kD" fragment), and pools #17-24 (derived from the amino-terminal sequence of the "90 kD" fragment). The most effective amplifications observed (based on the vi~ual yield of a discrete DNA band detected on gel electrophoretic analysis of the reaction products) are between oligonucleotide pools #4 and #18, and pools, #4 and #9,10,11, or 12. In general, the other pools, which differ by one nucleotide from these pools, also yield some amplification. A difference of two nucleotides result~, essentially, in no observed product. It is important to note, however, that the Anne~ling temperatures are deliberately kept at 60-62C to ~nh~nce such discrimination.
PCR amplifications using oligonucletide pools #4 and #18 yield a product of approximately 500 bp as estimated relative to size stAn~ds (pBR322 digested with MSP-1 (New England Biolabs, Beverly, MA) ranging from 30 to 700 bp on NuSieve~ agarose gels.
The product from the use of oligonucleotide pool #4 combined with pools #9, 10, 11, or 12 is approximately 350 bp in length. Furthermore, the larger product could be isolated from an agarose gel, diluted a thousand-fold, and then used as the template in a ~econd PCR reaction employing oligonucleotide pools #4 W094/25567 - ~CT~S94/0~95 2~L~ll lZ~

and #9 as primers, which yield a product of approximately 350 bp. That iB, the smaller PCR
product is synthesized from the larger one in agreement with what would be expected if these sequences were all derived from the P. vulqaris chon~noitinase I gene. This indicates that the desired region of the genome is amplified.
The larger PCR product is isolated from an agarose gel using a QiaexTM extraction procedure according to the manufacturer's instructions (Qiagen, Chatsworth, CA). The isolated DNA is then subjected to a "fill-in" reaction (11) to remove the extra, protruding A~n;ne residue that Taq DNA polymerase tends to add to the 3'-end of DNA in a template-independent reaction (12). The isolated DNA is then treated with T4 polynucleotide kinase to add a phosphate moiety to the 5'-ends of the PCR products to allow them to be joined to the ~ector DNA. After these treatments, the PCR product is ligated to pIBI24, a high copy ~ector cont~in;n~ a polylinker (IBI, New Haven, CT), that is first sequentially digested with PstI, n filled-in" and then treated with calf intestinal alkaline phosphatase (Boehringer-~nnheim) .
Once the PCR product is cloned into pIBI24, it is remo~ed as an EcoRI -HindIII fragment by virtue of the restriction sites within the polylinker carried by the plasmid. This fragment is then cloned into both M13mpl8 and M13mpl9 (13; New England Biolabs, Beverly, MA) after cleavage with both EcoRI and HindIII and then phosphatased. Single stranded DNA
correspQn~;ng to these constructions is then isolated and subjected to DNA sequence analysis using an Applied Biosystems (Foster City, CA) instrument and ~ sequencing kit. The results indicate that the W094/25567 PCT~S94/~95 larger PCR product is 455 bp in length. As expected, the ends of the fragment are derived from the oligonucleotide pools used as primers.
The DNA sequence is translated into an uninterrupted amino acid sequence that is in agreement (with one exception described below) with the available data obtained by amino acid sequence analysis on the native chn~oitinase I protein itself, including, for example, a twelve residue oligopeptide (SEQ ID N0:2, amino acids 133-144). An eight residue oligopeptide derived from the DNA
sequence (SEQ ID N0:2, amino acids 71-78) also matches a previously sequenced oligopeptide derived by a combination of trypsin digestion and cyanogen bromide treatment of the native protein. The only discrepancy between the two sequences is at amino acid residue #162 of the mature protein (SEQ ID N0:2, amino acid 186), where the DNA sequence codes for an arginine, while the native protein sequence indicates a leucine.
Since a single nucleotide alteration would change a leucine codon (CTT) to an arginine codon (CGT), an initial interpretation suggeste that this may be caused by a lack of perfect incorporation fidelity by the ~q DNA polymerase during the in vitro amplification process. However (see below) later results indicate that the DNA sequence is correct, rather than the amino acid sequence obt~; ne~ by analyzing the native enzyme. These results also indicate that the "18 kD" and the "90 kD" fragments are, in fact, contiguous pieces of the chQ~oitinase I protein that has been cleaved (presumably by a contaminating protease), predominately between residues #157 (Gln) and #158 (Asp) of the mature protein (SEQ ID N0:2, between amino acids 181 and 182). All of the above information supports the W094l2~567 PCT~S94/04495 2l6ll2s interpretation that the cloned DNA (at least that portion that is bracketed by the oligonucleotide primers) generated by PCR amplification represents part of the authentic c~Qn~oitinase I gene of P.
wlqaris and, therefore, can be used as a probe to identify cosmid clones that carry the intact gene.
Although it is possible to isolate the entire gene coding for a protein of interest using PCR
amplification (thereby avoiding construction of a gene bank and many of the other steps described below) by employing oligonucleotide primer~ derived from the amino-terminus of the protein coupled with primers derived from the carboxyl-terminal amino acid sequence, there are ~everal potential problems in this approach. In the ca~e of the P. w l~aris rho~oitina~e I, the problems include: (1) the assumption that the protein being sequenced has not been proces~ed at either end (not likely to be true, for example, with a secreted protein), (2) the occasional lack of fidelity exhibited by Taq DNA
polymerase during PCR reactions, and (3) the rather large size of the bracketed region of the DNA that is to be amplified which was expected to be approximately 3000 bp (deduced from the apparent molecular weight of approximately 110 kD). Consequently, the approach of constructing a gene bank is selected.

ExamPle 3 Generation Of A Labeled Probe, Colony Hybridization And Identification Of Positive Cosmid Clones From The P. wl~aris Gene Bank The cloned PCR product correspQn~; ng to the 455 bp near the amino-terminal coding portion of the P. w lqaris chon~oitinase I gene is released from the W094/25S67 - PCT~S94/0~95 ~ 1 6 1 1 2 ~

plasmid DNA into which it had been cloned by dige~tion with the restriction enconuclease SalI. This is a consequence of the presence of one SalI site within the polylinker sequence and a second SalI site within the cloned PCR amplification product (this is fortuitous in that the latter $alI site is derived from the nucleotide sequence of oligonucleotide pool #18 near its 5'-end; in fact, there is no recognition site for SalI within the P. w l~aris c~Qn~roitinase I
gene itself). A total of approximately 260 ~g of plasmid DNA is digested with SalI and the product~
separated by electrophoresis on a NuSieveT~ GTG agarose gel. The desired approximately 450 bp fragment is isolated using a Qiaex~ extraction protocol. The fragment is then denatured by heating at 95-100C for 5-15 minutes, followed by rapid cooling. The denatured fragment is then labelled with digoxigenin-labelled dUTP (Boehringer-Mannheim, Indianapolis, IN) in two 200 ~1 reactions.
Aliguots of the six P. vul~aris cosmid gene banks described in Example 1 above are used to infect the E. coli strain ER1562 described above and a total of a~plo~imately 10,000 colonies are obtaineA on the appropriate selective plates. These colonies (on a total of 50 plates) are replica plated onto two nylon m~hranes on selective agar as well as to a third selective plate. After overnight incubation, the colonies on the filters are lysed by sequentially treating with 10% sodium dodecyl ~ulfate (SDS) and 0.5 M NaOH for 5-30 minutes each. The cells from the lysed colonies are neutralized by being placed on sheets saturated with 1 M Tris-HCl (pH 7.4) (twice) and then on paper saturated with 2X stAn~AFd saline citrate prior to vacuum drying at 80C. The DNA from the lysed colonies is then fixed to the membranes.

W094/25567 PCT~S94/0~9 2~6~12S - 56 -The filters are then washed by incubation of the filters at 42C with agitation for 1-3 hour~, using at least 10 ml/filter of 0.05 M Tris HCl, 0.5-1 N
NaCl/0.001 M EDTA, pH 8, 0.1% SDS and 0.05 mg/ml proteinase K. The filters are then rinsed with 2 X
SSC and pre-hybridized by incubation with a hybridization buffer at 65C for 1-3 hours. The filters are then hybridized overnight at 65-68C using the digoxigenin-labeled probe described above (0.5-50 ng/ml in a hybridization solution). The hybridized filters are washed with SSC and SDS, re-blocked with a blocking reagent (Component #11 of DNA Labelling and Detection Rit, Nonradioactive, Boehringer M~nnheim, Indianapolis, IN) and exposed to polyclonal sheep anti-digoxigenin Fab fragments conjugated to alkaline phosphatase.
The positive clones are visualized by incubation of the antibody-labeled filters in the presence of BCIP (bromo-chloro-indolyl-phosphate) and NBT (nitro-blue tetrazolium). The presence of the desired DNA fragment within a colony will result in a dark brownish-purple spot in the filter after this hybridization procedure. After approximately four hour~, the developed filters are used as templates to guide the selection of a total of 117 clones which are then picked to selective media. A small-volume (10 ml) culture ("Miniprep") of each of these clones is grown in selective media and plasmid DNA is then isolated using materials and protocols supplied by Qiagen.

W094/25567 - 21 G 112 $ PCT~S94104495 ExamPle 4 Restriction Mapping And Southern Hybridization Used To Localize The Position Of The Cho~roitinase I Gene Within Individual Clones A number of approaches are used to guide the selection of particular cosmid clones for further study. One is to carry out Southern hybridization (8) using the same PCR-generated fragment as a probe against P. w lqaris genomic DNA that had been digested by a number of restriction enzymes and then fractionated on an agarose gel prior to transfer to a nylon membrane. The probe is labeled with digoxigenin-dUTP by including this nucleotide analogue in a PCR amplification. In this reaction, the gel-purified product of a previous PCR amplification (that using P. vulqaris genomic DNA as template) is diluted l0,000-fold and serves as the template in a second PCR
amplification.
This latter reaction is made up as a 0.5 ml mixture, which is then divided into ten individual tubes and amplified as described above for 25 cycles using oligonucleotide pools #2 and #l0 (see above) as the primers. The normal complement of deoxyr;ho~-~cleoside triphosphates is replaced with a digoxigenin-d~TP labeling mixture from the manufacturer (Boehringer-M~nnh~;m, Indianapolis, IN), which yields a final concentration of l00 ~M each of dATP, dCTP and dGTP, 65 ~M dTTP and 35 ~l digoxigenin-dUTP. The reactions are pooled and precipitated according to the manufacturer's recommendations. An aliquot of the resuspended product is examined by gel electrophoresis and exhibits a single band between approximately 300 and approximately 400 bp in length as expected for the ~smaller" PCR product described W094l~567 PCT~S94/0~95 above.
To avoid problems encountered with the highly viscous P. vulqaris genomic DNA preparation, the DNA (approximately 5 ~1) is diluted into large (0.35 ml) volumes for digestion with the various restriction enzymes. The DNA is then concentrated by ethanol precipitation prior to fractionation on agarose gels and transfer to nylon m~hranes. The data obtA; n~ in these experiments indicates that the rhon~roitinase I gene (at least that portion that hybridizes to the N-terminal coding region represented by the probe described above) is carried on a BstYI
fragment of a~.Gximately 2800 bp, an EcoRV fragment of 5400 bp, and on large (eaual to or greater than approximately lOkb) DNA fragments generated by NsiI, BalII, HindIII, and StYI.
Large scale cultures (500 ml) of a number of hybridizing cosmid clones are grown and plasmid DNA is isolated from these cultures for use in mapping the location of the rhQn~-oitinase I gene. The DNA of the gene is expected to represent only approximately 10 per cent of the P. vulqaris DNA carried within each cosmid. A number of these clones are digested with BstYI and NsiI and the products are fractionated on an agarose gel. Individual fragments are then isolated, a portion tested for the presence of rh~roitinase I
seauences by Southern hybridization, and then subcloned into appropriate vectors.
Two of these fragments are of special interest. The first, a BstYI fragment of approximately 2800 bp, is observed in a n~mher of cosmid clones, including those designated #2 and #45.
The DNA isolated from these two cosmid clones i~
designated Lp2 751 and Lp2 760. With Lp2 760, the approximately 2800 bp BstYI fragment is well separated W094l25567 PCT~S94/0~95 21~1125 from the other BstYI fragments and is therefore more readily subcloned into another vector designated pT660-3. The plasmid designated pT660-3 is a derivative of pBR322 in which the DNA from a point immediately down~tream of the promoter for tetracycline resistance (approximately bp 80) as far as the PvuII site (approximately bp 2070) is deleted and replaced with a BamHI linker. Similarly, the approximately 10 kb NsiI fragment (which hybridizes with the ~ho~roitinase probe described above) is readily isolated from a digest performed on Lp2 751.
These two fragments are referred to as the "2800 bp BstYIn fragment and the "10 kb NsiI" fragment.
The 2800 bp BstYI fragment is small enough to permit a second restriction enzyme digestion on this piece of DNA in order to obtain a fragment suitable for DNA sequence analysis. This is important because the hybridization experiments serve to identify the N-terminal coding region of the cho~roitinase I gene, due to how the probe is derived. This procedure does not, however, indicate to which side the rest of the gene is located. Given the relative size of the probe (less than 500 bp) compared to the predicted size of the intact gene (greater than 3000 bp), this is not a trivial consideration. The nucleotide sequence, however, clearly indicates in which direction the gene would be "read" and therefore, which restriction fragments should be cloned in order to obtain the entire gene.
The subcloned 2800 bp BstYI fragment contains two internal EcoRV sites, which suggests that the resulting fragments might be small enough for DNA
sequencing. However, the EcoRV sites are symmetrically placed within the 2800 bp BstYI
fragment; each EcoRV site is approximately 1200 bp wo 94/25567 ~6~ PCT~S94/0~95 from one end, with the space between them equal to approximately 400 bp. The subcloned fragment i8 digested asymmetrically by t~k; ng advantage of unique restriction sites present within the vector. In this manner, the "halves" of the 2800 bp BstYI fragment are distinguished physically and, by Southern hybridi-zation, the "end~ that contains the ch~n~roitinase I
N-terminal coding region is ascertained. Once this is done, the appropriate piece, which is a HindIII-EcoRV
fragment of approximately 1200 bp, is subcloned into both M13mpl8 and M13mpl9 vectors which are first digested with both HindIII and SmaI and subsequently treated with calf intestinal alkaline phosphatase.
The DNA sequence derived from these ~ubclones reveals a number of features that clearly establish the location of the rhQnAroitinase I gene, as well as the direction in which it is read.
Starting with nucleotide #183 in this sequence (SEQ ID NO:l, nucleotide 191), a coding region is observed which matches the first thirty previously-identified amino acids of the P. vulqaris c~n~roitinase I enzyme. Preceding this sequence, it is possible to discern a number of other features by their analogy to correspsn~;ng sequence motifs from previously analyzed E. coli genes. These features include: (1) nucleotides 32-37 (SEQ ID NO:l, nucleotides 40-45) which match in three of six positions with the consensus n_35" region of a promoter and, after a 17 nucleotide space, a n-10"
region of a promoter (matching in six of seven positions with the consensus n-10" region); (2) a putative "Shine-Dalgarno" sequence can be noted between nucleotides 98-103 (SEQ ID NO:l, nucleotides 106-111): and (3) there is an in-frame ATG initiation codon at nucleotides 111-113 (SEQ ID NO:l, nucleotides W094/25567 PCT~S94/~495 216112~ -119-121), which indicates that the P. vulqaris cho~roitinase I enzyme is synthesized with a 24 amino acid signal sequence which is, presumably, removed as .the protein is transported across the inner membrane.
The second fragment that is subcloned (into a pIBI24 derivative that is first modified to include an NsiI restriction site in place of the PstI site normally present in the polylinker of this vector) is the approximately 10 kb NsiI fragment. Digestion of this a~ o~imately 14 kb recombinant molecule (the approximately 10 kb NsiI fragment in pIBI24) with EcoRV yields four fragments of approximately 9 kb, 2.3 kb, 2.1 kb, and 0.4 kb. Southern hybridization analysis using the probe derived from the N-terminal amino acid sequence indicates that the related cho~roitinase gene sequences are contA;ne~ within the largest fragment (the a~ ~imately 9 kb EcoRV
fragment).
Since there is no other fragment larger than 2.9 kb (the size of pIBI24 which has no internal EcoRV
recognition sites), this approximately 9 kb EcoRV
fragment must contain the ~ector as well as P.
vulaaris DNA. A double digestion of this recombinant molecule with NsiI and EcoRV releases the pIBI24 vector as a 2.9 kb fragment; it also yields fragments of approximately 4.5 kb, 2.3 kb, 2.1 kb, 1.0 kb and 0.4 kb. Taken together (along with the information presented above on the 2.8 kb BstYI fragment which has two internal EcoRV sites separated by approximately 0.4 kb), an initial restriction map is constructed.
A double digestion with EcoRV and HindIII
releases fragments of approximately 4.1 kb, 2.3 kb, 2.1 kb, 2.0 kb, 1.3 kb, 1.1 kb and 0.4 kb. Three of these fragments (2.3 kb, 2.1 kb, and 0.4 kb) are apparently EcoRV fragments that have not been cut by W094/25567 - PCT~S94/0~95 X~ 62 -HindIII. Again, the only fragment larger than the vector (4.1 kb) indicates that this fragment includes pIBI24 (2.9 kb). The approximately 2.0 kb fragment hybridizes with the chQ~roitinase probe, thereby serving to place one of the HindIII sites. Since there is a HindIII site in the polylinker, it too can be placed, leaving the last HindIII site to be placed by deduction.
Double digestion of the cloned approximately 10 kb NsiI fragment with EcoRV and EcoRI yields six fragments (of approximately 4.2 kb, 3.5 kb, 2.3 kb, 2.1 kb, 1 kb, and 0.4 kb), indicating the presence of two EcoRI sitos -- one in the polylinkor and one in the cloned P. vulgaris DNA. Southorn hybridization roveals that the approximately 4.2 kb band in this double digest contains the ch~Aroitina~e I N-terminal coding sequence. ~; ng this information to the above data yields a preliminary restriction map for the subcloned a~lo~imately 10 kb NsiI fragment in pIBI24 (Figure 1).
It should be noted that, in further support of the placement and orientation of the chQ~roitinase I gene, in vitro chon~roitinase I assays in which the activity of the enzyme based on measuring the release of unsaturated disaccharide from ch~roitin sulfate C
at 232 nm are carried out on a small number of samples. In one case, an aliquot of an overnight culture used to prepare LP~ 751 (ER1562 carrying cosmid DNA selected from the colony hybridizations) is found to exprss 0.12 units/ml of chn~roitinase. In addition, one of the EcoRV-deletion constructions (to be described below) is grown overnight in the presence of ampicillin. This culture is then inoculated into fresh selective media either with or without isopropyl-beta-D-thiogalactopyranoside (IPTG) which i~

WOg4/25567 216112 ~ PCT~S94/04495 .

expected to increase the level of transcription from the lac promoter present in pIBI24. The assay results of 0.29 units/ml of chon~oitinase without and 0.36 units/ml with IPTG induction indicate that, even after the EcoRV deletion, the gene is still intact and possibly oriented in the same direction as that of the lac promoter.
Although the sizes of the fragments in the above discussion are approximate (especially the approximately 1 kb region between the EcoRI/NsiI in the polylinker and the nearest EcoRV site; in addition, there also might be another small EcoRV
fragment that is still unmapped), overall they suggest that the approximately 4.2 kb EcoRV-EcoRI fragment contains the entire cho~A~oitinase I gene. In order to facilitate the restriction mapping, an EcoRV
deletion is constructed using the approximately 10 kb NsiI fragment cloned into pIBI24 (LP2776). This DNA is digested with EcoRV, treated with calf intestinal alkaline phosphatase, and fractionated on an agarose gel. The largest (a~o~imately lOkb) fragment is extracted from the gel and ligated together in the presence of a phosphorylated EcoRI linker. The resulting construction (Lp2 786) is next digested with EcoRI to yield three fragments. Although it is not completely separated from the pIBI24-contA; ni ng, somewhat smaller fragment, an approximately 95%
homogenous, approximately 4.2 kb EcoRI fragment is obt~;ne~ after extraction from the gel. This EcoRI
fragment is then used for DNA sequence analysis.

