WO1994025567A1 - CLONAGE ET EXPRESSION DE GENES DE CHONDROITINASE I ET II A PARTIR DE $i(P. VULGARIS) - Google Patents

CLONAGE ET EXPRESSION DE GENES DE CHONDROITINASE I ET II A PARTIR DE $i(P. VULGARIS) Download PDF

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WO1994025567A1
WO1994025567A1 PCT/US1994/004495 US9404495W WO9425567A1 WO 1994025567 A1 WO1994025567 A1 WO 1994025567A1 US 9404495 W US9404495 W US 9404495W WO 9425567 A1 WO9425567 A1 WO 9425567A1
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enzyme
ser
chondroitinase
ala
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PCT/US1994/004495
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Michael Joseph Ryan
Kiran Manohar Khandke
Bruce Clifford Tilley
Jason Arnold Lotvin
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American Cyanamid Company
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Priority to JP6524437A priority Critical patent/JPH09500011A/ja
Priority to AU68183/94A priority patent/AU697156B2/en
Priority to EP94916561A priority patent/EP0702715A4/fr
Publication of WO1994025567A1 publication Critical patent/WO1994025567A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • 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) .
  • 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.
  • This invention also relates to the cloning and expression of the genes containing these DNA sequences and to the amino acid sequences of the recombinant chondroitinase 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 chondroitinase ABC, which is referred to as "chondroitinase I”, from Proteus vulgaris (P. vulgaris) .
  • This invention further relates to methods for the isolation and purification of the recombinantly expressed second protein component of chondroitinase ABC, which is referred to as "chondroitinase II", from P. vulgaris.
  • 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 chondroitinase I enzyme from P. vulgaris. Background of the Invention
  • Chondroitinases are enzymes of bacterial origin which have been described as having value in dissolving the cartilage of herniated discs without disturbing the stabilizing collagen components of those discs.
  • chondroitinase enzymes examples include chondroitinase ABC, which is produced by the bacterium P. vulgaris. and chondroitinase AC, which is produced by A. aurescens.
  • the chondroitinases function by degrading polysaccharide side chains in protein- polysaccharide complexes, without degrading the protein core.
  • Yamagata et al. describes the purification of the enzyme chondroitinase ABC from extracts of P. vulgaris (Bibliography entry 1) .
  • the enzyme selectively degrades the glycosaminoglycans chondroitin-4-sulfate, dermatan sulfate and chondroitin-6-sulfate (also referred to respectively as chondroitin sulfates A, B and C) at pH 8 at higher rates than chondroitin or hyaluronic acid.
  • the enzyme did not attack keratosulfate, heparin or heparitin sulfate. Kikuchi et al.
  • glycosaminoglycan degrading enzymes such as chondroitinase ABC
  • chondroitinase ABC glycosaminoglycan degrading enzymes
  • Brown describes a method for treating intervertebral disc displacement in mammals, including humans, by injecting into the intervertebral disc space effective amounts of a solution containing chondroitinase ABC (3) .
  • the chondroitinase ABC was isolated and purified from extracts of P. vulgaris. This native enzyme material functioned to dissolve cartilage, such as herniated spinal discs. Specifically, the enzyme causes the selective 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 membranes 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 chondroitin 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 chondroitinase ABC.
  • chondroitinase ABC obtained from Seikagaku Kogyo Co., Ltd., Tokyo, Japan.
  • isolating and purifying the chondroitinase ABC enzyme from the Seikagaku Kogyo material it was noted that there was a correlation between effective preparations of the chondroitinase 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 component of chondroitinase ABC.
  • the second protein is now designated “chondroitinase II", while the major protein component of chondroitinase ABC is referred to as “chondroitinase I.”
  • the chondroitinase I and II proteins are basic proteins at neutral pH, with similar isoelectric points of 8.30-8.45. Separate purification of the chondroitinase 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.
  • chondroitinase I and II forms of the native enzyme have 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. vulgaris 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 (chondroitin sulfate) , and the opportunistically pathogenic nature of P. vulgaris, has resulted in the requirement for a more efficient method of production.
  • the native forms of the enzyme produced by conventional techniques are subject to degradation by proteases present in the bacterial extract.