W094/25567 PCT~S94/04495 216~

Exam~le 5 DNA Sequence Analysis Of The Approximately 4.2 kb EcoRI Fraament The approximately 10 kb NsiI fragment, cloned into pIBI24, is digested with EcoRV (as described above) and ligated together in the presence of EcoRI linkers. The net result of this construction is the deletion of approximately 5 kb of P. vulaaris DNA from this subcloned piece of DNA and the simultaneous introduction of another EcoRI site into the molecule. One hundred micrograms of this "EcoRV
deletion" construction (LP~ 786) is digested with EcoRI
and fractionated on an agarose gel. The desired approximately 4.2 kb fragment is eluted from the gel, precipitated and resuspended in 150 ~1 TE described above. One-third of this material is then ligated to itself (polymerized) and, after destruction of the DNA
ligase by heating, the DNA is sonicated to generate random, small pieces suited to DNA sequence analysis.
The ends are rendered flush in a "fill-in"
reaction mediated by the "Rlenow fragment" (10; New England Biolabs, Beverly, MA) of the DNA polymerase I
of E. coli, and then ligated into SmaI-cut and phosphatased M13mpl9. This recombinant DNA is used to transform the male E. coli strain MV1190 and 500 of the phage plaques obtA;neA are picked into SM buffer (NaCl, 100 mM, MgSO~, 8 mM, Tris-HCl, pH 7.4, 50 mM and 0.01~ gelatin) to serve as stocks for the infection of small (less than or equal to 10 ml) cultures that are then used for the isolation of single stranded template DNA.
DNA sequencing is carried out at elevated temperatures using Taa DNA polymerase and fluorescently-labeled oligonucleotide primer~. The W094/25567 - PCT~S94/0~95 ~161 l 25 data are collected using a Model 370A DNA sequencing system ~Applied Biosystem~, Foster City, CA).
Sequence editing, overlap determinations and derivation of a consensus sequence are performed using a collection of computer programs obta;ne~ from the Genetics Computer Group at the University of Wisconsin (14). The resulting DNA sequence of this EcoRI
fragment is 3980 nucleotides in length (SEQ ID NO:l).
It is to be noted that the EcoRI site near the N-terminal coding sequence is derived from the linker ligated into this site; it i8 not present in the P.
vul~aris chromosome. This position actually is an EcoRV site in the cloned cosmid DNA.
Translation of the DNA sequence into the putative amino acid sequence reveals a continuous open rea~; ng frame encoding of 1021 amino acids (SEQ ID
NO:2), with a 24 residue signal sequence (SEQ ID NO:2, amino acids 1-24), followed by a 997 residue coding sequence for the mature (processed) chon~roitinase I
protein (SEQ ID NO:2, amino acids 25-1021). Computer analysis using the programs described above of this sequence predicts a molecular weight of 115,090.94 for the U~lO essed protein, a molecular weight of 112,507.82 for the mature "110 kD" (transported) protein, 17,503.43 for the first 157 amino acids (the nl8 kD~ fragment) (SEQ ID NO:2, amino acids 25-181) and 95,022.40 for the remaining 840 amino acids (the "90 kD" fragment) (SEQ ID NO:2, amino acids 182-1021) and a molecular weight of 2601.14 for the 24-residue signal sequence. One notable feature of the amino acid composition is the absence of cysteine which could be important if the protein has to be re-folded at any point.
In the nucleotide sequence, it was noted above that there is a unique SPhI restriction site W094/25567 - ; PCT~S94/~95 located approximately 230 bp beyond the end of the gene (SEQ ID NO:1, nucleotides 3414-3419), which pre~ents a unique target site that can be manipulated to allow the facile movement of the gene to achieve the overall goal of expressing chon~roitinase at high levels in E. coli. Although there are two recognition sites for ClaI (ATCGAT), one of them (SEQ ID NO:1, nucleotides 2702-2707) iB embedded within the E. coli dam recognition sequence (GATC) (SEQ ID NO:1, nucleotides 2701-2704). The resulting ~en;ne methylation by the dam-encoded enzyme blocks cleavage of this site by ClaI; therefore, there is, in effect, a "unique" ClaI site (SEQ ID NO:l, nucleotides 497-502) which is used, a~ described below, to reconstruct the chQ~roitinase I gene after the ~lv~riate site-specific mutageneses are carried out.

Exam~le 6 Site-specific Mutagenesis Of The Cloned P. vulqaris Cho~roitinase I Gene The site-specific mutagenesis method employed is based on that of Runkel (15), using materials purchaæed from Bio-Rad, Melville, N.Y.
(Muta-Gene7~ In Vitro Mutagenesis Rit). In this procedure, the target DNA to be mutagenized is first cloned into an appropriate M13-derived vector. In this case, the recombinant molecule used (M13mpl9 into which is cloned the ~lG~imately 1200 bp EcoRV-HindIII fragment as described above) encompasses the N-terminal coding region of the chQn~roitinase I gene.
This recombinant phage is replicated in the E. coli host ætrain CJ236 (Bio-Rad), a male strain that carries the dut and unq alleles. The combination of these two mutationæ, dut (dUTPase) and unq (uracil-N-W094/25567 - ~16112 ~ PCT~S94/0~95 glycosylase), results in the incorporation of some uracil, rather than thymine, residues into the DNA
synthesized in this organism. Single stranded template is then isolated after propagation on CJ236 and an appropriate, mutagenic, synthetic oligonucleotide is ~nn~l ed to this DNA.
This oligonucleotide serves as a primer for T7 DNA polymerase which copies the entire recombinant molecule. T4 DNA ligase i8 then used to seal the nick between the first residue of the mutagenic oligonucleotide a~d the last residue added i vitro.
The newly synthesized DNA (cont~ning the desired ba~e changes) therefore does not contain uracil, while the template DNA does. Transformation of a non-mutant (with respect to the dut and una alleles) male E. coli strain yields phage p Oye~y that are pr;marily derived from the mutagenized strand synthesized i vitro as a result of the inacti~ation of the uracil-cont~;n;ng template strand.
In this specific case, four resuspended plaques (aliquots of which had been used for DNA
sequencing which established the N-terminal co~;ng region of the c~Qn~oitinase I gene and included another 110 bp "upstream" of the presumed translation initiation site (see above)) are used to infect the male host strain CJ236 (dut una). Individual plagues are picked to 0.5 ml of phage dilution buffer (PDB).
One picked plague from each transformation is adsorbed to log phase CJ236 and the infected culture grown for 6.5 hours. The cells are pelleted by centrifugation, and the supernatant heated to 55C for 30 minutes and then stored at 4C. Single str~n~ DNA is isolated from 100 ml of each supernatant and resuspended in a total volume of 0.1 ml of TE.
The goal of the site-specific mutagenesis is wo94n5s67 - PCT~S94/0~95 ~16112~ 68 -to modify the "ends~ of this gene to allow it to be moved, precisely, into an appropriate high-level E.
coli expression system. The target vector chosen (pET9-A; ~ee above) is one derived from genetic regulatory elements present in the bacteriophage T7.
In this sytem, there is a unique NdeI site (CATATG) that includes the translation initiation codon as well as a downstream BamHI site that, together, allow the direct, unidirectional, insertion of a gene encoding the protein that is to be expressed. These two sites are preceeded by a T7-specifc promoter sequence and trailed by a transcription terminator that functions with the T7 RNA polymera~e. Accordingly, these two restriction sites (NdeI and BamHI) are introduced into the cloned gene for P. vulaaris chQn~roitinase I.
In order to introduce the NdeI ~ite (cont~;nin~ the ATG initiation codon) both before the signal sequence as well as, in a second construction, before the coding sequence for the mature protein (thereby deleting the signal sequence), two synthetic oligonucleotides are designed and synthesized (purchased from BioSynthesis, Inc., Denton, TX). The first, designated oligonucleotide # 25 (SEQ ID NO:37), retains the signal sequence while the second, oligonucleotide #26 (SEQ ID NO:38), deletes the signal sequence and allows the direct expression of the mature chQ~roitina~e I protein (which can have an additional methionine residue at the N-terminus (SEQ
ID NO:5, amino acid number 1)).
The native sequence, including the predicted initiation codon, is presented on line 1 below while the mutagenic oligonucleotide #25 (which differs in the three nucleotides immediately upstream of the initiation codon) is presented on line 2:

W094/25567 - PCT~S94/0~95 216112~

1) 5'-GCCAGC~l--L~ AAGr~ TAATGCCGATATT-TCGTTTTACTGC-3' (SEQ ID NO:1, nucleotides 94-141) 2) 5'-GCCAGC~ ~lAAGG~G~ CATATGCCGATATT-TC~ ~ -1 1-lACTGC-3' ( SEQ ID NO: 37) For the construction in which the signal sequence is deleted, the site-specific mutagenesis is carried out at the junction of the signal sequence and the start of the mature protein (line 3) using the mutagenic oligonucleotide # 26 (line 4) (which differs by six nucleotides, including the location of the initiation codon):
3) 5'-G~-AT~CGCGATGGCAGCCACCAGCAATCCTG-3' (SEQ ID NO:1, nucleotides 170-206) 4) 5'-GCGCCTT~T~vC~CATATGGCCACCAGCAATCCTG-3' (SEQ ID NO: 38) The underlined GCC in line 3 corre8pond8 to the codon for alanine which is the N-terminal amino acid for the mature, processed form of the P. vulqaris c~ oitinase I.
In order for these oligonucleotides to be used, their 5'-ends need to be phosphorylated. There-fore, oligonucleotide # 25 (5 O.D. units) is resuspended with 0. 5 ml of TE, while oligonucleotide #
26 (al80 5 O.D. units) is resuspended in 0. 65 ml TE to yield stocks that are approximately 20 nM, i.e., 20 pmole/~l. Three nanomoles (150 ~1 of stock solution) of each oligonucleotide are kinased in separate (0. 35 - ml) reactions cont~in;ng 35 ~1 10x ligase salts (New England Biolabs, Beverly, MA): 0.5 M Tris-HCl (pH

WOg4/25567 ~ 6 11 Z 5 PCT~S94/0~95 7.8), 0.1 M MgCl2, 0.2 M dithiothreitol, 10 mM ATP, 0.5 mg/ml bovine serum albumin), 35 ~1 0.1 M
dithiothreitol, 10 ~1 (100 units) T4 polynucleotide kinase (New England Biolabs) and made up to volume with 120 ~1 TE. The reactions are incubated at 37C
for 40 minutes and the enzyme inactivated at 70C for 20 minutes.
Template DNA (5 ~1 of the preparation de-scribed above) and phosphorylated mutagenic primer (approximately 2 pmole) are ~nneAled in a 20 ~1 volume contAin;ng 20 mM Tris-HCl (pH 7.4), 2 mM MgCl2, and 50 mM NaCl. The sample is heated at 70C for 45 minutes in a Perkin-Elmer/Cetus Thermalcycler~. The sample is then gradually cooled from 70C to 25C over a 45 minute period. The ~nnealed mixture is placed on ice and the following comro~nts added: 2 ~1 of 10 X
synthesis buffer (Bio-Rad): 5mM each of dATP, dGTP, dCTP, dTTP; 10 mM ATP; lOOmM Tris-HCl (pH 7.4); 50 mM
MgCl2; 20 mM dithiothreitol), 2 ~1 of T4 DNA ligase (6 units) and 1 ~1 of T7 DNA polymerase (1 unit). These reactions are incubated for 5 minutes each at 0C (on ice), 11C, 25C, and finally for 30 minutes at 37C.
The reactions are stopped by the addition of 75 ~1 of 10 mM Tris-HCl-10 mM EDTA (pH 8.0) and placed at -20C.
After the mutagenized DNA is thawed, it is used to transform the male E. coli strain MVll90 (dutt Ya~). Individual plaques obtained are picked and single-stranded DNA is isolated and sequenced. For those cases in which the desired sequence changes are introduced, another aliquot of the resuspended plaque is used to infect strain MV1190, but in this case the intracellular, double-stranded replicative form of the recombinant DNA is isolated from the infected cell pellets using the Mini-Prep procedure referenced above.

WOg4/~567 - 21~ I 12 a PCT~S94/~95 ExamPle 7 Reconstruction Of The Site-Specifically Mutagenized Cho~roitinase I Gene And Its Hiqh-~evel ExPression In E. coli Example 6 described the site-specific mutageneses that created an NdeI site immediately preceeding the signal sequence, as well as a second construction which placed the NdeI site adjacent to the triplet which codes for the N-terminal alanine found on the mature, processed P. vulqaris chQ~roitinase I gene. In each case, the ATG ~equence of the NdeI recognition site (CATATG) can function as the translation initiation codon for the protein (either with or without the signal sequence).
In order to transfer these alterations from the M13 vector in which they were constructed, to the full ch~n~oitinase I gene, the isolated replicative form is digested with KPnI and ClaI. The R~nI site is part of the M13mpl9 polylinker, while the ClaI site is found approximately 490 bp from the end of the cloned fragment of the chQ~roitinase I gene. The restriction digestion products obt~ne~ are fractionated on a 4% NuSieve~ GTG agarose gel run in 1/2 X Tris-Acetate buffer (TAE). The appropriate approximately 500 bp band is extracted from the gel using Qiaex~. Similarly, plasmid DNA (~p2 786) carrying the ch~n~roitinase I gene is also digested with KpnI and ClaI and then fractionated on a 0.8%
agarose gel run in 1/2 X TAE. In this case, the R~nI
site is part of the polylinker of pIBI24, while the ClaI site corresponds to the one described above. (A~
stated above, there is a second ClaI site in the cho~roitinase I gene, but it is not cleaved by ClaI
because this site is apparently blocked by dam W094l25567 PCT~S94/0~95 ~l6ll2~

methylation. The site-specific mutagenesis and recon~truction of the chon~roitinase I gene were carried out before the entire nucleotide sequence was ascertained).
The approximately 7 kb fragment containing the pIBI24 vector and the large fragment of the cho~Aroitinase I gene are isolated from the agarose gel by electroelution ~11), followed by ethanol precipitation. This 7 kb fragment is then treated with calf intestinal alkaline phosphatase, extracted first with phenol-chloroform, then with chloroform, and then precipitated twice with ethanol and finally resuspended with 0.1 ml TE. The two isolated N-terminal ~nroA;ng fragments (the two approximately 500 bp RDnI-ClaI pieces cont~ining the two site-specifically mutagenized sequences, one with and one without the signal sequence) are each ligated to the approximately 7 kb fragment encompassing the remainder of the ~ho~Aroitinase I gene and the pIBI24 vector.
The ligase reaction i8 then used to transform the E.
coli strain 294 and ampicillin resistant derivatives obtained. DNA is isolated from small (10 ml) cultures and digested with NdeI to verify the presence of this restriction site within the reconstructed DNA.
In order to remove the (apparent) P.
vulgaris promoter and ribosome b;nA;ng site, the modified rh~nA~oitinase I genes are isolated as a~.G~imately 4.5 kb NdeI-NsiI fragments and subcloned into a pBR322 variant in which the EcoRI Bite i8 first filled-in, then dephosphorylated, and finally a phosphorylated NsiI linker (New England Biolabs) inserted. The sequence of the linker used (TGCATGCATGCA) to place the NsiI site (ATGCAT) into pBR322 also includes an SphI site (GCATGC). In order to trim extra, non-coding DNA from the subcloned NdeI-W094/25567 PCT~S94/~95 216112~

NsiI fragments, as well as to introduce a unique restriction site to be used later, plasmid~
(representing two clones each with the signal sequenc retained [Lp2 861 and Lp2 863] and two with the signal sequence deleted [Lp2 865 and Lp2 867]) contA;nin~ the approximately 4500 bp NdeI-NsiI segments including the cho~roitinase I gene are first digested with SPhI, the ends "filled-in" with the "Klenow" fragment (11) of the E. coli DNA polymerase I and the resulting DNA
fragments fractionated on an agarose gel (0.8% in 1/2 X TAE). The appropriate bands (approximately 5200 bp) are eluted from the gel using Qiaex~ and then treated with calf alkaline phosphatase. After the ~ -v-l of this enzyme by phenol-chloroform and chloroform extractions, the DNA is precipitated twice and finally resuspended with 0.1 ml TE.
This DNA is then ligated in the pre~ence of a phosphorylated BamHI linker and the mixture used to transform the E. coli strain 294. Six representative, ampicillin resistant colonies from each of the four constructions are y~O. n in small (10 ml) cultures and plasmid DNA is isolated. Digestion of the DNA from the 24 clones examined with the enzymes NdeI and BamHI
indicates which contain the BamHI site and, simultaneously, releases the approximately 3400 bp NdeI-BamHI fragment which contains the chnn~oitinase I gene. Seventeen clones (eight with and nine without the signal sequence) yield the desired fragment which i8 extracted from the agarose gel with Qiaex~.
These approximately 3.4 kb NdeI-Bam-HI
rho~oitinase I gene-cont~;n;ng fragments (both with and without the signal sequence) are then used to construct a high-level expression system. The expression vector used, pET-9A (9; Novagen), is derived from elements of the E. coli bacteriophage T7.

WOg4/25567 - PCT~S94/0~95 21611~S

It contains an origin of replication derived from the Col E1 plasmid, a kanamycin resistance determinant, and the transcription and translation initiation determinants of the T7 gene 10. The naturally-occurring translation initiation codon for this gene is part of an NdeI site. This region is followed by a unique BamHI site and a T7 transcription terminator.
A sample of this expression vector is digested with the restriction enzymes NdeI and BamHI, dephosphorylated with calf intestinal alkaline phosphatase, and purified by agarose gel electrophoresis. Each of the c~on~oitinase I gene fragments (both with and without the signal sequence) is ligated to the expression vector fragment. The resulting recombinant DNA mixture is used to transform the E. coli R-12 host, HMS174 (Novagen). Ranamycin-resistant colonies obta; neA are grown in small scale (10 ml) and plasmid DNA is isolated and examined to confirm the predicted structure.
Samples of these constructions are then used to transform the expression host BL21(DE3)/pLysS (10).
This E. coli B strain carries the T7 RNA polymerase gene under lac control (and is therefore inducible by either lactose or IPTG) on a lambda phage integrated within the E. coli chromosome, as well as the Col E1-compatible plasmid pLysS. This latter replicon specifies resistance to chloramphenicol and contains the T7 lysozyme gene inserted into the tetracycline-re~istance determinant of pACYC184 (ATCC 37033, American Type Culture Collection, Rockville, MD) in the "silentn orientation (read in the opposite direction relative to the tetracycline resistance gene). The T7 lysozyme is expressed at a relatively low level in this construction and serves as an inhibitor of the T7 RNA Polymerase (16), thereby W094/25567 ~16112 ~ PCT~S94/0~95 minimizing the basal-level expression of the gene to be overexpressed.
Derivatives of BL21(DE3)/pLysS carrying the chon~-oitinase I gene (with the signal sequence retained and which have been subjected to the ~ite-directed mutagenesis described in Example 6 (SEQ ID
NO:3)) in pET9-A are designated LL2084, LL2085, LL2086 and LL2087. They are not tested for expression of the cho~noitinase I enzyme. The native chon~oitinase I
gene (with the signal sequence retA;ne~) (SEQ ID
NO:1), which has not been subjected to site-directed mutagenesis, is inserted into a different expression host. Expression of the chQ~oitinase I enzyme is achieved.
One of the derivatives of BL21(DE3)/pLysS
carrying the signal-less chon~oitinase I gene which has been subjected to the site-directed mutagenesis described in Example 6 (SEQ ID NO:4) inserted into pET9-A, is designated LL2088, tested and used to establish a master cell bank. The insertion of the gene into pET9-A yields the plasmid designated pTM49-6. Samples of the E. coli B strain BL21(DE3)/pLysS
carrying the plasmid pTM49-6 constitute the deposited strain ATCC 69234.
An overnight culture of this deposited strain is grown at 30C in the presence of 40 ~g/ml of kanamycin and 25 ~g/ml of chloramphenicol. A 0.5 ml aliquot of this culture is used to inoculate 100 ml of a rich "expression" medium cont~ining M9 salts (17) supplemented with 20 g/l tryptone, 10 g/l yeast extract, and 10 g/l dextrose in addition to the same level of kanamycin and chloramphenicol.
The culture is grown at 30C to an a~ o~riate density (a value of 1 at A600) and then chQ~oitinase I expression is induced by the addition W094/25567 - PCT~S94/04495 21~ Z`~ 76 -of IPTG to a final concentration of 1 mM. After three hours, samples are taken, centrifuged, and the cell pellets frozen on dry ice prior to assay. The frozen pellets are thawed, resuspended in buffer and sonicated. A value of 56 units/ml is obtained (relative to the original culture volume), which indicates that this expression system is functional.
A subsequent 10 liter fermentation under controlled conditions at a higher cell density yields a maximum value of approximately 600 units/ml of chQn~roitinase I. This represents a substantial improvement over fermentation of the original native P. vulqaris, which had not expressed chQn~roitinase I at a level above 2 units/ml.
ExamPle 8 Method For The Isolation And Purification Of The Native rhQn~roitinase I Enzyme As Ada~ted To The Recombinant EnzYme The native enzyme is produced by fermentation of a culture of P. vul~aris. The bacterial cells are first recovered from the medium and resuspended in buffer. The cell suspension is then homogenized to lyse the bacterial cells. Then a charged particulate such as 50 ppm Bioacryl (Toso Haas, Philadelphia, PA), is added to remove DNA, ayy.ey~tes and debris from the homogenization step.
Next, the solution is brought to 40% saturation of ammonium sulfate to precipitate out undesired proteins. The c~ho~roitinase I remains in solution.
The solution is then filtered using a 0.22 micron SP240 filter (Amicon, Beverly, MA), and the retentate is washed using nine volumes of 40% ammonium sulfate solution to recover most of the enzyme. The W094l25567 216112 ~ PCT~S94/0~95 filtrate is concentrated and subjected to diafiltration with a sodium phosphate buffer using a 30 kD filter to remove salts and small molecules.
The filtrate contA;n;ng chn~roitinase I is subjected to cation eYchAnge chromatography using a Cellufine~ cellulose sulfate column (Chisso Corporation, distributed by Amicon). At pH 7.2, 20 mM
sodium phosphate, more than 98% of the rhon~roitinase I binds to the column. The native cho~roitinase I is then eluted from the column using a 0 to 250 mM sodium chloride gradient, in 20 mM sodium phosphate buffer.
The eluted enzyme is then subjected to addittonal chromatography steps, such as anion eYrhAn~e and hydrophobic interaction col-~n chromatography. As a result of all of these procedures, chQ~roitinase I is obtA;neA at a purity of 90-97% as measured by SDS-PAGE scAnn;ng (see above). However, the yield of the native protein is only 25-35%, determined as described above. This method also results in the cleavage of the a~,oAimately 110 kD ~hQn~roitinase I protein into a 90 kD and an 18 kD fragment. Nonetheless, the two fragments remain non-ionically bound and exhibit rho~roitinase I activity.
When this procedure is repeated with lysed host cells carrying a recombinant plasmid encoding rho~roitinase I, significantly poorer results are obtA;ne~. ~ess than 10% of the chon~roitinase I binds to the cation ~YchAn~e column at stAn~Ard stringent conditions of pH 7.2, 20 mM sodium phosphate.
Under less stringent b;n~;ng conditions of pH 6.8 and 5 mM phosphate, an improvement of b;n~;ng with one batch of material to 60-90% is observed.
However, elution of the recombinant protein with the NaC1 gradient gives a broad activity peak, rather than WOg4/25567 216 112 PCT~S94/0~95 a sharp peak (see Figure 2). This indicates the product is heterogeneous. Furthermore, in subsequent fermentation batches, the recombinant enzyme binds poorly (1-40%), even using the less stringent binA;ng conditions. Batches that bind poorly are not completely processed, 80 their overall recovery is not quantified.

ExamPle 9 First Method For The Isolation And Purification Of Recombinant ~h~nAroitinase I
Accordinq To This Invention As a first step, the host cells which express the recombinant rhonA~oitinase I enzyme are homogenized to lyse the cells. This releases the enzyme into the supernatant.
In one embodiment of this invention, the supernatant is first subjected to diafiltration to remove salts and other small molecules. An example of a suitable filter is a spiral wound 30 kD filter made by Amicon (Beverly, M~). However, this step only removes the free, but not the bound form of the negatively charged molecules. The bound form of these charged species is removed by passing the supernatant through a strong, high capacity anion exchange resin-contA;n~ng col~n. An example of such a resin is the Macro-Prep~ High Q resin (Bio-Rad, Melville, N.Y.).
Other Qtrong, high capacity anion ~YchAn~e columns are also suitable. The negatively charged molecules bind to the column, while the enzyme passes through the column. It is also found that some unrelated, undesirable proteins also bind to the column.
Next, the eluate from the anion eYrhAnge column is directly loaded to a cation exchange resin-W094/25567 216112 ~ PCT~S94/0~95 containing coll~n. Examples of such resin~ are the S-Sepharose~M (Pharmacia, Piscataway, N.J.) and the Macro-PrepTM High S (Bio-Rad). Each of these two resin-cont~;n;ng columns has S03- ligands bound thereto in order to facilitate the eYchAnge of cations. Other cation eYch~nge col~ns are also suitable. The enzyme binds to the column and is then eluted with a solvent capable of releasing the enzyme from the colll~.
Any salt which increases the conductivity of the solution is suitable for elution. Examples of such salts include sodium salts, a~ well as potassium salts and ammonium salts. An aqueous sodium chloride solution of appropriate concentration i8 suitable. A
gradient, such as 0 to 250 mM sodium chloride is acceptable, as is a step elution using 200 mM sodium chloride.
A sharp peak is seen in the sodium chloride gradient elution (Figure 3). The improvement in enzyme yield over the prior method is striking. The recombinant chQ~oitinase I enzyme is recovered at a purity of 99% at a yield of 80-90%.
The purity of the protein is measured by sc~nn;ng the bands in SDS-PAGE gels. A 4-20%
gradient of acrylamide is used in the development of the gels. The band(s) in each lane of the gel is scanned using the procedure described above.
These improvements are related directly to the increase in b;n~;ng of the enzyme to the cation ~Y~h~nge column which results from first using the - 30 anion exchange column. In comparative experiments, when only the cation exchange column is used, only 1%
of the enzyme binds to the column. However, when the anion ~Ych~nge column is used first, over 95% of the enzyme binds to the column.

W094/25567 PCT~S94/0~95 216~Z5 Example 10 Second Method For The Isolation And Purification Of Recombinant Chon~roitinase I
Accordinq To This Invention In the second embodiment of this aspect of the invention, two additional steps are inserted in the method before the diafiltration step of the first embodiment. The supernatant is treated with an acidic solution, such as 1 M acetic acid, bringing the supernatant to a final pH of 4.S, to precipitate out the desired enzyme. The pellet is obtained by centrifugation at 5,000 x g for 20 minutes. The pellet ie then dissolved in an alkali solution, such as 20-30 mM NaOH, bringing it to a final pH of 9.8.
The solution is then subjected to the diafiltration and subseguent steps of the first embodiment of this invention.
In comparative experiments with the second embodiment of this invention, when only the cation c~nge column is used, only 5% of the enzyme binds to the col~n. However, when the anion ~Ych~nge column iB used first, essentially 100% of the enzyme binds to the column. The second embodiment provides comparable enzyme purity and yield to the first embodiment of the invention.
Acid precipitation removes proteins that remain soluble; however, these proteins are removed anyway by the cation and anion ~Ych~nge steps that follow (although smaller columns may be used). An advantage of the acid precipitation step i8 that the sample volume is decreased to about 20% of the original volume after dissolution, and hence can be handled more easily on a large scale. However, the additional acid precipitation and alkali dissolution W094/25567 PCT~S94/0~95 ~161125 steps of the second embodiment mean that the second embodiment i8 more time consuming than the first embodiment. On a manufacturing scale, the marginal improvements in purity and yield provided by the second embodiment may be outweighed by the simpler procedure of the first embodiment, which still provides highly pure enzyme at high yields.
The high purity of the recombinant enzyme obtAineA by the two emhoA;ments of this invention is depicted in Figure 4. A single sharp band is seen in the SDS-PAGE gel photograph: Lane 1 is the enzyme using the method of the first ~mhodiment; Lane 2 is the enzyme using the method of the second embodiment;
~ane 3 represents the supernatant from the host cell prior to purification -- many other proteins are present; and ~ane 4 represents molecular weight E~ tAn~A rd8 .

Exa le 11 Site-Specific Mutagenesis Of A Fragment Encoding The N-Terminal Reqion Of rhonAroitinase II

The approach taken in the case of the ~honAroitinase II gene is to modify the naturally-occurring ATG initiation codon to embed it within an NdeI site. This results in a construction in which the signal peptide is retA; neA, such that the expressed gene is processed and secreted to yield the mature native enzyme structure that has a leucine residue at the N-terminus. The mutagenized ba~es are upstream of the coding region.
The method used for this site-specific alteration is that described above for the expression of the c~nn~oitinase I gene and is based on the work of Runkel (15) using the Muta-Gene~ In Vitro W094/25567 PCT~S94/0~95 2161~2~

Mutagenesis Kit Version 2 (Bio-Rad, Melville, N.Y.).
In this procedure, the target DNA to be mutagenized is first cloned into a suitable M13-derived vector to generate single-stranded DNA. This recombinant phage is replicated in the E. coli host strain CJ236 ~Bio-Rad), a male strain that carries the dut and una alleles. The combination of these two mutations, dut ~duTPase) and una (uracil-N-glycosylase), results in the incorporation of some uracil, rather than thymine, residues into the DNA synthesized in this organism.
Single-stranded template is then isolated after propagation on CJ236 and the appropriate mutagenic, synthetic oligonucleotide ~SEQ ID NO:41) is ~nne~led to this DNA.
This oligonucleotide serves a~ a primer for T7 DNA polymerase which copies the entire recombinant molecule. T4 DNA ligase is then uset to seal the nick between the first residue of the mutagenic oligonucleotide and the last residue added i vitro.
The newly synthesized DNA (contAin;ng the desired base changes) therefore does not contain uracil while the template DNA (with the native sequences) does.
Transformation of a non-mutant (with respect to the unq and dut alleles) male E. coli strain yields phage p- Gye~ that are primarily deri~ed from the mutagenized strand synthesized i vitro as a result of the inactivation of the uracil-cont~;n;~g template strand.
In this specific case, the fragment to be cloned for the mutagenesis is a MunI-EcoRI fragment that spans the region between nucleotides 2943 to 3980 (SEQ ID NOS:1 and 39). The DNA digested to obtain this fragment is designated LP2783. This plasmid is constructed in the same way as LP2786 (described in 3S Example 4), except that a HindIII linker is inserted W094l25567 ~161~ 2 a PCT~S94tO~95 into the EcoRV deletion of LP2776 rather than the EcoRI
linker. This MunI-EcoRI fragment is ligated into the unique EcoRI site of LP2941, an M13mpl9 derivative in which the normal polylinker is replaced with that found in the pla~mid vector pNEB193 (New England Biolabs, Beverly MA). The four base overhang produced by MunI digestion can bè ligated to an EcoRI site, but the resulting recombinant sequence cannot be digested by either enzyme. The EcoRI digested LP2941 is also dephosphorylated with calf intestinal alkaline phosphatase (Boehringer M~nnh~; m, Indianapolis IN) prior to gel purification and use.
The ligated DNA mixture is used to infect the male E. coli strain MVll90 and the plaques obtA;ne~ are picked to 0.5 ml. of SM buffer and the phage allowed to elute by diffusion. These are then used to infect 10 ml. cultures of MV1190 and grown overnight. The cultures are centrifuged and the pellets used for the isolation of the double-stranded replicative forms of the recombinant virus. The supernatant~, which contain the correspQ~; ng phage particles, are stored under refrigeration until needed. The orientation of the cloned fragment is determined by digestion of the replicative form DNA
and HindIII, because there is one site within the polylinker and a second, aymmetrically placed site (SEQ ID NOS:1 and 39, nucleotides 3326-3331) within the above MunI-EcoRI fragment.
Once the desired orientation is identified, - 30 the correspo~;ng phage-cont~;n;ng supernatant is serially diluted, used to infect the E. coli strain CJ236, and then plated to obtain single plaques which are picked and eluted as above. One of these is then used to infect CJ236 and another 10 ml culture grown and the single-~tranded DNA is isolated from the W094l25567 PCT~S94/0~95 2l61125 phage-cont~ining supernatant using QiaexTM columns and materials and methods recommended by the manufacturer (Qiagen, Chatsworth, CA) and finally resuspended in a volume of 0.01 ml. The recombinant phage are grown on CJ236 (dut- un~~) for two rounds in order to maximize the accumulation of uracil residues in the template and strand prior to the actual site-specific mutagenesis.
The mutagenic oligonucleotide used is obtained from Bio-Synthesis (Denton, TX) and has the following sequence:

5'-ATT-TGC-AGG-AAA-TCT-GCA-TAT-GCT-AAT-AAA-AAA-CCC-3' (SEQ ID NO:41) This sequence differs from the correspon~;ng region of SEQ ID NOS:1 and 39 in that an AT sequence (base pairs 3235 and 3236) i8 replaced by a CA
sequence which creates the desired NdeI sequence (CATATG) at the start of the presumed leader sequence for the chQ~roitinase II gene. One optical density unit of this oligonucleotide is dissolved in 0.46 ml.
of TE 7.4 (O.OlM TrieHCl, pH 7.8-O.OOlM EDTA, pH 8.0), yielding an oligonucleotide concentration of ~5 approximately 6 pmol/~l. Three hundred picomoles of this oligonucleotide are phosphorylated in a 0.1 ml reaction cont~;ning 0.05 M TrisHCl, pH 7.8, 0.01 M
MgCl2, 0.02M dithiothreitol, 0.001 M ATP, 25 ~g/ml bovine eerum albumin and 100 units of T4 polynucleotide kinase (New England Biolabs) at 37C
for 30 minutes, followed by incubation at 75 for 20 minutes to inactivate the enzyme. The phosphorylated oligonucleotide is then stored frozen at -20 at a concentration of approximately 3 pmoles/~l.
For the site-specific mutagenesi~ 1 (3 W094/25567 PCT~S94/~95 21611~S

pmole) of the mutagenic oligonucleotide is mixed with 6 ~1 of the single-stranded DNA prepared above in a 10 ~1 volume of 0.02 M TrisHCl, pH 7.4, 0.002 M MgCl2, 0.05 M NaCl. The oligonucleotide is annealed to this template by first incubating the sample at 70C for 5 minutes and then cooling this sample at 25C over a 45 minute period in a DNA Thermal Cycler~M (Perkin-Elmer Cetus/Norwalk, CT). The sample is maintained at 25C
for another 5 minutes before being cooled to 20C and finally transferred to an ice bath.
The annealed primer i8 then extended after the addition of 1 ~1 of 10X synthesis buffer (Bio-Rad:
conta;n;ng 0.005 M of each of the dNTP's, 0.01 M ATP, 0.1 M TrisHCl, pH 7.4, 0.05 M MgCl2, 0.02 M DTT). One ~1 of T4 DNA ligase (3 units/~l Bio-Rad) and 1 ~1 of T7 DNA polymerase (0.5 units/~l Bio-Rad). The in vitro DNA synthesis is carried out on ice for 5 minutes, at 11C for ten minutes, and at 37C for 30 minutes prior to transfer to ice.
This sample is used directly to transform the male E. coli host MV1190 (dut~ unq~) and the resulting plaques, conta;n;ng the site-specifically mutagenized phage, are obta; n~, picked and eluted as described above. Aliquots of these phage stocks are used in infect 10 ml. cultures of MV1190 and allowed to grow overnight. The cultures are centrifuged and the replicative forms of the recombinant phage are isolated using Qiaex~ col~mns and methods recommended by the manufacturer (Qiagen, Chatsworth CA). The DNA
isolated is resuspended in 0.1 ml of TE 7.4. Initial digestions of a portion of each of these DNA samples with _deI reveals that at least four appeared to have acquired a new NdeI site, indicate that the site-specific mutagenesis is successful. Consequently, larger samples of these four clones (0.04 ml each) are W094l25567 - PCT~S94/04495 216112~ `~

digested with NdeI and EcoRI and fractionated on a 1.4% agarose gel run in a Tris-acetate-EDTA buffer system.
The desired approximately 740 base pair fragment is observed in each case and this band is excised from each pattern. The four samples are then combined and the DNA extracted from the gel using a QiaexTM resin and buffers according to the manufacturer's recommendations (Qiagen, Chatsworth CA) and resuspended in 0.05 ml. of TE, pH 7.4. This isolated, site-specifically mutagenized N-terminal coding region of the cloned P. vulqaris gene for the rhQ~roitinase II gene is then subcloned into the plasmid pNEB193 (New England Biolabs, Beverly MA) between the (dephosphorylated) unique NdeI and EcoRI
sites present in this plasmid. After transformation of the E. coli host strain 294, 10 ml cultures derived from the individual transformants are grown and the recombinant plasmid DNA isolated as above. The DNA
sample from one of the positive clones is designated m#l5-5712. This sample represents the modified N-terminal region that is to be joined to the C-terminal coding region for the rhQn~roitinase II gene, which is described in Example 12.
ExamPle 12 Isolation, Characterization And DNA Sequence Analysis Of A Fragment Encoding The C-terminal Region Of ~hon~roitinase II
The DNA sequence contained in SEQ ID N0: 39 indicates that chQn~roitinase II is encoded by a region that is downstream of that for rhn~roitinase I. This information is derived from a portion of a 10 kilobase N~iI fragment of P. vulqaris that is W094/25567 216112 ~ PCT~S94/0~95 subcloned originally from a cosmid clone designated LP2751. The combination of the DNA sequencing and the restriction map in Figure 1 reveals that the chon~oitinase II coding region initiates to the "left" of the EcoRI site that lies within the P.
vulqaris derived DNA and proceeds toward the NsiI site at the "right" end of the fragment depicted in Figure 1. Therefore, this restriction map should be eYpAn~ed to the "right" to find a suitable fragment that will include the C-terminal coding region for the chQn~roitinase II gene.
Digestion of LP~751 reveals three EcoRI
fragments of a~Gximately 20 kb, 13 kb, and 10 kb, and indicates that there are three EcoRI sites within LP2751. Because there are two EcoRI sites that bracket the cloning site, the conclusion is that there is one EcoRI site within the cloned P. vulqaris DNA in this clone. Furthermore, since the approximately 13 kb fragment corresponds to the size of the cosmid vector per se, this unique EcoRI site lies between the approximately 20 kb and the approximately 10 kb fragments noted above. Since it is known that the entire coding region for chon~roitinase I, as well as the N-terminal coding region for ch~n~roitinase II, are both contA;ne~ within the approximately 10 kb NsiI
fragment, restriction digestions that compare the patterns obtA; neA among the cloned 10 kb NsiI (present in the recombinant plasmid designated LP2770) and gel-purified sample~ of the _bove a~lo~imately 20 kb EcoRI and approximately 10 kb EcoRI fragments indicate which of these EcoRI fragments contain the chQ~roitinase I coding sequence and, therefore by deduction, which will carry the C-terminal coding region for ~ho~roitinase II. Consequently, digestions are carried using the restriction enzymes W094l25567 PCT~S94/~95 ~16112~ - 88 -AflIII, ClaI, EcoRV, and HindIII each of which has been noted by Applicants to yield eight to ten fragments upon digestion of the original cosmid clone designated LP2751.
The recombinant molecule carrying the subcloned approximately 10 kb NsiI fragment (LP2770) and the individually gel-purified approximately 20 kb EcoRI and approximately 10 kb EcoRI fragments are digested with each of these enzymes to yield patterns of fragments that are compared. These digestions re~eal that the approximately 20 kb EcoRI and the LP2770 patterns have a number of fragments in common.
This indicates that the rhon~roitinase I gene and the N-terminal co~; ng region of the chon~roitinase II gene are cont~; ne~ within the larger EcoRI fragment and, therefore, the C-terminal coding region for the rhQn~roitinase II gene is on the ~ G~imately 10 kb EcoRI fragment.
The approximately 10 kb EcoRI fragment is cloned into the unique EcoRI site of the derivative of pNEB193 (New England Biolabs, Beverly MA) that is designated lac~oA pNEB193. This vector carries two deletions relative to the parental molecule pNEB193.
The first ~ ve8 the sequences between the unique NdeI and EcoRI sites, ret~;n;ng the EcoRI site but removing the NdeI site (and one of the two PvuII
sites). The second deletion removes the region between the HindIII site at the other end of the polylinker and the (now unique) PvuII site, maint~;n;ng the HindIII site, while removing the PvuII
site. The recombinant DNA molecule carrying the subcloned a~lG~imately 10 kb EcoRI fragment in the vector lacpoA pNEB193 is designated LP21263. The orientation of the 112 kD C-terminal coding region within LP21263 is determined by restriction enzyme W094t25567 PCT~S94/~95 mapping. The results indicate that this region i8 positioned 80 as to proceed from the EcoRI site (defined as the "left" end) toward the HindIII site at the other end of the polylinker. Similarly, unique restriction sites for SmaI, XhoI, NocI and NdeI are found approximately 2.6, 4.6, 5.8 and 8.5 kb from the "left" end of the approximately 10 kb EcoRI fragment.
Digestion of LP21263 with SmaI, therefore, deletes a downstream region of approximately 7.4 kb from the site within the cloned P. vulqaris DNA to the second site within the polylinker region, leaving approximately 2.6 kb which should be enough to encode the missing region of the chQn~roitinase II gene.
This construction also "places" a BamHI site (present in the polyl;n~r) downstream of the coding region for the chon~roitinase II gene. This recombinant DNA
molecule which carries the chon~roitinase II gene from the EcoRI site to (and presumably just beyond) the termination codon for this gene has been designated m#25-5712.
DNA seguence analysis is initiated on the approximately lO kb EcoRI fragment derived from LP'1263 and is completed after the assembly of the intact gene for chon~oitinase II. The materials and methods for the DNA sequencing of this fragment are essentially the same as those used for the approximately 4 kb fragment cont~;n;ng the gene for chon~roitinase I.
Random fragments are derived from this approximately 10 kb EcoRI fragment by self-ligating the DNA and then fragmenting the polymerized DNA by sonication as well as by partial digestion with the restriction enzymes Sau3A or MseI. These pieces are then e~entually cloned into Ml3 deri~ed vectors and the single-stranded recombinant molecules sequenced using the st~n~d protocols described above.

W094l25567 PCT~S94/~95 Finally, with the two set of sequence data available, an approximately 300 base-pair BclI
fragment is identified that is predicted to contain the EcoRI site that i8 the junction between the two P.
vulqaris fragments of approximately 20 kb and approximately 10 kb obtained by digestion with EcoRI.
This small fragment is sequenced in both directions to verify the nucleotide sequence through this junction point used in the constructions described below.
Exam~le 13 Assembly Of The Entire Site-Specifically Mutagenized Gene For ChnnAroitinase II

During the DNA seguencing, the molecule designated m#25-5712 is digested with EcoRI and BamHI.
This releases a DNA fragment of approximately 2.6 kb.
Similarly, the construction designated m#l5-5712 i8 digested with EcoRI and BamHI and then dephosphorylated prior to purification by gel electrophoresis. The latter molecule therefore carries the N-terminal coding region of the chnnAroitinase II gene from the ATG initiation codon (now present as part of an NdeI site from the site-specific mutagenesis) to the EcoRI site.
These two fragments are ligated and then the mixture used to transform the _. coli strain 294.
Plasmid DNA is isolated from the transformants and positive clone~ identified. Restriction digestion with NdeI and BamXI releases the de~ired fragment ~ncoA;ng the c~QnAroitinase II gene (SEQ ID N0:39, nucleotides 3235-6518, followed by 14 nucleotides derived from the polylinker, which includes a BamXI
site). This fragment is then ligated to the expression ~ector pET9A (Novagen, Madison, WI) W094l25567 ~1 6 11 2 5 PCT~S94/04495 described in the expression of the chsn~roitinase I
gene.
The coding region of the chon~roitinase II
gene includes nucleotides 3238-6276 of the SEQ ID NO:
39, which encodes 1013 amino acids (SEQ ID NO:40). Of this region, nucleotides 3238-3306 encode the 23 amino acid signal peptide (SEQ ID NO:40, amino acids 1-23), while nucleotides 3307-6276 encode the mature 990 amino acid chon~roitinase II protein (SEQ ID NO:40, amino acids 24-1013).
Restriction analysis with four enzymes of the region spann;ng both cho~roitinase genes and fl~nk;ng sequences thereof reveals the following restriction sitos:
EnzymeNucleotide EnzYme Nucleotide EcoRI 2 MunI 4510 HindIII2046 HindIII 4530 MunI 2904 MunI 5176 MunI 2943 HindIII 5427 HindIII3326 SmaI 6515 EcoRI 3974 In addition, restriction analysis with Sau3AI reveals a multiplicity of sites, including those at SEQ ID NO:39, nucleotides 212, 602, 890, 1042, 1181, 1241, 1442, 1505, 1746, 2330, 2363, 2701, 2705, 2920, 3697, 3708, 3745, 3868, 4087, 4800, 4872, 5565, 5635, 5860, 6058 and 6467.
One of the recombinant molecules (the chon~roitinase II gene inserted into pET9A) obtA;ne~
in this experiment is y~.n in large scale (0.5 liter) and the expression system contA;n;ng the rhon~roitinase II gene isolated and designated LP21359.
An aliquot of this DNA is used to transform the expression host BL21(DE3)/pLysS described in the W094l25567 PCT~S94/0~95 216 11~5 - 92 -expression of the chon~roitinase I gene. The resulting strain is designated TD112 and is used for large-scale fermentation and isolation of the ch~ oitinase II enzyme.
A fermentation at a 10 liter scale carried out with this E. coli strain cont~;ning the plasmid expressing the chon~roitinase II protein, provides a maximum cho~roitinase II titer of approximately 0.3 mg/ml, which is approximately 25 times that of the approximately 0.012 mg/ml obtA;ne~ from the native P.
vul~aris fermentation process for chQn~roitinase II.