  • chondroitinase I and chondroitinase II in quantities not readily achievable using present non-recombinant bacterial fermentation and extraction techniques. It is a further object of this invention to produce chondroitinase I and chondroitinase II, each in a form substantially free of proteases which would otherwise degrade the enzyme and cause a loss of its activity.
  • this invention is directed to the cloning of the P. vulgaris gene for chondroitinase I and the high level expression of that enzyme in E. coli, as well as the cloning of the P. vulgaris gene for chondroitinase II and the high level expression of that enzyme in E. coli.
  • This invention provides a purified isolated DNA fragment of P. vulgaris which comprises a sequence encoding for chondroitinase I.
  • This invention further provides a purified isolated DNA fragment of P. vulgaris which hybridizes with a nucleic acid sequence encoding for amino acids as follows: (a) the chondroitinase I enzyme with its signal peptide (SEQ ID NO:2, amino acids 1-1021) or a biological equivalent thereof (encoded for example by: (1) 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, nucleotides 116- 118)); (b) the mature chondroitinase I enzyme (SEQ ID NO:2, amino acids 25-1021) or a biological equivalent thereof (encoded for example by: (1) nucleotides numbered 191-3181 of SEQ ID
  • nucleotides numbered 191-3181 of SEQ ID NO:3, where the three nucleotides immediately upstream of the initiation codon are changed SEQ ID NO:3, nucleotides 116-118)
  • the recombinant chondroitinase I is produced by transforming a host cell with a plasmid containing a purified isolated DNA fragment of P. vulgaris 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.
  • This invention also provides a purified isolated DNA fragment of P. vulgaris which comprises a sequence encoding for chondroitinase II.
  • This invention further provides a purified isolated DNA fragment from P. vulgaris which hybridizes with a nucleic acid sequence encoding for amino acids as follows:
  • the recombinant chondroitinase II is produced by transforming a host cell with a plasmid containing a purified isolated DNA fragment of P. vulgaris 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.
  • the first method comprises the steps of:
  • step (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.
  • step (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;
  • step (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;
  • step (g) concentrating the eluate from step (f) and crystallizing out the enzyme from the supernatant which contains an approximately 37 kD contaminant.
  • step (b) of the first method just described the following two steps are performed:
  • Figure 1 depicts a preliminary restriction map for the subcloned approximately 10 kilobase Nsi fragment in pIBI24.
  • the Nsi fragment contains the complete gene encoding chondroitinase I and a portion of the gene encoding chondroitinase II.
  • the restriction sites 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 set forth in Example 13 below.
  • Figure 2 depicts the elution of the recombinant chondroitinase I enzyme from a cation exchange 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 chondroitinase I enzyme activity. The fractions at right containing the enzyme are marked "eluted activity”. The gradient is from 0.0 to 250 mM NaCl.
  • Figure 3 depicts the elution of the recombinant chondroitinase I enzyme from a cation exchange column, after first passing the supernatant through an anion exchange 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 chondroitinase I activity.
  • the fractions at right containing the enzyme are marked "eluted activity”.
  • the gradient is from 0.0 to 250 mM NaCl.
  • Figure 4 depicts sodium dodecyl sulfate- polyacrylamide gel chromatography (SDS-PAGE) of the recombinant chondroitinase I enzyme before and after the purification methods of this invention are used. In the SDS-PAGE gel photograph.
  • Lane 1 is the enzyme purified using the method of the first embodiment of the invention
  • Lane 2 is the enzyme purified using the method of the second embodiment of the invention
  • Lane 3 represents the supernatant from the host cell prior to purification -- many other proteins are present
  • Lane 4 represents the following molecular weight standards: 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 chondroitinase II enzyme during various stages of purification using a method of this invention.
  • Lane 1 is the crude supernatant after diafiltration
  • Lane 2 the eluate after passage of the supernatant through an anion exchange resin-containing column
  • Lane 3 is the enzyme after elution through a cation exchange resin- containing column
  • Lane 4 is the enzyme after elution through a second anion exchange resin-containing column
  • Lane 5 represents the same molecular weight standards as described for Figure 4, plus 6.5 kD - aprotinin
  • Lane 6 is the same as Lane 4, except it is overloaded to show the approximately 37 kD contaminant
  • Lane 7 is the 37 kD contaminant in the supernatant after crystallization of the chondroitinase II enzyme
  • Lane 8 is first wash of the crystals
  • Lane 9 is the second wash of the crystals
  • Lane 10 is the enzyme in the washe
  • oligonucleotides designed to bracket part of the chondroitinase I gene
  • DNA synthesis is carried out in 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 exponentially with each cycle.