Exam~le 14 First Method For The Isolation And Purification Of Recombinant rhon~roitinase II
Accordin~ To This Invention The initial part of this method is the same as that used for the recombinant c~Qn~roitinase I
enzyme. As a first step, the host cells which express the recombinant chQ~roitinase II enzyme are homogenized to lyse the cells. This relea~es the enzyme into the supernatant.
In one embodiment of this invention, the supernatant is first subjected to diafiltration to remove salts and other small molecules. An example of a suitable filter is a spiral wound 30 kD filter made by Amicon (Beverly, MA). However, this step only removes the free, but not the bound form of the negatively charged molecules. The bound form of these charged species is removed by passing the supernatant (see the SDS-PAGE gel depicted in Figure 5, lane 1) through a strong, high capacity anion eYch~nge resin-cont~;n;ng column. An example of such a resin is the Macro-Prep~ High Q resin (Bio-Rad, Melville, N.Y.).

W094/25567 PCT~S94/0~95 ~16112~

Other strong, high capacity anion eYchAnge columns are also suitable. The negatively charged molecules bind to the column, while the enzyme passes through the col~mn with approximately 90% recovery of the enzyme.
It is also found that some unrelated, undesirable proteins also bind to the column.
Next, the eluate from the anion ~YchAnge column (Figure 5, lane 2) is directly loaded to a cation ~Ych~nge resin-contA;n;ng column. Examples of such resins are the S-SepharoseT~ (Pharmacia, Piscataway, N.~.) and the Macro-PrepT~ High S (Bio-Rad). Each of these two resin-contA;ning columns has S03- ligands bound thereto in order to facilitate the ~YchAnge of cations. Other cation eY~h~nge colnmns are also suitable. The enzyme binds to the column, while a significant portion of contaminating proteins elute unbound.
At this point, the method diverges from that used for the chQn~roitinase I protein. Instead of eluting the protein with a non-specific salt solution capable of releasing the enzyme from the cation eYch~nge column, a specific elution using a solution conta;n;n~ chQn~roitin sulfate is used. A 1%
concentration of chQn~roitin sulfate is used; however, a gradient of this solvent is also acceptable. The specific rho~roitin sulfate solution is preferred to the non-specific salt solution because the recombinant rhon~roitinase II protein is expressed at levels approximately several-fold lower than the recombinant cho~roitinase I protein: therefore, a more powerful and selective solution is necessary in order to obtain a final chQn~roitinase II product of a purity equivalent to that obt~;ne~ for the chon~oitinase I
protein.
The cation ~YrhAnge column is next washed W094l25567 - ~ PCT~S94/0~95 with a phosphate buffer, pH 7.0, to elute unbound proteins, followed by w~sh;ng with borate buffer, pH
8.5, to elute loosely bound contaminating proteins and to increase the pH of the resin to that required for the optimal elution of the cho~roitinase II protein using the substrate, cho~roitin sulfate.
Next, a 1% solution of rho~roitin sulfate in water, adjusted to pH 9.0, is used to elute the rhnn~roitinase II protein, as a sharp peak (recovery 65%) and at a high purity of approximately 95% (Figure 5, lane 3). However, the cho~roitin sulfate has an affinity for the rhn~Aroitinase II protein which is stronger than its affinity for the resin of the col~n, and therefore the r~on~roitin sulfate co-elutes with the protein. This ensures that only protein which recognizes rhon~roitin sulfate is eluted, which is desirable, but also means that an additional process step i8 necessary to separate the ~ho~roitin sulfate from the chQn~roitinase II
protein.
In this separation step, the eluate is adjusted to pH 7.0 and is loaded as is onto an anion eY~h~nge resin-cont~i n; ng column, such as the Macro-Prep~ High Q resin. The column is washed with a 20 mM
phosphate buffer, pH 6.8. The rhon~roitin sulfate binds to the column, while the rhnn~roitinase II
protein flows through in the unbound pool with greater than 95% recovery. At this point, the protein is pure, except for the presence of a single minor contaminant of approximately 37 kD (Figure 5, lanes 4 and 6). The contaminant may be a breakdown product of the rhnn~roitinase II protein.
This contaminant is effectively removed by a crytallization step. The eluate from the anion ~Ych~nge column is concentrated to 15 mg/ml protein wo 94n5s67 - 216112 5 pcTluss4lo44ss using an Amicon stirred cell with a 30 kD cutoff. The solution is maintained at 4C for several days to crystallize out the pure chon~-oitinase II protein.
The supernatant contains the 37 kD contA~;nAnt (Figure 5, lane 7). Centrifugation causes the crystals to form a pellet, while the supernatant with the 37 kD
contaminant is removed by pipetting, and the crystals washed twice with water. After the first wash, some of the contaminant remains (Figure 5, lane 8), but after the second wash, only the chQn~roitinase II
protein is visible (Figure 5, lane 9). The washed crystals are redissolved in water and exhibit a single protein band on SDS-PAGE, with a purity of greater than 99% (Figure 5, lane 10).
Exam~le 15 Second Method For The Isolation And Purification Of Recombinant rhQn~roitinase II
Accordinq To This Invention In the second embodiment of this aspect of the invention, two additional steps are inserted in the method for purifying the chon~roitinase II enzyme before the diafiltration step of the first embodiment.
The supernatant is treated with an acidic solution, such as 1 M acetic acid, bringing the supernatant to a final pH of 4.5, to precipitate out the desired enzyme. The pellet is obtA; ne~ by centrifugation at 5,000 x g for 20 minutes. The pellet is then dissolved in an alkali solution, such as 20-30 mM
NaOH, bringing it to a final pH of 9.8. The solution is then subjected to the diafiltration and subsequent steps of the first embodiment of this aspect of the invention.

W094l2~567 PCT~S94/0~9~

Z16112~ 96 -BiblioqraPhy 1. Yamagata, T., et al., J. Biol. Chem., 243, 1523-1535 (1968).
2. R;~ch;, H., et al., U.S. Patent Number 5,198,355.
3. Brown, M. D., U.S. Patent Number 4,696,816.
4. Hageman, G. S., U.S. Patent Number 5,292,509.
5. Malitschek, B., and Schartl, M., Biotechniques, 7, 177-178 (1991).
6. Sanger, F., et al., Proc. Natl. Acad.
Sci. USA, 74, 5463-5467 (1977).
7. Innis, M. A., and Gelfand, D. H., "Optimization of PCRs", pages 3-12 in PCR Protocols, A
Guide to Methods and A~lications, Academic Press, New York, N.Y. (1990).
8. Southern, E., J. Mol. Biol., 98, 503-517 (1975).
9. Studier, F. W., et al., Methods in EnzYmoloqY, 185, 60-89 (1990).
10. Studier, F. W., and Moffatt, B. A., J.
Mol. Biol., 189, 113-130 (1986): Moffatt, B. A., and Studier, F. W., Cell, 49, 221-227 (1987).
11. Sambrook, J., et al., Molecular Clonin~: A LaboratorY 2-n~al, 2nd ed., Cold Spring Harbor ~aboratory Press, Cold Spring Harbor, N.Y.
(1989).
12. Clark, Nucleic Acids Research, 16, 9677 (1988).
13. Yanisch-Perron, C., et al., Gene, 3, 103-119 (1985).
14. Devereaux, et al., Nucleic Acids Research, 12, 387-395 (1984).
15. Kunkel, T. A., Proc. Natl. Acad. Sci., W094t25567 PCT~S94/04495 ~16112~

USA, 82, 488-492 (1985).
16. Studier, F. W., J. Mol. Biol., 219, 37-44 (1991).
17. Miller, J. H., Experiment~ in Molecular Genetics, Cold Spring Harbor Laboratory Pre~R, Cold Spring Harbor, N.Y. (1972).

S_QU_N OE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: American Cyanamid Company (ii) TIT~E OF INV_NTION: Cloning And Rxpression Of The Chondroitinase I
and II Genes From P. Vulgaris (iii) NUMB_R OF SEQUBNCES: 41 (iv) COP~`POND_NCR ~nDPR~S:
(A) ~n~PR-eSRR: American Cyanamid Company (B) STRBET: One Cyanamid Plaza (C) CITY: Wayne (D) STAT_: New Jersey (E) Cuu~nY ~. s .A.
(F) ZIP: 07470-8426 (v) COMP~TBR ~R--~ FORM:
(A) MEDIUM TYPE: Floppy di8k (B) COMP~TER: IBM PC compatible (C) OPBRATING SYSTBM: PC-DOS/MS-DOS
(D) SOFTWAR_: PatentIn Rele-os #1.0, Version #1.25 (vi) C~RRBNT APPLICATION DATA:
~A) APPLICATION NUMBBR: PCT/~S94/
(B) FILING DATE:
(C) ~T-~C~ ;ATION:
(viii) ATTORNBY/AaBNT INFORMATION:
(A) NAME: Gordon, Alan M.
(B) RBGISTRATION NUMBBR: 30,637 (C) k~r~bNCB/~O~ NIMBBR: 31,726-00/PCT
(ix) TBL_COMMnNICATION INFORMATION:
(A) TBLBPHON_: 201-831-3244 (B) TBLBFAX: 201-831-3305 (2) INFORMATION FOR S_Q ID NO:l:
(i) SBQUBNCB rU~T'~TT~DTeTICS:
(A' LBNGTH: 3980 base pairs (B TYPE: nucl-ic acid (C sTp~TnnNBss: single (D) TOPOLOGY: lin-ar (ii) MOTRC~TR TYPE: DNA (genomic) (iii) ~rG,A~,~AL: NO
(iv) ANTI-S_NS_: NO

(ix) FBAT~RB:
(A) NAMB/REY: CDS
(B) LOCATION: 119..3181 (xi) SBQ~BNCB DR-c~TPTIoN S_Q ID NO:l:

SUBS Tl I U . S~ET (RULE 26) 21~1~2~

GGAATTCCAT CACTCAATCA TTAAATTTAG Gr~r~rr~ GGGCTATCAG CGTTATGACA 60 AATTTAATGA ~ CGr~TT G~.,, Q CTG TT~Cr~GCG TTTCT~G~ G~ ~A 118 ~et Pro Ile Phe Arg Phe Thr Ala Leu Ala Met Thr Leu Gly Leu Leu 1 , 5 10 15 Ser Ala Pro Tyr Asn Ala Met Ala Ala Thr Ser Asn Pro Ala Phe A~p Pro Lys Asn Leu Met Gln Ser Glu Ile Tyr His Phe Ala Gln Asn Asn Pro Leu Ala ABP Phe ger Ser Asp Lys Asn Ser Ile Leu Thr Leu S-r Asp Lys Arg Ser Il- ~et Gly Asn Gln 8er L-u Leu Trp Lys Trp Lys aly Gly Ser 8er Phe Thr L-u His Lys Lys L-u Il- Val Pro Thr A-p Lys Glu Ala 8er Lys Ala Trp Gly Arg Ser Ser Thr Pro Val Phe S-r TTT TGG CTT TAC AAT GAA AAA CCG ATT GAT GGT TAT CTT ACT ATC aAT 502 Phe Trp L-u Tyr Asn alu Lys Pro Il- Asp Gly Tyr L-u Thr Ile A~p Ph- Gly Glu Lys L-u Ile Ser Thr Ser Glu Ala Gln Ala Gly Phe Lys Val Lys Leu Asp Phe Thr Gly Trp Arg Ala Val Gly Val 8er Leu Asn Asn Asp Leu Glu Asn Arg Glu ~et Thr Leu Asn Ala Thr Asn Thr Ser Ser Asp Gly Thr Gln A~p Ser Il- Gly Arg Ser Leu Gly Ala Lys Val Asp Ser Il- Arg Phe Ly- Ala Pro Ser Asn Val Ser Gln Gly Glu Ile Tyr Ile ABP Arg Il- ~et Phe S-r Val Asp Asp Ala Arg Tyr Gln Trp Ser Asp Tyr Gln Val Lys Thr Arg Leu Ser Glu Pro Glu Ile Gln Phe SUBSTI~UT~ SH~T (~UL~ 26) 1-2~ - 1 ~i8 A~n Val Lys Pro Gln Leu Pro Val Thr Pro Glu A-n Leu Ala Ala ATT GAT CTT ATT CGC CAA CqT CTA ATT AAT GAA TTT GTC GGA GGT GAA 934 Ile Asp Leu Ile Arg Gln Arg Leu Ile Asn Glu Phe Val Gly Gly Glu Ly- Glu Thr Asn Leu Ala L-u Glu Glu Asn Ile Ser Lys Leu Lys 8-r Asp Phe ABP Ala Leu Asn Ile Hi- Thr Leu Ala Asn Gly Gly Thr Gln Gly Arg His Leu Il- Thr Asp Ly- Gln Ile Ile Ile Tyr Gln Pro Glu Asn Leu Asn Ser Gln Asp Lys Gln Leu Phe Asp A n Tyr Val Ile Leu Gly Asn Tyr Thr Thr Leu Met Phe Asn lle Ser Arg Ala Tyr Val Leu Glu Lys A-p Pro Thr Gln Ly- Ala Gln Leu Lys Gln ~et Tyr Leu Leu ~et Thr Ly- ~i- L-u Leu Asp Gln Gly Ph Val Lys Gly S-r Ala L-u Val Thr Thr His His Trp Gly Tyr Ser Ser Arg Trp Trp Tyr Ile Ser Thr Leu Leu ~et Ser A-p Ala Leu Lys Glu Ala Asn L-u Gln Thr Gln Val Tyr Asp 8er Leu Leu Trp Tyr Ser Arg Glu Ph- Lys Ser 8-r Phe GAT ATG AAA GTA AGT GCT GAT AGC TCT GAT CTA GAT TAT TTC A~T ACC 1462 Asp Met Lys Val Ser Ala Asp Ser Ser Asp Leu Asp Tyr Ph- Asn Thr Leu Ser Arg Gln ~is Leu Ala Leu Leu Leu Leu Glu Pro Asp Asp Gln Lys Arg Ile Asn Leu Val A-n Thr Phe Ser Hi- Tyr Ile Thr Gly Ala SUBST~UT~ SHEET (RULE 26) WO 94/25567 21611 2 5 PCTtUS94/04495 `

Leu Thr Gln Val Pro Pro Gly Gly Lys ABP Gly L-u Arg Pro Asp Gly Thr Ala Trp Arg His Glu Gly Asn Tyr Pro Gly Tyr S-r Phe Pro Ala Phe Lys Asn Ala S-r Gln Leu Ile Tyr Leu Leu Arg Asp Thr Pro Phe ger Val Gly Glu S-r Gly Trp Asn Asn L-u Lys Lys Ala ~et Val Ser Ala Trp Ile Tyr S-r Asn Pro Glu Val Gly Leu Pro Leu Ala Gly Arg His Pro Phe Asn 8er Pro 8er Leu Lys S-r Val Ala Gln Gly Tyr Tyr Trp Leu Ala Net 8-r Ala Lys 8er 8er Pro Asp Lys Thr Leu Ala 8er Ile Tyr Leu Ala Ile 8-r Asp Ly~ Thr aln Asn Glu S-r Thr Ala Il-Ph- Gly Glu Thr Il- Thr Pro Ala S-r L-u Pro Gln Gly Phe Tyr Ala Ph- Asn Gly Gly Ala Ph- Gly Il- ~is Arg Trp Gln Asp Lys N-t Val Thr Leu Lys Ala Tyr Asn Thr Asn Val Trp 8er Ser Glu Ile Tyr Asn Lys Asp Asn Arg Tyr Gly Arg Tyr Gln S-r H~s Gly Val Ala Gln Ile Val 8er Asn Gly S-r Gln L-u Ser Gln Gly Tyr Gln Gln Glu Gly Trp Asp Trp Asn Arg ~et Gln Gly Ala Thr Thr Ile His Leu Pro L-u Lys Asp L-u Asp S-r Pro Lys Pro his Thr Leu Met Gln Arg Gly Glu Arg Gly Phe S-r Gly Thr S-r S-r L-u Glu Gly Gln Tyr Gly Met M-t Ala SUBSTITUTE SHEET (~11 E 26) W 0 94/25567 PCTrUS94/04495 i 6 ~ i 2 ~ - 102 -Phe ABP Leu Ile Tyr Pro Ala Asn Leu Glu Arg Phe Asp Pro Asn Phe Thr Ala Lys Lys Ser Val Leu Ala Ala Asp Asn His Leu Ile Phe Ile Gly Ser Asn Ile Asn Ser Ser Asp Lys Asn Lys Asn Val Glu Thr Thr Leu Phe Gln His Ala Ile Thr Pro Thr Leu Asn Thr Leu Trp Ile A~n Gly Gln Lys Ile Glu Asn Met Pro Tyr Gln Thr Thr Leu Gln Gln Gly Asp Trp Leu Ile Asp 8er Asn Gly A~n Oly Tyr L-u Ile Thr Oln Ala Glu Lys Val A~n Val Ser Arg Gln His Gln Val Ser Ala Glu Asn Lys Asn Arg Gln Pro Thr Glu Gly Asn Phe 8er 8er Ala Trp Il- Asp His Ser Thr Arg Pro Lys Asp Ala 8er Tyr Glu Tyr Met Val Phe Leu ABP

Ala Thr Pro Glu Lys ~et Gly Glu ~et Ala Gln Ly~ Phe Arg Glu Asn Asn Gly Leu Tyr Gln Val Leu Arg Lye Asp Lys Asp Val His Ile Ile Leu Asp Lys Leu Ser A~n Val Thr Gly Tyr Ala Phe Tyr Gln Pro Ala Ser Ile Glu Asp Lys Trp Ile Lys Lys Val Asn Lys Pro Ala Ile Val ATG ACT CAT CGA ~ AAA GAC ACT CTT ATT GTC AGT GCA GTT ACA CCT 2998 ~et Thr His Arg Gln Lys Asp Thr Leu Ile Val Ser Ala Val Thr Pro Asp Leu Asn Met Thr Arg Gln Lys Ala Ala Thr Pro Val Thr Ile Asn SU~S 111 UTE ~E~T (RULE 26) ~161125 Val Thr Ile Asn Gly Lys Trp Gln Ser Ala Asp Lys Asn Ser Glu Val Lys Tyr Gln Val S-r Gly A~p Asn Thr Glu Leu Thr Phe Thr Ser Tyr Phe Gly Ile Pro Gln Glu Ile Lys Leu Ser Pro Leu Pro ~ CGC ~,,GC~.,C ~......... ~ATT TG~ T CTGATTATGC T~T~ 3251 CC~...AGCC r~GC~A QTTAAGCCT ~ .AT Q TT~CCGr~r AAG Q TTACC 3311 Q ~. ~.~-. Q TGAAGCTT -.CGGC~'AT~T TTA-~--~-- GAAGGTGAAT T~Cr~T~C 3371 CCTT~r~rT Tr~T~T~ ATCAATTATC GCT~GG~ CAGCATGCTA AAGATGGTGA 3431 Ar~T Q CTC AAATGGCAAT ATr~r~ ar~TTA ~CT~T~ ATATTGTTAA 3491 TT-~r~AT ~'T~ T~ r~cr~ c ACT Q CTTTT ATGATGTGGA TTTATAATGA 3551 ~ C r~ --CCC~-AT TAACGTTAGC ATTT~ T--T~ TTGr~rT~ 3611 -r~-rr~A-AATGCT GAACTTAATT TT~C~G6h--G GCGAGGTATT G~-~--C~-- -~,~ATAT 3671 Gr~GrTCT GC~-A~TC AACTTGAT Q ATTAGTGATC ACCG~-C~AA ~Cr~CCCC 3731 TGACTAT QA A Q CCTTACG T~T~GC AGT~ CG ATGGTTAGTA AAAACTGGAG 3851 TG QTTATTG ATGT~C~'TC AGA.~..~CA ~GCCr~TTAC CCTACTTTAA ACTTCGATAC 3911 TGAATTTCGC GAT~'~r~ r~ TGGC TTCGATTTAT Q GCG~-,,,G AATATTATCA 3971 (2) INFORMATION FOR SEQ ID NO:2:
li) SEQVBNCB CHARACTERISTICS:
(A) LENGTH: 1021 amino aclds (B) TYP8: ~ino ac~d (D) TOPOLOGY: lin-ar (ii~ MOT.~T.R TYPB: prote~n (Xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:2:
Net Pro Il- Phe Arg Phe Thr Ala Leu Ala ~et Thr Leu Gly Leu L-u Ser Ala Pro Tyr Asn Ala Met Ala Ala Thr Ser Asn Pro Ala Phe A~p Pro Lys Asn Leu Met Gln Ser Glu Ile Tyr His Phe Ala Gln Asn Asn Pro Leu Ala Asp Phe Ser Ser Asp Lys Asn Ser Ile Leu Thr Leu Ser S~IBST!TUTE S~EET ~RU~E 26) 216112~