  • a putative nucleotide sequence of the appropriate oligonucleotides is constructed from available amino acid sequence information derived from the protein purified from P. vulgaris bacteria.
  • the DNA fragment produced by PCR is cloned and its DNA sequence determined to verify that it is part of the chondroitinase I gene. It is then labeled and used as a probe to indicate which members of the gene bank actually contain the chondroitinase I gene. Subsequent restriction mapping and Southern hybridization narrows the location to a piece of DNA of approximately four thousand base-pairs (bp) . This is then sequenced using the Sanger dideoxy chain termination method (6) to reveal the exact position of the gene and guide the subsequent manipulations used to place the gene into a high-level expression system in E. coli. A fermentation at a 10 liter scale carried out with this E.
  • coli strain containing a recombinant plasmid expressing the P. vulgaris chondroitinase I gene yields a maximum chondroitinase I titer of approximately 600 units/ml (which is the same as 1.2 mg/ml) . This yield far exceeds that of the native P. vulgaris fermentation process which had not achieved a titer of more than 2 units/ml.
  • genomic DNA is obtained. DNA is separated from protein and other material contained in a P. vulgaris 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 appropriate restriction endonuclease, such as Sau3A, and then Iigated into a cosmid vector.
  • the packaged recombinant cosmids containing the P. vulgaris DNA fragments are introduced into an appropriate 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 chondroitinase I gene.
  • Applicants have conducted some amino acid sequencing of the native chondroitinase I enzyme. Samples of the enzyme are generated by fermentation of P. vulgaris. Samples may also be obtained from Seikagaku Kogyo Co., Ltd., Tokyo, Japan. The amino acid sequence information is used to design oligonucleotides for use in screening for the chondroitinase I gene.
  • oligonucleotides are designed for use in PCR.
  • a first set of oligonucleotides is designed so as to encode a heptapeptide that has minimal degeneracy of its genetic code. Seven amino acids near the amino terminus of the chondroitinase 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.
  • the approximately 110 kD chondroitinase I enzyme is cleaved proteolytically into an 18,000 MW ("18 kD”) fragment and an approximately 90,000 MW (“90 kD”) 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.
  • the complementary strand has the same number of potential sequences (SEQ ID NOS:27 and 28; see Example 2) .
  • 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 amplifications are observed as discrete bands on electrophoretic gels. Products approximately 500 and 350 base pairs (bp) in size are obtained. 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 chondroitinase 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.
  • the PCR amplification fragment is used as a probe to identify the cosmid gene banks prepared in the first stage which contain the chondroitinase I gene.
  • the PCR fragment is denatured and labelled with, for example, digoxigenin- labelled dUTP (Boehringer-Mannheim, Indianapolis, IN) .
  • the cosmid gene banks are then used to infect a 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.
  • Southern hybridization (8) and restriction mapping are used to localize the position of the chondroitinase I gene within individual clones.
  • the PCR-generated fragment described above is used as a Southern hybridization probe against P. vulgaris genomic DNA that is first digested by restriction enzymes and fractionated.
  • several of the oligonucleotides described above are used as primers.
  • the results indicate that the portion of the chondroitinase I gene that hybridizes to the probe is carried on several large DNA fragments. These large DNA fragments are digested to yield individual fragments which are isolated, tested for the presence of chondroitinase 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 vitro chondroitinase I assays in which the activity of the enzyme based on measuring the release of unsaturated disaccharide from chondroitin sulfate C at 232 nm are conducted on several samples to assist in the placement and orientation of the chondroitinase I gene. The results of these procedures suggest that a 4.2 kb EcoRV-EcoRI fragment of a larger 10 kb Nsil fragment could contain the entire chondroitinase I gene.
  • the above-mentioned 4.2 kb fragment is subjected to DNA sequence analysis.
  • the resulting DNA sequence is 3980 nucleotides in length (SEQ ID NO:l) .
  • Translation of the DNA sequence into the putative amino acid sequence reveals a continuous open reading frame (SEQ ID NO:l, nucleotides 119-3181) encoding 1021 amino acids (SEQ ID NO:2) .
  • 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) chondroitinase I enzyme (SEQ ID NO:2, amino acids 25-1021) .
  • Signal sequences are required for a complex series of post-translational processing steps which result in secretion of a protein from a host cell.
  • the signal sequence 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 chondroitinase 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 containing a purified isolated DNA fragment encoding for chondroitinase I enzyme.
  • the host cell is then cultured under conditions which permit expression of the enzyme by the host cell.
  • 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 chondroitinase I enzyme at high levels.
  • site-directed mutagenesis to introduce unique restriction sites.
  • Example 6 two different constructs are prepared.
  • the three nucleotides immediately upstream of the initiation codon are changed (SEQ ID NO:3, nucleotides 116-118) through the use of a mutagenic oligonucleotide (SEQ ID NO:37) .
  • the coding region and amino acid sequence encoded by the resulting construct are not changed, and the signal sequence is preserved (SEQ ID NO:3, nucleotides 119-3181; SEQ ID NO:2) .
  • the second construct is used.
  • 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 NO:38) which differs at six nucleotides from those of the native sequence (SEQ ID NO:l, 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 NO:4, nucleotides 188-3181; SEQ ID NO:5) .
  • the enzyme In the absence of a signal sequence, the enzyme is not secreted. Fortunately, it is not retained within the cell in the form of insoluble 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.
  • 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.
  • chondroitinase I enzyme expressed 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. vulgaris.
  • the supernatant from the host cells is treated to isolate and purify the enzyme.
  • Initial attempts to isolate and purify the recombinant chondroitinase I enzyme do not result in high yields of purified protein.
  • the previous method for isolating and purifying native chondroitinase I from fermentation cultures of P. vulgaris is found to be inappropriate for the recombinant material.
  • the native enzyme is produced by fermentation of a culture of P. vulgaris.
  • the bacterial cells are first recovered from the medium and resuspended in buffer.
  • the cell suspension is then homogenized to lyse the bacterial cells.
  • a charged particulate such as Bioacryl (Toso Haas,
  • the solution is then filtered and the retentate is washed to recover most of the enzyme.
  • the filtrate is concentrated and subjected to diafiltration with a phosphate to remove the salt.
  • the filtrate containing the chondroitinase I is subjected to cation exchange chromatography using a cellulose sulfate column. At pH 7.2, 20 mM sodium phosphate, more than 98% of the chondroitinase I binds to the column.
  • the native chondroitinase I is then eluted from the column using a sodium chloride gradient.
  • chondroitinase I is obtained at a purity of 90-97%.
  • the level of purity is measured by first performing SDS-PAGE. The proteins are stained using Coomassie blue, destained, and the lane on the gel is scanned using a laser beam of wavelength 600 nm. The purity is expressed as the percentage of the total absorbance accounted for by that band.
  • the yield of the native protein is only 25-35%.
  • the yield is measured as the remaining activity in the final purified product, expressed as a percentage of the activity at the start (which is taken as 100%) .
  • the activity of the enzyme is based on measuring the release of unsaturated disaccharide from chondroitin sulfate C at 232 nm.
  • This purification method also results in the extensive cleavage of the approximately 110,000 dalton (110 kD) chondroitinase I protein into a 90 kD and an 18 kD fragment. Nonetheless, the two fragments remain non-covalently bound and exhibit chondroitinase I activity.
  • 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 exchange column.
  • cation exchange resins bind to proteins better at lower pH' s than higher pH's.
  • a protein which is not very basic, and hence does not bind at a high pH can be made to bind to the cation exchanger by carrying out the operation at a lower pH.
  • the native enzyme binds completely to a cation exchange resin.
  • the recombinant-derived enzyme due to the lowered basicity as a result of binding of the negatively charged molecules, 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% binding level; typically, it is much less (less than 10%) even at pH 6.8. This level of binding varies dramatically between different fermentation batches. This hypothesis and a possible solution to the problem are then tested. If negatively charged molecules are attaching non-covalently to chondroitinase I, thus decreasing its basicity, it should be possible to remove these undesired molecules by using a strong, high capacity anion exchange resin. Removal of the negatively charged molecules should then restore the basicity of the enzyme.