Asp Lys Arg Ser Ile Met Gly Asn Gln Ser Leu Leu Trp Lys Trp Lys ly Gly Ser Ser Phe Thr Leu His Lys Lys Leu Ile Val Pro Thr Asp ys Glu Ala Ser Lys Ala Trp Gly Arg Ser Ser Thr Pro Val Phe S-r Phe Trp Leu Tyr Asn Glu Lys Pro Ile Asp Gly Tyr Leu Thr Ile Asp Phe Gly Glu Lys L-u Ile Ser Thr Ser Glu Ala Gln Ala Gly Phe Lys Val Lys Leu Asp Phe Thr Gly Trp Arg Ala Val Gly Val Ser Leu Asn sn Asp Leu Glu Asn Arg Glu Met Thr Leu Asn Ala Thr A~n Thr 8-r er Asp Gly Thr Gln Asp Ser Ile Gly Arg Ser L-u Gly Ala Lys Val Asp Ser Ile Arg Phe Lys Ala Pro Ser Asn Val 8er Gln Gly Glu Ile Tyr Ile Asp Arg Il- Met Ph- 8er Val Asp A~p Ala Arg Tyr Gln Trp 8er Asp Tyr Gln Val Lys Thr Arg Leu 8er Glu Pro Glu Ile aln Ph-is Asn Val Lys Pro Gln Leu Pro Val Thr Pro Glu Asn Leu Ala Ala le Asp Leu Ile Arg Gln Arg Leu Ile Asn Glu Phe Val Gly Gly Glu Lys Glu Thr Asn Leu Ala Leu Glu Glu Asn Ile 8er Lys Leu Lys 8er Asp Phe Asp Ala Leu Asn Ile His Thr Leu Ala Asn Gly Gly Thr Gln Gly Arg His Leu Ile Thr Asp Ly~ Gln Ile Ile Ile Tyr Gln Pro Glu sn Leu Asn Ser Gln Asp Lys Gln L-u Phe Asp Asn Tyr Val Ile Leu ly Asn Tyr Thr Thr Leu Met Phe Asn Ile Ser Arg Ala Tyr Val Leu Glu Lys Asp Pro Thr Gln Lys Ala Gln Leu Lys Gln Met Tyr Leu Leu Met Thr Lys His Leu Leu Asp Gln Gly Phe Val Lys Gly Ser Ala Leu SUBSTITUT~ SHEET (RU~E 26~

-- WO 94/25567 21 61 1 2 ~ PCT/US94/04495 Val Thr Thr His His Trp Gly Tyr Ser Ser Arg Trp Trp Tyr Ile Ser hr Leu Leu Met Ser Asp Ala Leu Lys Glu Ala Asn Leu Gln Thr Gln al Tyr Asp Ser Leu Leu Trp Tyr Ser Arg Glu Phe Lys Ser Ser Phe Asp Met Lys Val Ser Ala Asp Ser Ser Asp Leu Asp Tyr Phe Asn Thr Leu Ser Arg Gln His Leu Ala Leu Leu Leu Leu Glu Pro Asp Asp Gln Ly~ Arg Ile Asn Leu Val Asn Thr Phe Ser His Tyr Ile Thr Gly Ala eu Thr Gln Val Pro Pro Gly Gly Ly~ Asp Gly Leu Arg Pro Asp Gly hr Ala Trp Arg His Glu Gly Asn Tyr Pro Gly Tyr Ser Ph- Pro Ala Phe Lys A~n Ala 8er Gln Leu Ile Tyr Leu Leu Arg Asp Thr Pro Ph-Ser Val Gly Glu 8er Gly Trp A~n Asn L-u Lys Ly~ Ala ~ t Val S-r Ala Trp Ile Tyr Ser A~n Pro Glu Val Gly L-u Pro Leu Ala Gly Arg i~ Pro Phe A~n S-r Pro S-r Leu Lys 8er Val Ala Gln Gly Tyr Tyr rp Leu Ala Met Ser Ala Lys Ser Ser Pro A~p Ly~ Thr Leu Ala Ser Ile Tyr Leu Ala Ile Ser Asp Lys Thr Gln A~n Glu Ser Thr Ala Ile Phe Gly Glu Thr Ile Thr Pro Ala Ser Leu Pro Gln Gly Phe Tyr Ala Phe Asn Gly Gly Ala Phe Gly Ile His Arg Trp Gln Asp Ly~ Met Val hr Leu Lys Ala Tyr Asn Thr Asn Val Trp Ser Ser Glu Ile Tyr A~n ys Asp Asn Arg Tyr Gly Arg Tyr Gln Ser His Gly Val Ala Gln Ile Val Ser Asn Gly Sor Gln Leu Ser Gln Gly Tyr Gln Gln Glu Gly Trp Asp Trp Asn Arg Met Gln Gly Ala Thr Thr Ile His Leu Pro Leu Lys Asp Leu Asp Ser Pro Lys Pro His Thr Leu Met Gln Arg Gly Glu Arg SUB~i I I I I JTE SHEET (RULE 26) 2~61~25 Gly Phe Ser Gly Thr Ser Ser Leu Glu Gly Gln Tyr Gly Met Met Ala he Asp Leu Ile Tyr Pro Ala Asn Leu Glu Arg Phe A~p Pro Asn Phe Thr Ala Lys Lys Ser Val Leu Ala Ala ABP Asn His Leu Ile Phe Ile Gly Ser Asn Ile Asn Ser Ser Asp Lys Asn Lys Asn Val Glu Thr Thr Leu Phe Gln His Ala Ile Thr Pro Thr Leu Asn Thr Leu Trp Ile Asn ly Gln Lys Ile Glu Asn Met Pro Tyr Gln Thr Thr Leu Gln Gln Gly sp Trp Leu Ile Asp Ser Asn Gly Asn Gly Tyr Leu Ile Thr Gln Ala Glu LYB Val Asn Val Ser Arg Gln H~B Gln Val Ser Ala Glu A~n Lys Asn Arg Gln Pro Thr Glu Gly A~n Phe 8er 8er Ala Trp Ile ABP His 8-r Thr Arg Pro Lys Asp Ala 8er Tyr Glu Tyr Met Val Phe Leu A~p la Thr Pro Glu Lys Met Gly Glu Met Ala Gln Lys Phe Arg Glu A~n sn Gly Leu Tyr Gln Val Leu Arg Ly~ Asp Lys Asp Val ~is Ile Ile Leu Asp Lys Leu Ser Asn Val Thr Gly Tyr Ala Phe Tyr Gln Pro Ala 8er Ile Glu Asp Lys Trp Ile Lys Lys Val Asn LYB Pro Ala Ile Val Met Thr His Arg Gln LYB ABP Thr Leu Ile Val 8er Ala Val Thr Pro BP Leu Asn Met Thr Arg Gln Lys Ala Ala Thr Pro Val Thr Ile Asn al Thr Ile Asn Gly LYB Trp Gln 8er Ala Asp LYB Asn 8er Glu Val LYB Tyr Gln Val 8er Gly Asp Asn Thr Glu Leu Thr Phe Thr 8er Tyr Phe Gly Ile Pro Gln Glu Ile Lys Leu 8er Pro Leu Pro (2) INFORMATION FOR SEQ ID NO 3 (i) SEQUENCE ~U~T~T~TICS
(A) LBNGTH 3980 base pairs (B) TYPE n~ eic ac~d SlJBSTITlJTE SHEET (RULE 26) (C) STRANDEDNESS: single ~D) TOPOLOGY: line~r (ii) MOTR~ R TYPE: DNA (genomic) (iii) ~rO,~h~lCAL: NO
(iv) ANTI-SENSE: NO

(xi) SBQ~NCR DR~CT~TPTION: SEQ ID NO:3:
GGAATTCCAT CACTCAATCA TTAAATTTAG Gr~ ra~T GGGCTATCAG CGTTATGACA 60 AA m AATGA ~n-~Gr~TT G~.~ACTG TT~GCr~rCG TTTCTAAGGA -~ T~T 120 GCCC~T~TTT C~,,,,ACTG CACTTGCAAT GACATTGGGG CTATTATCAG cac~- .ATAA 180 CGCGATGGCA aC~r~C~~ A,C~ ATT TGATC AAA AATCTGATGC AGTr~ T 240 TTACCATTTT Gr~a~T~ ACCCATTAGC AGA~-,,~ `A Tr~T~ ACTCAATACT 300 AACGTTATCT C~T~C~TA GCATTATGGG ~ Cr~TCT CTTTTATGGA AATGG~-C 360 TGGTAGTAGC TTTACTTTAC ~T~ a~CT GA-,,~.CCCC ~ccr~T~c AAGC~TCTAA 420 AGCATGGGGA CGCTCATCTA CCCCC~...- CTCATTTTGG C-rTTACAATG ~ rC~T 480 TGATGGTTAT CTTACTATCG A~CG~AGA AAAACTCATT Tr~Cr~GTG AGGCTCAGGC 540 AGG~-~..AAA GTAAAATTAG ATTTCACTGG ~GaC~G~-~ GTGGGAGTCT CTTT~T~a 600 CGATCTTGAA AATcr~ TGACCTTAAA Tar~r~T AC~C~ G ATGGTA CA 660 ~ TT GaGC~.~ TA¢~ AA AGTCGATAGT A~C~ A ~CGC~- -C 720 TAATGTGAGT CAGGGTGAAA T ATATCGA CCGTATTATG ....~-~CG ATGA,~,CG 780 CT~rr~TGG TCTGATTATC AAGT~ r ,CG~-,ATCA GAACCTGAAA TTCAATTTCA 840 r~CGTAAAG Cr~ ~T~r CTGT~ rC TGAAAATTTA GCGGC~-~TTG ATCTTATTCG 900 CCAACGTCTA ATTAATGAAT .-~CG4AGG TC~ rCTCG CATT~ C~ 960 GAATATCAGC AAATTAAAAA GTGATTTCGA ~ AAT ATTCACACTT T~G-~TGG 1020 TG-~rGr~ GGr~-~r~TC TGATCACTGA T~r~TC ATTATTTATC ~cr~ 1080 TCTTAACTCC r~ T~ AACTATTTGA TAATTATGTT ATTTTAGGTA ATT~rC~r 1140 ATTAATGTTT AATATTAGCC ~-G~--ATGT GCTG-~ GATCCr~r~r A~CGCC-~ 1200 ~CT~C~~r ATGTACTTAT TAATG'r~ GCA m ATTA GATCAAGGCT TTGTTAAAGG 1260 GA~.G~.~A GTG~ra~CC ATCACTGGGG ~T~r~-TTCT C~,,¢~. ATATTTCCAC 1320 GTTATTAATG TCTGATGCAC T~ GC C~arCT~ ACTCAAGTTT ATGATTCATT 1380 A~.~.AT TCACGTGAGT TTAAAAGTAG TTTTGATATG ~rT~CTG CTC~T~GrTC 1440 TGATCTAGAT TATTTCAATA CCTTATCTCG Cr~r~TTTA GCCTTATTAT T~rT~-~-CC 1500 SUBSTI~IJTE SHEET (RULE 26) WO 94/25567 ~12' PCT/US94/04495 TGATGATCAA AAGCGTATCA ACTTAGTTAA TACTTTCAGC CATTATATCA ~GGCG~TT 1560~rGr~rTG cr~rCGg~G aTA~r~TGG TTT~CGCCCT GATGGTACAG CATGGC~a~C~ 1620 TC~ 3~r TA~CC~GG~ A~ CCC AGC~AAA AA~GCL~C AGCTTATTTA 1680 TTTATTACGC ~T~r~Cr~T TTTCAGTGGG TGAAAGTGGT TGC~T~rC T~ ~C 1740 GA~G~ A GC~GGATCT ACAGTAATCC AGAAGTTGGA TTACCG~G ra~ C~ 1800 CC~-....AAC TCAC~--C~- TAAAATCAGT CGCTCAAGGC TATTACTGGC TTGCCATGTC 1860 TGr~ TCA -~CGC~:ATA ~ar~CTTGC ATCTATTTAT ~.~GC~ATTA GTG~T~ r 1920 ~r~aTGAA TCAACTGCTA ~ G~AGA AACTATTACA CCAG~,-~ TACCTCAAGG 1980 TTTCTATGCC TTTAATGGCG ~,~,,,,GG TATTCATCGT TGGr~ T~ AAATGGTGAC 2040 ACTG~GCT T~T~r~rra A~ .G~' ATCTGAAATT T~T~ra~ Ta~GCGTTA 2100 ~CC~AC CAAAGTCATG ~CGC~A AATAGTGAGT AA-a~CGC AG~.~ACA 2160 G~GCT~TCAG r~a~GCTT GGGATTGGAA TAGAATGCAA G~GCG~rr~ CTATTCACCT 2220 ~C~ AAA GACTTAGACA GTCCT~rC T~aT~C~TTA ATGCAACGTG ~GCGTGG 2280 ATTT~CGG~ ACATCATCCC TTGAAGGT Q ATATGGCATG ATGGCATTCG ATCTTATTTA 2340 ~CCCGC~ T CTTGAGCGTT TTGATCCTAA TTT Q CTGCG ~a~'~TG TATT~CCGC 2400 TGATAATCAC TTAATTTTTA TTGGTAGCAA TaTa~aTa~T AGT~Taaaa aT~a~aaTGT 2460 T~aC~C TTATTCCAAC ATGCCATTAC TCC~ TTA A~T~rCCTTT GGATTAATGG 2520 ~r'~ T~ C~ TGC CTTATCAAAC AACACTTCAA CAAGGTGATT GGTTAATTGA 2580 T~ TGGC AATGGTTACT TAATTACTCA ~ a~ GTAAATGTAA ~-CGC~r~ 2640 TCAG~A GCGr~aa~T~ AAAATCGCCA arcr~r~ G~a~TTTA G~,C~CATG 2700 GATCGATCAC AGCACTCGCC Cr~ TGC CAGTTATGAG TATATGGTCT TTTTAGATGC 2760 GACACCTGAA AAAATGGGAG AGATeC~ AAAA~ G~ TG GGTTATATCA 2820 G~ CG~ a~r~T~a~ ACGTTCATAT TA--C-C~AT AAACTCAGCA ATGTa~CGGG 2880 ATA~GC~ TATr~Gcra~ CATCAATTGA ~ TGG AT~aa~G~ TTa~T~a~C 2940 TGCAATTGTG ATGACTCATC C~C~a~ CACTCTTATT GTCAGTGCAG TT~a~CTGA 3000 TTT~T~TG A~.CGC Aa~ ~C~r ~C~.~ ACC ATCAATGTCA CGATTAATGG 3060 CAAATGGCAA ~.G--~ATA ~a~aT~-TGA AGTC~T~T CA~---~-G GT'T~r~C 3120 TGAACTGACG TTTACGAGTT A~.a~.AT TCr~r~ ATCAAACTCT CGCr~rTCCC 3180 TTGATTTAAT r~a~ rG ~-- GCG~ ~C~ AT TTGr~ a TCTGATTATG 3240 CT~T~ ACC~AGC Cr~CGC~ ACATTAAGCC .~.~.~.ATC ATT~CCCG~ 3300 ra~ TTAC CCA~ ~-C TCATGAAGCT .C~GCt'~T~ TTTATCTTTT TGAAGGTGAA 3360 SIJB~T~TUTE S~EET (RULE 26) _ W 0 94/25567 216112 5 PCTrUS94/044g5 TT~rCr~T~ CC~--r~ACCAC TTr~TA~T AATCAATTAT CGCT~ ACAGCATGCT 3420 AAAGATGGTG AACAATCACT CAAATGGCAA TATr~Cr~C ~r~ TT AACACTAAAT 3480 AATATTGTTA ATT~ TG~T~ T ~r~GC~r CACTCACTTT TATGATGTGG 3540 ATTTATAATG ~ ~CTCA A~ ~CCC~A TTAACGTTAG CATTTAAACA ~T~T~ 3600 ATTGr~CT~ GTTTTAATGC TGAACTTAAT TTT~CGGGGT GGCr~G~TAT G~--~-.~- 3660 ...C~.~ATA TG~GGCTC TGC~GGT CAACTTGATC AATTAGTGAT CACCG~--~-CA 3720 ~5r~GCCG C~r~TCTT TTTTGATCAA ATCATCATGA GTGTACCGTT ~ ~TCGT 3780 TGGGCAGTAC CTGACTATCA ~ r~-TTAC GT~T~G CAGT~r~ GATGGTTAGT 3840 ~ CTGGA GTGCATTATT GATGTACGAT CAGA.~...C ~GCC~aTTA CCCT~rTTTA 3900 AACTTCGATA CTGAATTTCG CGATG~r~a ~r~ TGG CrTCGATTTA TCAGCG~ . 3960 GAATATTATC ~r-~TTCC 3980 (2) INFORMATION FOR 8BQ ID NO:4:
(i) 8BQUBNCB r~rT~T~TICS:
(A) LBNGT~: 3980 ba~ pais~
(B) TYPB: nucl-lc acld (C) STRANDBDNBSS: ~lngl-(D) TOPOLOGY: lin-ar (li) MOT~C~T~ TYPE: DNA (g-nomic) (iii) ~rO,A~, CAL: NO
(lv) ANTI-SBNSB: NO

(ix) FBAT~RB:
(A) NAMB/~BY: CDS
(B) LOCATION: 188..3181 (xi) SBQUBNCB DBSCRIPTION: SBQ ID NO:4:
GGAATTCCAT CACTCAATCA TTAAATTTAG G-~r~Cr~T ~GGCT~TCAG CGTTATGACA 60 AATTTAATGA A~r~rG~aTT G~,,,~ACTG TTaGcra- cG TTTcraa~r~ TaaT 120 GCCr~TaTTT C~ ACTG CACTTGCAAT GACA,,aGGG CTATTATCAG CGC~-~ATAA 180 CGCG-~T ATG GCC ACC AGC AAT CCT GCA m GAT CCT AAA AAT CTG ATG 229 Met Ala Thr Ser Asn Pro Ala Ph- Asp Pro Ly~ Asn Leu ~et Gln Ser Glu Ile Tyr Ais Phe Ala Gln Asn Asn Pro Leu Ala Asp Phe Ser Ser Asp Ly~ Asn Ser Ile Leu Thr L-u Ser Asp Ly~ Arg S-r Ile SUBSTITUTE SHEE~ (RULE 26) WO 94nss67 PCT/US94/04495 21~112~ - llo -Met Gly Asn Gln Ser Leu Leu Trp Lys Trp Lys Gly Gly Ser Ser Phe Thr Leu His Lys Lys Leu Ile Val Pro Thr Asp Lys Glu Ala Ser Lys Ala Trp Gly Arg Ser Ser Thr Pro Val Phe Ser Phe Trp Leu Tyr Asn alu Lys Pro Ile Asp Gly Tyr Leu Thr Ile Asp Ph~ Gly Glu ~ys L-u Ile Ser Thr Ser Glu Ala Gln Ala Gly Phe Lys Val Lys Leu Asp Phe Thr Gly Trp Arg Ala Val Gly Val Ser Leu Asn Asn Asp Leu Glu Asn CGA GAG ATG ACC TTA AAT GCA ACC AAT ACC TCC TCT GAT GGT ACT r~ 661 Arg Glu Met Thr Leu Asn Ala Thr Asn Thr ger Ser Asp Gly Thr Gln Asp 8er Ile Gly Arg Ser Leu Gly Ala Ly~ Val Asp 8er Ile Arg Phe Lys Ala Pro Ser Asn Val Ser Gln Gly Glu Ile Tyr Ile Asp Arg Ile Met Phe Ser Val Asp Asp Ala Arg Tyr Gln Trp Ser Asp Tyr Gln Val Lys Thr Arg Leu Ser Glu Pro Glu Ile Gln Phe His Asn Val Lys Pro Gln Leu Pro Val Thr Pro Glu Asn Leu Ala Ala Ile Asp Leu Il- Arg Gln Arg Leu Ile Asn Glu Phe Val Gly Gly Glu Lys Glu Thr Asn Leu Ala Leu Glu Glu Asn Il- Ser Lys Leu Lys Ser Asp Ph- Asp Ala Leu Asn Ile His Thr Leu Ala Asn Gly Gly Thr Gln Gly Arg his Leu Ile Thr Asp Lys Gln Ile Ile Ile Tyr Gln Pro Glu Asn Leu Asn Ser Gln SUB~ I ~ I lJTE SI~EET (R~I~E 26) . WO 94/2S567 PCT/US94/04495 216112~i Asp Lys Gln Leu Phe Asp Asn Syr Val Ile Leu Gly A~n Syr Thr Thr TTA ATG TTT AAT ATT AGC CGT GCT TAT GTG G GAA AaA GAT CCC ACA 1189 L-u Met Phe Asn Ile Ser Arg Ala Tyr Val Leu Glu Lys Asp Pro Thr Gln Lys Ala Gln Leu Lys Gln ~et Tyr Leu Leu Met Thr Ly~ His Leu Leu Asp Gln Gly Phe Val Lys Gly Ser Ala Leu Val Thr Thr His His Trp Gly Tyr 8er 8er Arg Trp Trp Tyr Ile Ser Thr Leu Leu ~et 8er A~p Ala Leu Ly~ Glu Ala Asn Leu Gln Thr Gln Val Tyr Asp 8er Leu Leu Srp Tyr 8er Arg Glu Phe Ly~ 8er 8es Phe A~p Met Ly~ Val Ser Ala Asp 8er Ser A-p Leu Asp Tyr Phe A-n Thr Leu 8er Arg Gln ~8 Leu Ala Leu Leu Leu Leu Glu Pro A~p A~p Gln Lys Arg Ile A~n L-u Val Asn Thr Phe 8er Hi~ Tyr Ile Thr Gly Ala Leu Thr Gln Val Pro CCG GGT GGT AaA GAT GGT TSA CGC C GAT GGT ACA GCA TGG CGA CAT 1621 Pro Gly Gly Ly~ Asp Gly Leu Arg Pro Asp Gly Shr Ala Srp Arg H~s Glu Gly Asn Tyr Pro Gly Tyr 8er Phe Pro Ala Phe Ly~ Asn Ala 8er Gln Leu Ile Tyr Leu Leu Arg A~p Thr Pro Phe Ser Val Gly Glu 8er Gly Trp Asn Asn Leu Lys Lys Ala Met Val 8er Ala Trp Ile Syr 8er Asn Pro Glu Val Gly Leu Pro Leu Ala Gly Arg ~is Pro Phe Asn 8er SUBSTIT~TE SHEET (RU~E 26) Pro Ser Leu Lys Ser Val Ala Gln Gly Tyr Tyr Trp Leu Ala Met Ser Ala Lys S-r S-r Pro Asp Lys Thr L-u Ala S-r Ile Tyr Leu Ala Il-S-r ABP Lys Thr Gln Asn Glu Ser Thr Ala Ile Phe Gly Glu Thr Ile Thr Pro Ala Ser Leu Pro Gln Gly Ph- Tyr Ala Ph- Asn Gly Gly Ala m GGT ATT CAT CGT TGG CAA GAT AAA ATG GTG ACA CTG AAA GCT TAT 2053 Ph- Gly Ile H$s Arg Trp Gln Asp Lys M-t Val Thr L-u Lys Ala Tyr Asn Thr Asn Val Trp Ser g-r Glu Il- Tyr Asn Lys Asp Asn Arg Tyr Gly Arg Tyr Gln g-r His Gly Val Ala Gln Il- Val S-r Asn Gly g-r Gln Leu g-r Gln Gly Tyr Oln Gln Glu Gly Trp Asp Trp Asn Arg ~-t Gln Gly Ala Thr Thr Il- His L-u Pro Leu Ly- Asp Leu Asp g-r Pro AAA CCT CAT ACC TTA ATG CAA CGT GGA GAG CGT GGA TTT AGC aGA ACA 2293 Lys Pro Hi~ Thr Leu Met Gln Arg Gly Glu Arg Gly Phe Ser Gly Thr TCA TCC C-r~ GAA GGT CAA TAT GGC ATG ATG GCA TTC GAT CTT ATT TAT 2341 Ser g-r L-u Glu Gly Gln Tyr Gly M t M-t Ala Ph- A~p L-u Ile Tyr CCC GCC AAT CTT GAG CGT TTT GAT CCT AAT TTC ACT GCG AAA AAG AGT a 389 Pro Ala A~n Leu Glu Arg Phe A~p Pro A~n Ph- Thr Ala Ly~ Ly~ S-r Val Leu Ala Ala A-p A~n His L-u Il- Ph- Il- Gly g-r A~n Il- A~n g-r Ser A~p Ly~ Asn Ly~ A~n Val Glu Thr Thr L-u Phe Gln Hi~ Ala Il- Thr Pro Thr L-u Asn Thr L-u Trp Il- Asn Gly Gln Lys Il- Glu A~n M t Pro Tyr Gln Thr Thr L-u Gln Gln Gly Asp Trp Leu Il- Asp SUBSTITUTE SHEET (RULE 2~