  • chondroitinase 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 suitable for use in purifying either of these forms of the enzyme.
  • the host cells which express the recombinant chondroitinase 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.
  • this step only removes the free, but not the bound form of the negatively charged molecules.
  • the bound form of these charged species i ⁇ next removed by passing the supernatant through a strong, high capacity anion exchange resin-containing column.
  • a strong, high capacity anion exchange resin-containing column is the Macro-PrepTM High Q resin (Bio-Rad, Melville, N.Y.) .
  • Other strong, high capacity anion exchange columns are also suitable.
  • Weak anion exchangers containing a diethylaminoethyl (DEAE) ligand also are suitable, although they are not as effective.
  • 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 column.
  • the eluate from the anion exchange column is directly loaded to a cation exchange resin- containing column.
  • cation exchange resin- containing column examples include the S- Sepharo ⁇ eTM (Pharmacia, Piscataway, N.J.) and the Macro-PrepTM High S (Bio-Rad) .
  • S- Sepharo ⁇ eTM Pulsoa, Piscataway, N.J.
  • Macro-PrepTM High S Bio-Rad
  • the enzyme binds to the column and is then eluted with a solvent capable of releasing the enzyme from the column.
  • Any salt which increases the conductivity of the solution is suitable for elution.
  • 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.
  • the purity of the protein is measured by scanning 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.
  • 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 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 anyway by the cation and anion exchange 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.
  • the additional acid precipitation and alkali dissolution steps of the second embodiment mean that the second embodiment is more time consuming than the first embodiment.
  • 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 chondroitinase 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 material depo ⁇ ited with the ATCC can al ⁇ o be used in conjunction with the sequences disclo ⁇ ed herein to regenerate the native chondroitinase I gene sequence (SEQ ID NO:l) or the modified chondroitinase I gene sequence which includes the signal sequence (SEQ ID NO:3) using conventional genetic engineering technology.
  • the present invention further comprises DNA sequences which, by virtue of the redundancy 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.
  • the invention contemplates those DNA sequences which are sufficiently duplicative of the sequences of SEQ ID NOS:l, 3 or 4 so as to permit hybridization therewith under standard 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 chondroitinase 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 conservative substitutions to the enzyme sequence, such that the tertiary configurations of the sequences are essentially unchanged from those of the enzyme.
  • 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.
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for gl tamic 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.
  • the nucleotide sequence determined above for the region encoding the chondroitinase I gene includes an additional approximately 800 base pairs beyond the translation termination codon (SEQ ID NOS:l and 39, nucleotides 3185-3980) .
  • An inspection of this region reveals that the sequence between nucleotides 3307 and 3372 (SEQ ID NOS:l and 39) encodes the identical 22 amino acids in the same order as the first 22 amino acids of native chondroitinase II.
  • 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 chondroitinase II (SEQ ID NO:40, amino acids 1-23) .
  • a Shine-Dalgarno sequence (AGGA; SEQ ID N0S:1 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. vulgaris chondroitinase 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 N0:1, 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 reading frame which then extends through the end of SEQ ID NO: 39 (SEQ ID N0S:1 and 39 include the inserted T as nucleotide 3594) . (Thus, the three bases TAA at base-pairs 3608-3610, properly numbered, do not constitute a termination codon.)
  • the cloning and expression of the P. vulgaris chondroitinase II gene is performed in three stages.
  • 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 chondroitinase I gene.
  • the mutagenized bases are upstream of the coding region (an AT sequence (SEQ ID NOS:l 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 chondroitinase II gene.
  • the available DNA sequence information is adequate to account for approximately 220 amino acids of an estimated 1000 for the entire chondroitinase II 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 chondroitinase II that has been modified to include the initiation codon as part of an Ndel site and to be followed by a BamHl 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.
  • Sequencing 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 chondroitinase II protein at residues numbered 24-1013 (SEQ ID NO:40) .
  • the signal peptide is retained, 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 gene encoding the chondroitinase II protein is inserted into pET9A and the resulting recombinant plasmid is designated LP 2 1359.
  • the plasmid i ⁇ then used to transform an appropriate expres ⁇ ion host cell, such a ⁇ the E. coli B strain BL21(DE3)/pLysS (which is also used for the expres ⁇ ion of the chondroitinase I gene.