WO 94125567 PCTtUS94tO4495 216112 j~

8er Asn Gly Asn Gly Tyr Leu Ile Thr Gln Ala Glu LYB Val Asn Val Ser Arg Gln His Gln Val Ser Ala Glu Asn Lyo Asn Arg Gln Pro Thr Glu Gly Asn Phe Ser Ser Ala Trp Ile Asp His Ser Thr Arg Pro Lys Asp Ala Ser Tyr Glu Tyr ~et V-l Phe Leu Asp Ala Thr Pro Glu Lys Met Gly Glu Met Ala Gln Lys Phe Arg Glu Asn Asn Gly Leu Tyr Gln Gll CTT CGT AAG GAT AAA GAC GTT CAT ATT ATT CTC GAT AAA CTC AGC 2869 Val L-u Arg LYB Asp Ly~ ABP Val His Ile Ile L-u Asp Lys Leu 8-r A n Val Thr Gly Tyr Ala Ph- Tyr Gln Pro Ala S-r Il- Glu ABP Ly~

Trp Il- LYB Ly~ Val Asn LYB Pro Ala Il- Val ~ t Thr H~B Arg aln LYB ABP Thr Leu Ile Val 8er Ala Val Thr Pro Asp Leu Asn Met Thr Arg Gln LYB Ala Ala Thr Pro Val Thr Ile Asn Val Thr Ile Asn aly Lys Trp Gln ger Ala Asp Lys Asn Ser Glu Val LYB Tyr Gln Val 8-r GGT GAT AAC ACT GAA CTG ACG TTT ACG AGT TAC m GGT ATT CCA CAA 3157 Gly ABP Asn Thr Glu Leu Thr Phe Thr ger Tyr Phe Gly Ile Pro Gln GAA ATC AAA CTC TCG CCA CTC CCT TGA m AATC ~ rGC '~ C~ ~C 3211 Glu Ile Lys Leu Ser Pro Lou Pro .ATT T~r~ T CTGAT~ATGC ~ CC~--.AGCC ~CGC~A 3271 CATT~CCCT ~.v...ATCA TT~rCCG-~r AAGCATTACC CA~-~ CATGAAGCTT 3331 .~'~GC~AT~ TTA.~.... GAAGGTGAAT ~rCr~ r CCTTACCACT TC~T~TA 3391 ATCAATTATC GCT~C~ CAGCATGCTA AAGATGGTGA ACAATCACTC AAATGGCAAT 3451 SUBSTITUTE SHEET (RULE 26) WO 94/25567 - PCTrUS94/04495 21~ 114-ATr~r~r~ ~n~ TTA Ar~CT~T~ ATATTGTTAA TT~rr~ T ~T~ TA 3511 r~C~ rC ACTCAC m T ATGATGTGGA TTTATAATGA AAAACCTCAA ~ CCC~AT 3571 TAACGTTAGC ATTT~r~ A~T~T~ TTGC~ ~G TTTTAATGCT GAACTTAATT 3631 TT~'GGG~G GCGAGGTATT G~ C~ C~GATAT GC~GCTCT GCr~GGTC 3691 AACTTGATCA ATTAGTGATC ACCG~C~AA ~ GCCGG AACACTCTTT TTTGATCAAA 3751 T~T~rGC AGT~ CG ATGGTTAGTA AAAACTGGAG TGCATTATTG ATGTACGATC 3871 AGA,~,,,~A ~CCC~TTAC CCTACTTTAA ACTTCGATAC TGAATTTCGC GATC~ 3931 r~ TGGC TTCGATTTAT r~GCG~ G AATATTATCA AGGAATTCC 3980 2) INFORMATION FOR SEQ ID NO 5 (i) SEQ~ENCE rU~T ~T~T -CTICS
(A) LENGT~ 998 amino acids (B) TYPE mino acid ~D) TOPOLOGY lin-ar ~ii) MOT~C~T~ TYP~ prot-in ~xi) SEQUENCE ~T~TPTION SEQ ID NO 5 t Ala Thr Ser Asn Pro Ala Phe Asp Pro Ly~ Asn L-u M-t Gln 8er lu Il- Tyr ~is Ph- Ala Gln Asn Asn Pro L-u Ala Asp Ph- S-r S-r sp Lys Asn S-r Il- Leu Thr Leu Ser Asp Lys Arg Ser Ile Net Gly A~n Gln S-r L-u Leu Trp Ly~ Trp Lys Gly Gly ger Ser Phe Thr Leu His Lys Lys L-u Ile Val Pro Thr Asp Lys Glu Ala 8er Lys Ala Trp ly Arg ger Ser Thr Pro Val Phe Ser Phe Trp Leu Tyr Asn Glu Lys ro Il- Asp Gly Tyr Leu Thr Ile Asp Phe Gly Glu LYB L-u Ile ger Thr Ser Glu Ala Gln Ala Gly Phe Lys Val Lys Leu Asp Phe Thr Gly Trp Arg Ala Val Gly V-l Ser Leu Asn A~n Asp Leu Glu Asn Arg Glu Net Thr Leu Asn Ala Thr Asn Thr Ser Ser Asp Gly Thr Gln Asp ger Ile Gly Arg 8er Leu Gly Ala Lys Val Asp Ser Ile Arg Phe Lys Ala SIJBSTITUTE SHEET (RU~E 26) ~161125 Pro Ser Asn Val Ser Gln Gly Glu Ile Tyr Ile Asp Arg Ile Met Phe Ser Val Asp Asp Ala Arg Tyr Gln Trp Ser ABP Tyr Gln Val Lys Thr Arg Leu Ser Glu Pro Glu Ile Gln Phe His Asn Val Lys Pro Gln L-u Pro Val Thr Pro Glu Asn Leu Ala Ala Ile Asp Leu Ile Arg Gln Arg eu Ile Asn Glu Phe Val Gly Gly Glu Lys Glu Thr Asn Leu Ala Leu lu Glu Asn Ile Ser Lys Leu Lys Ser Asp Phe Asp Ala Leu Asn Ile His Thr Leu Ala Asn Gly Gly Thr Gln Gly Arg His Leu Ile Thr A~p Lys Gln Ile Ile Ile Tyr Gln Pro Glu Asn Leu Asn Ser Gln Asp Lys Gln Leu Phe Asp Asn Tyr Val Ile Leu Gly A~n Tyr Thr Thr Leu ~et he Asn Ile Ser Arg Ala Tyr Val Leu Glu Lys Asp Pro Thr Gln Lys la Gln Leu Lys Gln Met Tyr Leu Leu Met Thr Lys ~is Leu Leu Asp Gln Gly Phe Val Lys Gly Ser Ala ~eu Val Thr Thr His ~is Trp Gly Tyr Ser Ser Arg Trp Trp Tyr Ile Ser Thr Leu Leu Met Ser Asp Ala Leu Lys Glu Ala Asn Leu Gln Thr Gln Val Tyr Asp Ser Leu Leu Trp yr Ser Arg Glu Phe Lys Ser Ser Phe Asp Met Lys Val Ser Ala Asp er Ser Asp Leu Asp Tyr Phe Asn Thr Leu Ser Arg Gln His Leu Ala Leu Leu Leu Leu Glu Pro Asp Asp Gln Lys Arg Ile Asn Leu Val Asn Thr Phe Ser His Tyr Ile Thr Gly Ala Leu Thr Gln Val Pro Pro Gly Gly Lys Asp Gly L-u Arg Pro Asp Gly Thr Ala Trp Arg His Glu Gly sn Tyr Pro Gly Tyr Ser Phe Pro Ala Phe Lys Asn Ala Ser Gln Leu le Tyr Leu Leu Arg Asp Thr Pro Phe Ser Val Gly Glu Ser Gly Trp SUB~TITUTE SHEET (RlJLE 26) W 0 94l25567 PCTrUSg4/04495 q ~6~5 116 ~

sn Asn Leu Lys Lys Ala Met Val Ser Ala Trp Ile Tyr Ser Asn Pro Glu Val Gly Leu Pro Leu Ala Gly Arg Hio Pro Phe Asn Ser Pro Ser Leu Lys Ser Val Ala Gln Gly Tyr Tyr Trp Leu Ala Met Ser Ala Lys er Ser Pro Asp Lys Thr Leu Ala Ser Ile Tyr Leu Ala Ile Ser Asp ys Thr Gln Asn Glu Ser Thr Ala Ile Phe Gly Glu Thr Ile Thr Pro la Ser Leu Pro Gln Gly Phe Tyr Ala Phe Asn Gly Gly Ala Phe Gly Ile H$8 Arg Trp Gln Asp Ly~ Met Val Thr L-u Lys Ala Tyr Asn Thr Asn Val Trp Ser Ser Glu Ile Tyr Asn Lys Asp Asn Arg Tyr Gly Arg yr Gln 8er His Gly Val Ala Gln Ile Val 8-r Asn Gly S-r Gln L-u 6~5 650 655 -r Gln Gly Tyr Gln Gln Glu Gly Trp Asp Trp Asn Arg M-t Gln Gly la Thr Thr Ile Hls L-u Pro Leu Lys Asp L-u Asp Ser Pro Lys Pro His Thr L-u Met Gln Arg Gly Glu Arg Gly Ph- S-r Gly Thr S-r S-r L-u Glu Gly Gln Tyr Gly Met Met Ala Phe Asp Leu Ile Tyr Pro Ala sn Leu Glu Arg Phe Asp Pro Asn Phe Thr Ala Lys Lys S-r Val L-u la Ala Asp Asn Hls Leu Il- Ph- Il- Gly S-r Asn Ile Asn Ser S-r sp Lys Asn Lys Asn Val Glu ~hr Thr L-u Phe Gln His Ala Il- Thr Pro Thr Leu Asn Thr Leu Trp Il- Asn Gly Gln Lys Il- Glu Asn M t Pro Tyr Gln Thr Thr Leu Gln Gln Gly Asp Trp L-u Ile Asp Ser Asn ly Asn Gly Tyr Leu Il- Thr Gln Ala Glu Lys Val Asn Val Ser Arg ln His Gln Val Ser Ala Glu Asn Lys Asn Arg Gln Pro Thr Glu Gly sn Ph- Ser S-r Ala Trp Il- Asp His Ser Thr Arg Pro Lys Asp Ala SUB~TITUTF ~5EET (RULE 26) WO 94/25567 2 ¦ 61~ 2 5 PCT/US94/04495 Ser Tyr Glu Tyr Met Val Phe Leu Asp Ala Thr Pro Glu Lys Met Gly Glu ~et Ala Gln Lys Phe Arg Glu Asn Asn Gly Leu Tyr Gln Val Leu rg Ly~ Asp Lys Asp Val His Ile Ile Leu Asp Lys Leu Ser Asn Val hr Gly Tyr Ala Phe Tyr Gln Pro Ala Ser Ile Glu Asp Lys Trp Ile Lys Lys Val Asn Lys Pro Ala Ile Val Met Thr His Arg Gln Lys Asp Thr L-u Ile VA1 Ser Ala Val Thr Pro Asp L-u Asn Met Thr Arg Gln Lys Ala Ala Thr Pro Val Thr Ile Asn Val Thr Ile Asn Gly Lys Trp ln Ser Ala ABP Lys Asn ger Glu Val Lys Tyr Gln Val Ser Gly Asp sn Thr Glu Leu Thr Phe Thr Ser Tyr Phe Gly Ile Pro Gln Glu Il-Lys Leu Ser Pro LQU Pro995 ~2) INFORMATION FOR SEQ ID NO 6 (1) SBQ~BNCB ~R~T~TBRI8TICg A) L D ~TH 21 base pairs B) TYP~ nucleic acid C) STRAND~DNBgS s~ngle D) TOPOLOGY lin-ar (ii) MOT~CI~R TYPE DNA (g-nomic) (i~l) n~r~.A~.~CAL NO
(Iv) ANTI-SFNSE NO

(xi) SBQ~ENCB nT~-erPTPTION SEQ ID NO 6 CA~ ~NC ~T~Y~YCC N 21 (2) INFORMATION FOR SEQ ID NO 7 (i) SBQ~FNCB r~rTERISTICS
(A) LBNGTH 20 base pairs (B) m E nucleic acid (C) STRANDBDN~SS single (D) TOPOLOGY linear (ii) ~OTT~C~T~ TYPE DNA (g-nom~c) ~iii) n~r~.A~.lCAL NO

SUBSTITUTE SHEET tRULE 26) W O 94/25567 PCTrUS94/04495 2161.125 ~iv) ANTI-SBNSE: NO

(xi) SBQVENCE DESCRIPTION: SEQ ID NO:7:
CACTTCGCNC ~ T~ ~Cc , 20 (2) INFORMATION FOR SEQ ID NO:8:
(i) SBQVBNCE CHARACTERISTICS:
(A) LBNGTH: 20 base pairs (B) TYPE: nuclelc acid (C) STP~n~NESS: singl-(D) TOPOLOGY: linear (ii) MOT-RC~TR TYPE: DNA (g-nomic) (iii) n~r~,A~.lCAL: NO
(iv) ANTI-SBNSB: NO

(xi) SBQVBNCB ~ C Y~ ON: 8BQ ID NO:8:
CACTTCGCNC ~ ~CC 20 (2) INFORMATION FOR SBQ ID NO:9:
(i) SBQVBNCB ~U~D~TBRISTICS:
(A) LENGTH: 20 base pair~
(B) TYPB: nucleic acid (C) STRANDBDNBgS: single (D) TOPOLOGY: linear (ii) MOT-RC~T-R TYPE: DNA (g-nomic) (iii) n~rG.A~.~CAL: NO
(iv) ANTI-SBNSB: NO

(xi) SBQVBNCB DBSCRIPTION: SBQ ID NO:9:
CA~-~ ~N~ r~TCC 20 (2) INFORMATION FOR SBQ ID NO:10:
(i) SBQUBNCE rU~--YDrT~TSTICS
(A) LBNGTH: 20 bas- pairs ~B) TYPE: nucleic acid (C) STRAND_DNBSS: singl-(D) TOPOLOGY: lin-ar (ii) ~OT-Rcc~-R TYPE: DNA (9_ ~ 'c) ( iii ) A~rO~A~ ~r~T~: NO
(iv) ANTI-SBNSB: NO

SUBSTITUTE SHEET (RULE 26~

WO 94/25567 2l6ll~ PCT/US94/04495 - 119 _ . ~, ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CAu,.CG~NC ~T~CCC 20 (2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUBNCB rU~CTERISTICS:
(A) LBNGTH: 20 base pair~
(B) TYPE: nucleic acid (C) STRAND~DNESS: ~ingle (D) TOPOLOGY: linear (ii) MOTRC~nR TYPE: DNA (genomic) (iii) ~rO~n~.lCAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUBNCB ~ ON: SBQ ID NO:ll:
CACTTCGCNC A~T~CC 20 (2) INFORMATION FOR SEQ ID NO:12:
(i) S_QUFNCB rU~T~T-CTICS:
(A) TFNGT~: 20 ba~e pairs (B) TYPB: nucl-ic acid (C) STRANDRDNFSS: ~lngle (D) TOPOLOGY: lin-ar (ii) M~rRC~R TYP~: DNA (genomic) (iii) h ru.A~.lCAL: NO
(iv) ANTI-SBNSB: NO

(xi) SBQ~BNCB D~C~lv~loN SBQ ID NO:12:
CA~--CG~C A~C~CCC 20 (2) INFORMATION FOR SEQ ID NO: 13:
(i) SBQ~BNC8 rU~CTERISTICS:
~A) ~NGTH: 20 ba~e pair~
(B) TYP~: nucl-ic acid (C) SSRAND_DN_SS: Bingle ~D) TOPOLOGY: lin-ar ~ii) MOTRC~R TYPE: DNA ~genomic) (iii) nYrO~n~lCAL: NO

(iv) ANTI-S_NS_: NO

(xi) SEQ~NCF r~c~DTDTIoN: SEQ ID NO: 13:

SUBSTITUTE SHEET ~RULE 26~

216112~ - 120 -CA---~CG~NC ~ TCC 20 (2) l~OR~ATION FOR SEQ ID NO:14:
(i) SFQVRNCR ~U~rTERISTICS:
(A) LENGTH: 20 ba~e p~ir~
(B) TYPE: nucleic acid (C) ST~ S: single (D) TOPOLOGY: linear (ii) MOT-RCUTR TYPE: DNA (genomic) (iii) hYrO,~h~lCAL: NO
(lv) ANTI-SRNSR: NO

(xi) SEQ~ENCE n~r~TPTION: SEQ ID NO:14:
CACTTCGCNC ~ T~CCC 20 (2) ~NFORMATION FOR SFQ ID NO:15:
(i) SFQ~ENCR ~U~TFRISTICS:
(A) LENGTH: 21 ba~e pairs (B) TYPE: ~ c acid (C) STRANDEDN~SS: ~ingle (D) TOPO OGY: lin-~r (ii) MOT-R~ R TYPE: DNA (genomic) (~ii) h~rO~A lCAL: NO
(iv) ANTI-SENSR: NO

(xi) SRQUENCR DR-SCT7TPTION: SEQ ID NO:15:

(2) INFORMATION FOR S_Q ID NO:16:
(i) SEQ~ENCE ~U~P~TERISTICS:
(A) LENGTn: 21 base pairs (B) TYPE: ~ucle~c acid (C) STRANDRDNESS: ~ingle (D) TOPOLOGY: lin-ar (li) MoTRc~T-R m_: DNA (genomic) (iii) n~rO,A~.lCAL: NO
(iv) ANTI-S_NSE: Y~S

(xi) SEQUENCE DR.5r~TPTION: S_Q ID NO:16:

SIJBSTITUTE SHEET (RULE 26) ~161125 (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQVENCE CHARACTERISTICS:
(A) ~ENGTH: 20 base pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: Q ingle (D) TOPOLOGY: linear (ii) MOT-T~C~TR TYPE: DNA (genom~c) (iii) ~YrG.~,lCAL: NO
(iv) ANTI-SENSE: YES

(xi) SEQ~LNCE DTCc~pTpTIoN: SEQ ID NO:17:
TTC~VC~ ~ ~-.~GG~--,C 20 (2) INFORMATION FOR SEQ ID NO:18:
(i) SBQUBNCB rU~D~CTBRISTICS:
(A) LENGTH: 20 base pairs (B) TYPB: nucl-ic acid (C) STRANDBDNBSS: single (D) TOPOLOGY: lin-ar ( ii ) MOT.~T.~ TYPE: DNA (, lc) (iii) ~rO~A~l~AL: NO
(iv) ANTI-SBNSE: YBS

(xi) SBQ~BNCB DBSCRIPTION: S8Q ID NO:18:

(2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQ~ENCE r~T~TT~DT~TICS:
(A) LENGTH: 20 bas- pairs (B) TYPE: nucleic acid (C) STRANDBDNESS: singlQ
(D) TOPOLOGY: lin-ar (ii) MOT-TeC~T-TC TYPE: DNA (genomic) (iii) ~ru.A~,~CAL: NO
(iv) ANTI-SBNSE: YES

(xi) SEQVENCE DESCRlr.lC.: SEQ ID NO:l9:

TTC~T7~rNG ~.~.G~-.. C 20 (2) INFORMATION FOR SEQ ID NO:20:

SUBS 1 l-l IJT~ SHEET (RULE 26~

21611~

(i) SEQ~ENCE CHARACTERISTICS:
(A) LENGTH: 20 base pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPODOGY: linear (ii) MOT-RC~T-R TYPE: DNA (genomic) (iii) nY~O~A~-~CAL: NO
(iv) ANTI-SENSE: YES

(xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:20:
TTGAARCCNG ~ GCG~-.. C 20 (2) I~OK~ATIoN FOR SEQ ID NO:21:
~i) SBQ~BNCB rU~D~TBRISTICS:
(A) LBNGTH: 20 ba~e pair~
(B) TYP~: nucl-ic acid (C) STRANDBDNBSS: ~ingl-(D) TOPOLOGY: lin-ar (ii) MOT-~C~T-~ TYPE: DNA (genomic) (iii) h~G.~.lCAL: NO
(iv) ANTI-SBNSB: YBS

(xi) SBQUBNCB DRC~TPTION: SEQ ID NO:21:
TTGAARCCNG ~-.a~GC~' 20 ~2) INFORMATION FOR SEQ ID NO:22:
(i) SBQUBNCB CU~CTBRISTICS:
'A' LENGTH: 20 base pair~
~B~ TYPB: nucleic ~cid C~ STRANDEDNESS: ~ingle ,D~ TOPO~OGY: lin-ar (ii) MOT-RC~T-~ TYPE: DNA (genomic) (iii) n~u.~.~AL: NO
(iv) ANTI-SENSE: YES

(xi) SEQ~ENCB ~R-~r~TPTION: SEQ ID NO:22:

(2) INFORMATION FOR SEQ ID NO:23:
(i) ~u~_~ ~u~ ERISTICS:
(A) LENGTH: 20 ba~e pair~

SUBSTITUTE ~SHEET ~RIJLE 26) ~16112~ -(B) TYPE: nucleic acid (C) STD~N~DNBSS: single (D) TOPOLOGY: linear (ii) MOrRCCrR TYPB: DNA (genomic) (iii) nYrO.n~.lCAL: NO
($v) ANTI-SBNSE: YES

(Xi) S~ ~rR D~Q~DTPTION: SEQ ID NO:23:
TTGAARCCNG ~ .GC~.C 20 (2) INFORMATION FOR SEQ ID NO:24:
(i) SBQ~BNC8 ~U~D~T~vTSTICS:
(A' LENGTH: 20 base pairs (B TYPB: nucleic acid (C STRANDBDNB8S: ~ingle (D, TOPOLOGY: lin-ar (ii) MOT-~Cr-R TYP~: DNA (, tc) (iii) ~YrG-~r~r: NO
(iv) ANTI-SBNSB: Y8S

(xi) SBQ~8NCB D-C~ ON: S_Q ID NO:24:
TTC~DC~NG ~ GC~C~.C 20 (2) INFORMATION FOR SBQ ID NO:25:
(i) SBQ~BNCB rR~V~CT8RISTICS:
~A) LENGTH: 18 ba~e pairs B) TYPB: nucleic acid C) STRAND D N8SS: single ~D) TOPOLOGY: lin-ar ($i) ~orR~ -R TYPE: DNA (g_ ~c) (iii) ~YrO,A~CAL: NO
(iv) ANTI-S_NSE: NO

(xi) SBQUBNC_ DBSCRIPTION: SBQ ID NO:25:

(2) INFORNATION FOR SEQ ID NO:26:

(i) SRQ~BNCB r~V~T~DTSTICS:
(A) L8NGTH: 18 basQ pair~
(B) TYPE: nucleic acid (C) STPU~NDBDN8SS: ~ingle SUBSTITUTE SHEET (RULE 26) WO 94125567 ` PCT/US94/04495 216112~
~D) TOPOLOGY: linear ($i) M~T-D~C~D~ TYPE: DNA (genomic) (~ii) AYrO,Ah.lCAL: NO
(iv) ANTI-SBNSB: NO

(xi) SEQUENC~ DRCrpTpTIoN: SBQ ID NO:26:
GGl.~ ~G TNGAYAGY 18 (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE rU~rT~DT~TICS:
(A) L~NGTA: 18 base pair~
(B) TYPE: nucl-ic ac~d (C) STRANDFDN~SS: singl-(D) TOPOLOGY: lin-ar ($i) MOT~C~D TYPE: DNA ~g-nomic) (lii) A~rG.A~lCAL: NO
(~v) ANTI-SBNSE: YBS

(xi) S~Q~NC~ DR~r~TPTIoN: SBQ ID NO:27:

(2) INFOR~ATION FOR SEQ ID NO:28:
(i) S~QUFNC~ ~r-DDT~TICS:
~A) L~NGTA: 18 bace pa~rc B) TYP~: nucl-ic acid C) STKANDBDN~SS: ~ingle ~D) TOPOLOGY: lin-ar ($i) MOTDC~ TYP~: DNA (genomic) (lii) h~ru~A lCAL: NO
(~v) ANTI-S~NSo: Y~S

~xi) S~Q~BNCE ~c~-~ ON: SBQ ID NO:28:
K~.~.~NACY TTNGCNCC 18 (2) INFOKMATION FOR S~Q ID NO:29:
(i) SFQVFNC~ ~U~ T~RISTICS:
(A) LFNGl~A: 17 base pair~
(B) TYPE: nucleic acid (C) STKAND~DN~DSS: ~ingle (D) TOPOLOGY: linear SUBSTI~UTE SHEET (RULE 26) WO 94/25567 ~161125 PCTIUS94/04495 (ii) MOT~C~T~ TYPE: DNA (geno~ic) (ii~) ~YrG.~.lCAL: NO
(~v) ANTI-SBNSE: YBS

(xi) SEQVENCB DR-e~TPTIoN SEQ ID NO:29:
GAGTCNACYT , nvCGCC I7 (2) INFORMATION FOR SEQ ID NO:30:
(i) SBQ~ENCB rU~CTR~TSTICS:
(A) LBNGTH: 17 base pairs (B) TYPE: nuel-ic aeid (C) STRANDBDNBSS: single (D) TOPOLOGY: lin-~r ( i ~ ) MO~ ~C~T ~ TYPB: DNA (, ~e) ($ii) ~rv~n~lcAL: NO
(lv) ANTI-SBNSB: YBS

(xl) SBQ~BNCB DBSCRIPTION: SBQ ID NO:30:
GAGTCNACYT T~a~c 17 (2) INFORMATION FOR SEQ ID NO:31:
(i) SBQV8NCB rU~D~TBRISTICS:
(A) L8NGTH: 17 b-s- pa~rs (B) TYPB: nuel-ie aeid (C) STRANDBDNBSS: single (D) TOPOLOGY: l~n-ar ( ii ) MOT-~C~T-~ TYPE: DNA (~_ ~e) rv.~.~AL: NO
(~v) ANTI-SBNSE: YBS

(xi) SEQ~_NCB DBSCRIPTION: SEQ ID NO:31:

(2) INFORMATION FOR SEQ ID NO:32:
~i) SBQVBNCB r~TBRISTICS:
(A) ~BNGTH: 17 base pairs (B) m E: nueleie aeid (C) STRANDEDNBSS: single (D) TOPO~OGY: linear (ii) MOLEC~LB TYPE: DNA (genomic) SUBSTITUTE SHEET (RULE 26) 216112~ 126-(iii) nYrG~ CAL: NO
(iv) ANTI-SENSE: YES

(xi) SEQUENCB DRCr~TpTIoN: SEQ ID NO:32:
GAGTCNACYT .~CCCC 17 (2) ~ N~ OR~ATION FOR SEQ ID NO:33:
~Qu~d~ CHARACT_RISTICS:
~A) LBNGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNBSS: 8ingl-(D) TOPOLOGY: lin-ar (ii) MOT-~CUT-~ TYP_: DNA (genomic) - (lii) ~rO.~,~CAL: NO
(iv) ANTI-8BNg_: YBS

(xi) SBQUBNCB DR~rT~TPTIoN SBQ ID NO:33:
GAGTCNACYT ~CGCC 17 (2) INFORMATION FOR SEQ ID NO:34:
(i) SBQ~BNCB r~T~TBRISTICS:
(A) LBNGTH: 17 bas- palra (B) TYPB: nucl-$c acid (C) ST~T~N~SS: singl-(D) TOPOLOGY: lin-ar (ii) ~OT-~C~T-T~ TYPE: DNA (g-nomic) (iii) ~ro~n~cAL: NO
(iv) ANTI-SBNSE: YBS

(xi) SEQ~ENCB DT~r~TPTION: S_Q ID NO:34:

(2) INFORMATION FOR SEQ ID NO:35:
(i) SBQ~FNCB ru~T~ rTT~ T~TICS:
A LBNGT_: 17 bas- pair~
B TYPB: nucl-ic acid .C~ STRANDBDNBSS: singl-~D TOPOLOGY: lin-ar (ii) ~OT-~ -R TYPB: DNA (g-n~mic) (iii) h~r~,A~,lCAL: NO

SUBSTITUTE SHEET (RULE 26) - 2161l2s (iv) ANTI-SBNSE: YES

~xi) SEQ~ENCB D~CrpTpTIoN: SEQ ID NO:35:
GAGTCNACYT -.~.CC 17 (2) INFORMATION FOR SFQ ID NO:36:
(i) SEQVBNCE rU~TF~TSTICS:
(A) LBNGT~: 17 ba~e palr~
(B) TYPE: nucl-ic acid (C) STRANDBDNBSS: single (D) TOPOLOGY: lin-ar (li) MOT~ T-~ TYPB: DNA (~ c) (lil) ~rO.A~.lCAL: NO
(iv) ANTI-SBNSE: YBS

(xl) SBQ~BNCF DBSCRIPTION: SEQ ID NO:36:
GAGTCNACYT .~CC~C 17 (2) INFORMATION FOR SEQ ID NO:37:
(i) SEQ~BNCE rU~P~rT~TSTICS:
(A) LBNGTH: 48 bas- palr-(B) TYPB: ~lcl-~c acld (C) STRANDBDNBSS: ~ingle (D) TOPOLOGY: lln-ar (il) MOT-~C~T-~ TYPE: DNA (g-nomlc) (lli) h~ru,A~.lCAL: NO
(lv) ANTI-SBNSB: NO

(xi) SBQUBNCB D~rDTPTION: SEQ ID NO:37:
GCr~GTTT CT~ a~ r~T~GCC GATA~....... ~. TTTACTGC 48 (2) INFORMATION FOR SBQ ID NO:38:
(1) SBQ~BNCB ~T'~u-~ CS:
(A) L~NG~H: 37 bas- palr~
(B) TYPB: nucl-ic acld (C) STRA~D~DNBSS: ~lngle (D) TOPOLOGY: lin-ar (ii) MOT-~C~T-~ TYPE: DNA (genomic) (lil) n~rO~ ~AL: NO
(~v) ANTI-SBNSE: NO

SUBSTITUTE SHEET tRULE 26) 21~1125 (x$) SEQ~BNCB DBSCRIPTION: SEQ ID NO:38:
GCGC~.. ATA ~rGCGr~T~T GGCr~Cr~GC AATCCTG 37 ~2) INFORMATION FOR SBQ ID NO:39:
(i) SEQUBNCB CHARACTERISTICS:
(A) ~BNGTH: 6519 b-s- pairs (B) TYPE: nucleic acid (C) ST~Nn-DNESS: single (D) TOPO~OGY: linear (ii) MOT-RC~T~T~ TYPB: DNA (genomic) (iii) ~rO-A~-lC~L: NO
(iv) ANTI-SENSE: NO

(ix) FBATURB:
(A) NAMB~BY: CDS
(B) T-OCATION: 3238..6276 (xi) SEQ~ENCE DES~KI~.lON: SBQ ID NO:39:
GGAATTCCAT CACTCAATCA TTAAATTTAG G~ C~T GGG ~TCAG CGTTATGACA 60 AATTTAATGA ~ CGr~TT G~--~ ACTG TT~Cr~qCG TTTCT~C' C ~ T~T 120 GCC~T~TTT C~...~ACTG CACTTG Q AT GA Q-.-~GGG CTATTAT Q G CGC~.ATAA 180 CGCGATGG Q GCr~rr~ A.C~.G~-ATT TGATC AAA AAT GATGC AGTr~ T 240 TTACCATTTT Gr~r~aT~ ACC QTTAGC AGA~ A Tr~T~ ACTr~T~CT 300 AACGTTAT C~T~rGTA G QTTATGGG ~Cr~T CTTTTATGGA AATG~C 360 TGGTAGTAGC TTTACTTTAC ~T~ rT GA..~. CCC ~Cr~T~ AAGCATCTAA 420 AGCATGGGGA CGCTCAT A CCCCC~.--- CTCA~ GG CTTTACAATG ~ arCC~T 480 TGATGGTTAT CTTACTATCG A..CG~AGA AAAA CATT Tr~rr~TG AGGCTCAGGC 540 AGG~...AAA GTAAAATTAG ATTTCACTGG ~-.GGC~.~- GTGGGAGT CTTT~T~ 600 CGAT~-rTGAA AATC~' TGACCTTAAA TGr~rr~T A~.~.~-.G ATGGTA CA 660 ~ TT GGGC~--~-- TAG~-G~-~AA AGTCGATAGT A-~C~ A ~CGC~-.~C 720 TAATGTGAGT CAGGGTGAAA TCTATATCGA CCGTATTATG ,,.- ~.CG ATGATGCTCG 780 CT~r~TGG TCTGATTATC AAGT~ r .~'G~..ATCA GAAC GAAA TTCAATTTCA 840 CAACGTAAAG Cr~ ~T~r CTGT~ rC TGAAAATTTA GCGGC~-~TTG ATCTTATTCG 900 CCAACGTCTA ATTAATGAAT .-~.CG~AGG TC~ G ~ rCTCG CATT~ 960 GAATATCAGC AAATTAAAAA GTGATTTCGA ~-~---AAT ATTCACACTT T~ TGG 1020 TG~rGr~ GGr~ TC TGATCACTGA T~ ~TC ATTATTTATC ~rr~ 1080 SUBSTITUTE SHEET (RULE 26~

TCTTAACTCC r~'~T'~C AACTATTTGA TAATTATGTT ATTTTAGGTA ATT~C~r 1140 ATTAATGTTT AATATTAGCC ~G~ATGT GCTG- '''' GATCCr'-~C ~ rCCGr~ 1200 ~T'~-C~G ATGTACTTAT TAATG'~ GCA m ATTA GATCAAGGCT TTGTTAAAGG 1260 GA~.~...A GTG'~rCC ATCACTGGGG ~T~GTTCT C~--G~-G~- ATATTTCCAC 1320 GTTATTAATG TCTGATGCAC T~ GC r~-r ~-- A CAAGTTT ATGATTCATT 1380 A~ AT TCACGTGAGT TTAAAAGTAG TTTTGATATG AAAGTAAGTG CTG'T~GCTC 1440 TGATCTAGAT TATTTCAATA CCTTAT CG Cr~ TTTA GCCTTATTAT T~CT~- ~CC 1500 TGATGATCAA AAGCGTATCA ACTTAGTTAA TACTTTCAGC CATTATATCA ~,G~C~-~TT 1560 ~'CGr'~-TG Cr~CGa~G GT~-'TGG TTT~rGCCCT GATGGTACAG cATGG5r~ 1620 T-~CC~r TA.~CGGG~. A~ ,,.CCC AGC~ AAA AA~GC~ C AGCTTATTTA 1680 TTTATTACGC C~T~C~T TTTCAGTGGG TGAAAGTGGT TGC'~T~aCC Ta'~ C 1740 GA~ ..~A GC~.O~ATCT ACAGTAATCC AGAAGTTGGA TT~Ca~-..G r---~ -- 1800 CC~-..-AAC TCA~---..C~. TAAAATCAGT CGCTCAAGGC TATTACTGGC TTGCCATGTC 1860 TGC~ TCA .CGC~.~ATA ''~'~TTGC ATCTATTTAT ~-,,GC~ATTA GTG'T~'~ 1920 AA TCAACTGCTA ----~A Q AACTATTACA CCAGC~.~. TACCTCAAGG 1980 TTTCTATGCC TTTAATGGCG ~.~-,,.,~4 TATTCATCGT T -~'-'T~ AAATGGTGAC 2040 ACTGAAAGCT T~T~'Cr' A.~.,,~,C ATCTGAAATT T-T~ 'T~'rCGTTA 2100 .aGCC~..AC CAAAGTCATG ~.~.cac. `A AATAGTGAGT AA.GG~-CGC AG~,,,~ACA 2160 CCGCT~TCAG r~ TT GGGATTGGAA TAGAATGCAA CCGG~-r~ ATTCACCT 2220 -~C~ AAA GACTTAaACA GTCCT'''~C Tr~T~CTTA ATGCAACGTG C'-'GCGTGG 2280 ATTT~GCC~' ACATCATCCC TTGAAGGTCA ATATGGCATG ATGGCATTCG ATCTTATTTA 2340 .CCCGC ~ T CTTGAGCGTT TTGATCCTAA TTTCACTGCG ''''~-TG TATT'-CCGC 2400 TGATAAT Q C TTAATTTTTA TTGGTAG Q A T-T-~T~ - T AGTC~T-~ AT~ TGT 2460 TC'''-C'CC TTATTC QAC ATGC Q TTAC TCr~ TTA ~T'~CCTTT GGATTAATGG 2520 - -~'T- C'~ TGC CTTAT QAAC AA Q CTT QA QAGGTGATT GGTTAATTGA 2580 T'CC~TGGC AATGGTTACT TAATTACT Q A~C'~''''' GTAAATGTAA ~ 2640 T Q G~,,, `A acc- ~T~ AAAATCGC Q ~Ccr~r~ G~ TTTA G~.C4a~ATG 2700 GATCaAT Q C AG Q CTCGCC Cr~ ~GC Q GTTATGAG TATA.a~.~-. TTTTAGATGC 2760 C'-~rCTaAA AAAATGGGAG AGATGGr~Ca AAAA--CC~- C'~T~TG GGTTATAT Q 2820 G~...-..C~. ' - - 'T'''C ACGTT Q TAT TA..~AT AAACT Q G Q ATGT'~GGG 2880 ATA~C~ TATr~-Cr~G Q T Q ATTGA A~'-~TGG ATr~ 'GC TT~'T~'~C 2940 SUBSTITUTE SI~EET (RULE 26) W O 94/25567 ~CTrUS94/04495 TGCAATTGTG ATGACTCATC ~ cAcT~rsATT GTCAGTGCAG TTACACCTGA 3000 TTT~T~TG A~.CGC~~C~ ~C~ACC ATCAATGTCA CGATTAATGG 3060 CAAATGGCAA .~ ATA ~ T~GTGA AGT~T~T CA~...-.G GTc~T~r~ 3120 TGAACTGACG TTTACGAGTT A~...G~.AT TCr~r~a~ ATCAAACTCT CGCCACTCCC 3180 TTGATTTAAT r~a~ rG ~ ..... GC~. C~....... AT TTGr~ TCTGATT 3237 ~et Leu Ile Ly~ Asn Pro Leu Ala His Ala Val Thr Leu Ser LQU Cys l 5 10 15 Leu S-r Leu Pro Ala Gln Ala L-u Pro Thr Leu S-r His Glu Ala Phe Gly Asp Ile Tyr Leu Ph- Glu Gly Glu Leu Pro Asn Thr L-u Thr Thr 8-r Asn A~n Asn Gln L-u S-r Leu 8-r Lys Gln Hi~ Ala Ly~ Asp Gly GAA CAA TCA CTC AAA TGa CAA TAT CAA CCA CAA GCA ACA TTA ACA CTA 3477 Glu Gln 8-r Leu Ly~ Trp Gln Tyr Gln Pro Gln Ala Thr L-u Thr L-u AAT AAT ATT ~TT AAT TAC CAA GAT GAT AAA AAT ACA GCC ACA CCA CTC 3525 A~n Asn Il- Val Asn Tyr Cln A~p A~p Ly~ Asn Thr Ala Thr Pro L-u Thr Ph- ~-t ~-t Trp Il- Tyr Asn Glu Ly~ Pro Gln Ser 8-r Pro L-u Thr Leu Ala Ph- Lys Gln Asn Asn Ly~ Il- Ala Leu Ser Ph- A~n Ala Glu L-u A~n Ph- Thr Gly Trp Arg Gly Il- Ala Val Pro Ph- Arg A~p ~-t Gln Gly Ser Ala Thr Gly Gln Leu A~p Gln Leu Val Ile Thr Ala CCA AAC ~ GCC GGA ACA CTC TTT TTT GAT CAA ATC ATC ATG AGT GTA 3765 Pro A~n Gln Ala Gly Thr Leu Ph- Ph- Asp Gln Il- Il- ~et 8-r V~l Pro Leu Asp Asn Arg Trp Ala Val Pro Asp Tyr Gln Thr Pro Tyr Val Asn Asn Ala Val Asn Thr Met Val Ser LYB Asn Trp Ser Ala Leu Leu SUBSTlTUTE SHEET (RULE 26) Met Tyr Asp Gln M-t Phe Gln Ala Hi~ Tyr Pro Thr Leu Asn Phe Asp Thr Glu Phe Arg ABP Asp Gln Thr Glu Met Ala Ser Ile Tyr Gln Arg Phe Glu Tyr Tyr Gln Gly Ile Arg Ser Asp Lys Lys Ile Thr Pro Asp ~et Leu Asp Lys His Leu Ala Leu Trp Glu Lys Leu Val Leu Thr Gln His Ala Asp Gly Ser Ile Thr Gly Lys Ala Leu Asp His Pro A~n Arg r~ CAT TTT ATG AAA GTC GAA GGT GTA TTT AGT GAG GGG ACT CAA AAA 4149 Gln His Ph- ~et Lys Val Glu Gly Val Ph- 8er Glu Gly Thr Gln Lys GCA TTA CTT GAT GCC AAT ATG CTA AGA GAT GTG GGC AAA ACG CTT C-~-r 4197Ala L-u L-u Asp Ala A~n M-t L-u Arg Asp Val Gly Lys Thr Leu L-u Gln Thr Ala Il- Tyr Leu Arg 8-r Asp 8er L-u 8er Ala Thr Asp Arg Lys Ly~ Leu Glu Glu Arg Tyr L-u Leu Gly Thr Arg Tyr Val Leu Glu Gln Gly Phe Thr Arg Gly 8er Gly Tyr Gln Il- Ile Thr His Val Gly Tyr Gln Thr Arg Glu Leu Phe Asp Ala Trp Ph- Il- Gly Arg His Val L-u Ala Lys Asn Asn L-u Leu Ala Pro Thr Gln Gln Ala ~et Met Trp Tyr Asn Ala Thr Gly Arg Il- Phe Glu Lys Asn Asn Glu Ile Val Asp Ala Asn Val Asp Ile Leu Asn Thr Gln Leu Gln Trp Met Ile Lys 8-r Leu Leu M t Leu Pro Asp Tyr Gln Gln Arg Gln Gln Ala Leu Ala Gln Leu Gln 8er Trp Leu Asn Lys Thr Ile Leu 8er 8er Lys Gly Val Ala SUB~TITU~ SHEET (RU~E 26) 216112~

Gly Gly Phe Lys Ser Asp Gly Ser Ile Phe His His Ser Gln His Tyr Pro Ala Tyr Ala Lys Asp Ala Phe Gly Gly Leu Ala Pro Ser Val Tyr Ala Leu Ser Asp Ser Pro Phe Arg Leu Ser Thr Ser Ala His Glu Arg Leu Lys ABP Val Leu Leu Lys Met Arg Ile Tyr Thr Lys Glu Thr Gln Ile Pro Val Val Leu Ser Gly Arg Hi~ Pro Thr Gly Leu His Lys Ile GGO ATC GCG CCA TTT AAA TGG ATG GCA TTA GCA GGA ACC CCA GAT aGC 4917 Gly Il- Ala Pro Ph- Lys Trp Met Ala L-u Ala Gly Thr Pro A~p Gly Ly~ Gln Lys L-u Asp Thr Thr L-u 8-r Ala Ala Tyr Ala Lys L-u A~p Asn Lys Thr His Phe Glu Gly Ile Asn Ala Glu g-r Glu Pro Val Oly Ala Trp Ala M-t Asn Tyr Ala Ser Met Ala Il- Gln Arg Arg Ala g-r Thr Gln Ser Pro Gln Gln Ser Trp L-u Ala Il- Ala Arg Gly Ph- 8-r Arg Tyr Leu Val Gly Asn Glu Ser Tyr Glu Asn Aan Asn Arg Tyr Gly Arg Tyr Leu Gln Tyr Gly Gln Leu Glu Ile Ile Pro Ala Asp Leu Thr Gln Ser Gly Ph- Ser Hi~ Ala Gly Trp Asp Trp Asn Arg Tyr Pro Gly Thr Thr Thr Il- His Leu Pro Tyr Asn Glu Leu Glu Ala Lys Leu Asn Gln Leu Pro Ala Ala Gly Il- Glu Glu Met Leu Leu Ser Thr Glu Ser S~BST~TUT~ SHE~T (RULE 26~

WO 94t25567 21 61~ 2 S PCTtUS94/04495 Tyr Ser Gly Ala Asn Thr Leu Asn Asn Asn Ser Met Ph- Ala Met Lys Leu His Gly His Ser Lys Tyr Gln Gln Gln Ser Leu Arg Ala Asn Lys Ser Tyr Phe Leu Phe Asp Asn Arg Val Ile Ala Leu Gly Ser Gly Ile Glu Asn Asp Asp Lys Gln His Thr Thr Glu Thr Thr Leu Phe Gln Phe Ala Val Pro Lys Leu Gln Ser Val Ile Ile Asn Gly Lys Lys Val Asn Gln Leu Asp Thr Gln Leu Thr Leu Asn Asn Ala Asp Thr Leu Il- Asp CCT GCC GGC AAT TTA TAT AAG CTC ACT AAA GGA CAA ACT GTA AAA m 5685 Pro Ala Gly Asn L-u Tyr Lys L-u Thr Lys Gly Gln Thr Val Lys Ph-8-r Tyr Gln Lys Gln ~18 8-r L-u Asp Asp Arg Asn 8-r Lys Pro Thr GAA CAA TTA TTT GCA ACA GCT GTT ATT TCT CAT GGT AAG GCA cca AGT 5781 Glu Gln Leu Ph- Ala Thr Ala Val Il- 8-r H$s Gly Lys Ala Pro 8-r Asn Glu Asn Tyr Glu Tyr Ala Ile Ala Il- Glu Ala Gln Asn Asn Lys GCT CCC GAA TAC ACA GTA TTA CAA CAT AAT GAT CAG CTC CAT GCG aTA 5877 Ala Pro Glu Tyr Thr Val L-u Gln His Asn Asp Gln L-u H$s Ala Val AAA GAT AAA ATA ACC CAA GAA GAG GGA TAT GCT m m GAA GCC ACT 5925 Lys Asp Lys Ile Thr Gln Glu Glu Gly Tyr Ala Ph- Ph- Glu Ala Thr Lys L-u Lys Ser Ala Asp Ala Thr Leu Leu Ser Ser Asp Ala Pro Val M t Val Met Ala Lys Ile Gln Asn Gln Gln Leu Thr Leu Ser Ile Val A~n Pro Asp Leu Asn Leu Tyr Gln Gly Arg Glu Lys Asp Gln Phe Asp Asp Lys Gly Asn Gln Il- Glu Val Ser Val Tyr Ser Arg H$s Trp Leu SUBSTITUTE SHEET (RU~E 26) W O 94/25567 PCTrUS94/04495 6 1 1 2 ~ - 134 -Thr Ala Glu Ser Gln Ser Thr Asn Ser Thr Ile Thr Val Lys Gly Ile Trp LYB Leu Thr Thr Pro Gln Pro Gly Val Ile Ile Lys His His Asn A~n Asn Thr Leu Ile Thr Thr Thr Thr Ile Gln Ala Thr Pro Thr Val ATT AAT TTA GTT AAG TAAATTTCGT AACTTTTAAA CT~ GTC TCC~CAT~ 6316 Ile Asn Leu Val Lys AATATCGAGA ~..... AT T~ TTA ~a~r~CTT ~rr~TGAA TTAATTATTT 6376 C~ T~ T~TCG ATAG~TTTAT TATTGATAAT A~ ~.~--G TGCTCAATGG 6436 TTA-...~-. A.~ .~.GCG CGGA.;~.~G GATCAAT G GTTr~C~T ATCGr~C~ 6496 cr~ r~r~ ~CCCCG GGT 6519 (2) INFORMATION ~OR SEQ ID NO:40:
(i) S~QU%NCE rU~rT~RISTICS:
(A) L~NGT~: 1013 am~no acids (B) TYPE: amino acid (D) TOPOLOGY: l~near (ii) MOTRC~ TYPE: prot-ln (xi) SEQVENCE DESCRIPTION: SEQ ID NO:40:
et Leu Ile Lys Asn Pro L-u Ala His Ala Val Thr Leu Ser Leu Cys eu Ser Leu Pro Ala Gln Ala Leu Pro Thr Leu Ser His Glu Ala Phe Gly Asp Ile Tyr Leu Phe Glu Gly Glu Leu Pro Asn Thr Leu Thr Thr Ser Asn Asn Asn Gln Leu Ser Teu Ser Lys Gln His Ala Lys Aop Gly G1U Gln Ser ~oU Ly8 Trp Gln Tyr Gln Pro Gln Ala Thr Leu ~hr Leu sn Asn Ile Val Asn Tyr Gln Asp Asp Lys Asn Thr Ala Thr Pro L-u hr Phe Met Met Trp Ile Tyr Asn Glu Lys Pro Gln Ser Ser Pro Leu Thr Leu Ala Phe Lys Gln Asn Asn Lys Ile Ala Leu Ser Phe Asn Ala SUBSTITUTE SHEET (RlJL~ 26) WO 94125567 ~151 l2 ~ PCT~S94/04495 Glu Leu Asn Phe Thr Gly Trp Arg Gly Ile Ala Val Pro Phe Arg A~p Met Gln Gly Ser Ala Thr Gly Gln Leu Asp Gln Leu Val Ile Thr Ala 145 lS0 155 160 ro Asn Gln Ala Gly Thr Leu Phe Phe Asp Gln Ile Ile Met Ser Val ro Leu Asp Asn Arg Trp Ala Val Pro Asp Tyr Gln Thr Pro Tyr Val Asn A~n Ala Val Asn Thr Met Val Ser Lys Asn Trp Ser Ala Leu Leu Met Tyr Asp Gln Met Phe Gln Ala His Tyr Pro Thr Leu Asn Phe Asp Thr Glu Phe Arg Asp Asp Gln Thr Glu Met Ala Ser Ile Tyr Gln Arg he Glu Tyr Tyr Gln Gly Ile Arg Ser Asp Lys Lys Ile Thr Pro Asp et Leu Asp Lys H$s Leu Ala Leu Trp Glu Lys Leu Val Leu Thr Gln His Ala Asp Gly Ser Ile Thr Gly Lys Ala Leu Asp His Pro Asn Arg Gln His Phe Met Lys Val Glu Gly Val Phe Ser Glu Gly Thr Gln Lys Ala Leu Leu Asp Ala Asn Met Leu Arg Asp Val Gly Lys Thr Leu Leu ln Thr Ala Ile Tyr Leu Arg Ser Asp Ser Leu Ser Ala Thr Asp Arg ys Lys Leu Glu Glu Arg Tyr Leu Leu Gly Thr Arg Tyr Val Leu,Glu Gln Gly Phe Thr Arg Gly Ser Gly Tyr Gln Ile Ile Thr His Val Gly Tyr Gln Thr Arg Glu Leu Phe Asp Ala Trp Phe Ile Gly Arg His Val Leu Ala Lys Asn Asn Leu Leu Ala Pro Thr Gln Gln Ala Met Met Trp yr Asn Ala Thr Gly Arg Ile Phe Glu Lys Asn Asn Glu Ile Val Asp la Asn Val Asp Ile Leu Asn Thr Gln Leu Gln Trp Met Ile Lys Ser Leu Leu Met Leu Pro Asp Tyr Gln Gln Arg Gln Gln Ala Leu Ala Gln Leu Gln Ser Trp Leu Asn Lys Thr Ile Leu Ser Ser Lys Gly Val Ala SlJBS~ E SHEET (RlJ~E 26) 216112~ - 136 -Gly Gly Phe Lys Ser Asp Gly Ser Ile Phe His His Ser Gln His Tyr ro Ala Tyr Ala Lys Asp Ala Phe Gly Gly Leu Ala Pro Ser Val Tyr la Leu Ser Asp Ser Pro Phe Arg Leu Ser Thr Ser Ala ~is Glu Arg Leu Lys Asp Val Leu Leu Lys Met Arg Ile Tyr Thr Lys Glu Thr Gln Ile Pro Val Val Leu Ser Gly Arg ~is Pro Thr Gly Leu His Lys Ile Gly Ile Ala Pro Phe Lys Trp Met Ala Leu Ala Gly Thr Pro Asp Gly YB Gln Lys Leu Asp Thr Thr Leu Ser Ala Ala Tyr Ala Lys L-u Asp sn Lye Thr His Phe Glu Gly Ile Asn Ala Glu Ser Glu Pro Val Gly Ala Trp Ala Met Asn Tyr Ala Ser Met Ala Ile Gln Arg Arg Ala S-r Thr Gln Ser Pro Gln Gln Ser Trp Leu Ala Il- Ala Arg Gly Phe 8-r Arg Tyr Leu Val Gly Asn Glu Ser Tyr Glu Asn Asn Asn Arg Tyr Gly rg Tyr Leu Gln Tyr Gly Gln Leu Glu Ile Ile Pro Ala Asp Leu Thr ln Ser Gly Phe Ser ~is Ala Gly Trp Asp Trp Asn Arg Tyr Pro Gly Thr Thr Thr Ile ~is Leu Pro Tyr Asn Glu Leu Glu Ala Lys Leu A~n Gln Leu Pro Ala Ala Gly Ile Glu Glu Met Leu Leu Ser Thr Glu Ser Tyr Ser Gly Ala Asn Thr Leu Asn Asn Asn Ser Met Phe Ala Met Lys eu ~is Gly His Ser Lys Tyr Gln Gln Gln Ser Leu Arg Ala Asn Lys er Tyr Phe Leu Phe Asp Asn Arg Val Ile Ala Leu Gly Ser Gly Ile Glu Asn Asp A~p Lys Gln ~is Thr Thr Glu Thr Thr Leu Phe Gln Phe Ala Val Pro Lys Leu Gln Ser Val Ile Ile Asn Gly Lys Ly~ Val Asn Gln Leu Asp Thr Gln Leu Thr Leu Asn Asn Ala ABP Thr Leu Ile Asp SUBSTITUTE SHEET (RULE 26) WO 94/25567 - 2 1 a 1 I ~ ~ PCT/US94/04495 - 137 - . ~

ro Ala Gly Asn Leu Tyr Lys Leu Thr Ly~ Gly Gln Thr Val Lys Phe er Tyr Gln Lys Gln His Ser Leu Asp Asp Arg Asn Ser Lys Pro Thr Glu Gln Leu Phe Ala Thr Ala Val Ile Ser His Gly Lys Ala Pro Ser Asn Glu Asn Tyr Glu Tyr Ala Ile Ala Ile Glu Ala Gln Asn Asn Lys Ala Pro Glu Tyr Thr Val Leu Gln ~is Asn Asp Gln Leu His Ala Val ys Asp ~ys Ile Thr Gln Glu Glu Gly Tyr Ala Phe Phe Glu Ala Thr ys Leu Lys Ser Ala Asp Ala Thr Leu Leu Ser Ser Asp Ala Pro Val Met Val Met Ala Ly~ Ile Gln Asn Gln Gln Leu Thr Leu Ser Ile Val Asn Pro Asp Leu Asn Leu Tyr Gln Gly Arg Glu Lys Asp Gln Phe Asp Asp Lys Gly Asn Gln Ile Glu Val Ser Val Tyr Ser Arg Bis Trp Leu hr Ala Glu Ser Gln Ser Thr Asn Ser Thr Ile Thr Val Lys Gly Ile rp Lys Leu Thr Thr Pro Gln Pro Gly Val Ile Ile Lys His His Asn sn Asn Thr Leu Ile Thr Thr Thr Thr Ile Gln Ala Thr Pro Thr Val Ile Asn Leu Val Lys (2) INFORNATION FOR SBQ ID NO:41:
(i) SEQUENCE r~D~r~DT.STICS:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) ST-D~r~R~NESS: single (D) TOPOLOGY: linear (ii) M~TRCC~TC TYPE: DNA (genomic) (iii) ~YrG,A~,lCAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:41:
ATTTGCAGGA AATCTGCATA TGCT~AT~A ~A~rCC 36 SUB~TITUTE SHE~T (RULE 26)

Claims (26)

What is claimed is:

1. A purified isolated DNA fragment of Proteus vulgaris (P. vulgaris) comprising a sequence encoding for the chondroitinase I enzyme.

2. A purified isolated DNA fragment of P.
vulgaris, wherein the fragment comprises a nucleic acid sequence encoding amino acids numbered 1-1021 of SEQ ID NO:2 or a biological equivalent thereof.

3. The purified isolated DNA fragment of Claim 2, wherein the fragment has the sequence of (a) the nucleotides numbered 119-3181 of SEQ ID NO: 1, or (b) the nucleotides numbered 119-3181 of SEQ ID NO: 3.

4. A purified isolated DNA fragment of P.
vulgaris, wherein the fragment comprises a nucleic acid sequence encoding amino acids numbered 25-1021 of SEQ ID NO:2 of a biological equivalent thereof.

5. The purified isolated DNA fragment of Claim 4, wherein the fragment has the sequence of (a) the nucleotides numbered 191-3181 of SEQ ID NO:1, or (b) the nucleotides numbered 191-3181 of SEQ ID NO:3.

6. A purified isolated DNA fragment of P.
vulgaris, wherein the fragment comprises a nucleic acid sequence encoding the amino acids numbered 24-1021 of SEQ ID NO:5 or a biological equivalent thereof.

7. The purified isolated DNA fragment of Claim 6, wherein the fragment has the sequence of nucleotides numbered 188-3181 of SEQ ID NO:4.

8. A purified isolated DNA fragment of Proteus vulgaris (P. vulgaris) comprising a sequence encoding chondroitinase II enzyme.

9. A purified isolated DNA fragment of P.

vulgaris, wherein the fragment comprises a nucleic acid sequence encoding (a) the amino acids numbered 1-1013 of SEQ ID NO:40 or a biological equivalent thereof, or (b) the amino acids numbered 24 - 1013 of SEQ ID NO: 40 or a biological equivalent thereof.

10. The purified isolated DNA fragment of Claim 9, wherein the fragment has the sequence of nucleotides (a) numbered 3238-6276 of SEQ ID NO:39, or (b) numbered 3307-6276 of SEQ ID NO:39.

11. A plasmid containing a purified isolated DNA fragment of P. vulgaris comprising the sequence of (a) Claim 1 or (b) Claim 8.

12. The plasmid of Claim 11 wherein the plasmid is that designated pTM49-6 or that designated LP21359.

13. A host cell transformed with the plasmid of Claim 11.

14. The host cell of Claim 13 wherein the plasmid is that designated pTM49-6 (ATCC 69234) of that designated Lp21359 (ATCC 69598).

15. A purified isolated recombinant chondroitinase I enzyme.

16. The chondroitinase I enzyme of Claim 15, whose amino acid sequence is depicted for (a) the amino acids numbered 1-1021 of SEQ ID NO:2 or a biological equivalent thereof, (b) the amino acids numbered 25-1021 of SEQ ID NO:2 or a biological equivalent thereof, or (c) the amino acids numbered 24-1021 of SEQ ID NO:5 or a biological equivalent thereof.

17. A purified isolated recominant chondroitinasse II enzyme.

18. The chondroitinase II enzyme of Claim 17, whose amino acid sequence is depicted for (a) the amino acids numbered 1-1013 of SEQ ID NO:40 or a biological equivalent thereof, or (b) the amino acids numbered 24-1013 of SEQ ID NO:40 or a biological equivalent thereof.

19. A method of producing chondroitinase I
enzyme which comprises transforming a host cell with the plasmid of Claim 11 (a) and culturing the host cell under conditions which permit expression of said enzyme by the host cell.

20. A method of producing the chondroitinase II enzyme which comprises transforming a host cell with the plasmid of Claim 11 (b) and culturing the host cell under conditions which permit expression of said enzyme by the host cell.

21. A method for the isolation and purification of the recombinant chondroitinase I
enzyme of Proteus vulgaris from host cells, said method comprising the steps of:
(a) lysing by homogenization the host cells to release the enzyme into the supernatant;
(b) subjecting the supernatant to diafiltration to remove salts and other small molecules;
(c) passing the supernatant through an anion exchange resin-containing column;
(d) loading the eluate from step (c) to a cation exchange resin-containing column so that the enzyme in the eluate binds to the cation exchange column; and (e) eluting the enzyme bound to the cation exchange column with a solvent capable of releasing the enzyme from the column.

22. The method of Claim 21, wherein prior to step (b), the following two steps are performed:
(1) treating the supernatant with an acidic solution to precipitate out the enzyme;
and (2) recovering the pellet and then dissolving it in an alkali solution to again place the enzyme in a basic environment.

23. A recombinant chondroitinase I enzyme isolated and purified by the method of Claim 21 or by the method of Claim 22.

24. A method for the isolation and purification of the recombinant chondroitinase II
enzyme of Proteus vulgaris from host cells, said method comprising the steps of:
(a) lysing by homogenization the host cells to release the enzyme into the supernatant;
(b) subjecting the supernatant to diafiltration to remove salts and other small molecules;
(c) passing the supernatant through an anion exchange resin-containing column;
(d) loading the eluate from step (c) to a cation exchange resin-containing column so that the enzyme in the eluate binds to the cation exchange column; and (e) obtaining by affinity elution the enzyme bound to the cation exchange column with a solution of chondroitin sulfate, such that the enzyme is co-eluted with the chondroitin sulfate;
(f) loading the eluate from step (e) to an anion exchange resin-containing column and eluting the enzyme with a solvent such that the chondroitin sulfate binds to the column; and (g) concentrating the eluate from step (f) and crystallizing out the enzyme from the supernatant which contains an approximately 37 kD contaminant.

25. The method of Claim 24, wherein prior to step (b), the following two steps are performed:
(1) treating the supernatant with an acidic solution to precipitate out the enzyme;
and (2) recovering the pellet and then dissolving it in an alkali solution to again place the enzyme in a basic environment.

26. A recombinant chondroitinase II enzyme isolated and purified by the method of Claim 24 or by the method of Claim 25.