  • an appropriate expres ⁇ ion host cell such a ⁇ the E. coli B strain BL21(DE3)/pLysS (which is also used for the expres ⁇ ion of the chondroitinase I gene.
  • coli B strain designated TD112 which is BL21 (DE3) /pLy ⁇ S carrying the recombinant pla ⁇ mid LP 2 1359, were depo ⁇ ited by Applicant ⁇ on April 6, 1994, with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A., and have been a ⁇ signed ATCC accession number 69598.
  • 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 the recombinant chondroitinase I protein is adapted for isolating and purifying the recombinant chondroitinase 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 chondroitinase II protein is expressed at levels approximately several-fold lower than the recombinant chondroitinase I protein; therefore, a more powerful and selective solution is neces ⁇ ary in order to obtain a final chondroitina ⁇ e II product of a purity equivalent to that obtained for the chondroitina ⁇ e I protein.
  • the first several step ⁇ of the method for the chondroitinase II protein are the same as those used to isolate and purify the chondroitinase I protein. Initially, the host cells which express the recombinant chondroitinase II 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 charged species is next removed by passing the supernatant through a strong, high capacity anion exchange resin-containing column.
  • Such a resin is the Macro-PrepTM High Q resin (Bio-Rad, Melville, N.Y.) .
  • Other strong, high capacity anion exchange columns are also suitable.
  • Weak anion exchangers containing a diethylaminoethyl (DEAE) ligand also are suitable, although they are not as effective.
  • low capacity resins are also suitable, although they too are not as effective.
  • the negatively charged molecules bind to the column, while the enzyme pas ⁇ es through the column. It is also found that some unrelated, undesirable proteins also bind to the column.
  • a cation exchange resin- containing column examples include the S- Sepharo ⁇ eTM (Pharmacia, Pi ⁇ cataway, N.J.) and the Macro-PrepTM High S (Bio-Rad) .
  • S- Sepharo ⁇ eTM Pulcoa, Pi ⁇ cataway, N.J.
  • Macro-PrepTM High S Bio-Rad
  • Each of these two resin-containing columns has S0 3 " ligands bound thereto in order to facilitate the exchange of cation ⁇ .
  • Other cation exchange columns are also suitable.
  • the enzyme binds to the column, while a significant portion of contaminating proteins elute unbound.
  • the method diverges from that used for the chondroitinase I protein.
  • a specific elution using a solution containing chondroitin sulfate is used.
  • This procedure utilizes the affinity the positively charged chondroitinase II protein has for the negatively charged chondroitin sulfate.
  • the affinity is larger than that accounted for by a simple positive and negative interaction alone. It i ⁇ an enzyme- ⁇ ub ⁇ trate interaction, which is similar to other specific biological interactions of high affinity, ⁇ uch a ⁇ antigen-antibody, ligand-receptor, co-factor-protein and inhibitor/activator-protein.
  • the chondroitin sulfate is able to elute the enzyme from the negatively charged resin.
  • the resin-enzyme interaction is a simple po ⁇ itive and negative interaction.
  • affinity elution chromatography i ⁇ as easy to practice as ion-exchange chromatography, the elution is specific, unlike salt elution. Thus, it has the advantages of both affinity chromatography (specificity) , as well as ion-exchange 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-exchange chromatography without a diafiltration/dialysi ⁇ ⁇ tep, which i ⁇ required when a salt is used. Note, that this is not a consideration in the method for the chondroitinase I protein, because no further ion- exchange chromatography i ⁇ needed in order to obtain the purified chondroitinase I protein. There is another reason for not using the method for purifying recombinant chondroitinase I.
  • Chondroitinase II obtained using the chondroitinase I salt elution purification method has poor stability; there is extensive degradation at 4°C within one week.
  • chondroitina ⁇ e II obtained by affinity elution i ⁇ stable The reason for this difference in stability is not known. It is to be noted that chondroitinase I obtained by salt elution is stable.
  • the cation exchange column is next washed with a phosphate buffer to elute unbound proteins, followed by washing with borate buffer to elute loo ⁇ ely bound contaminating protein ⁇ and to increa ⁇ e the pH of the re ⁇ in to that required for the optimal elution of the chondroitina ⁇ e II protein using the substrate, chondroitin sulfate.
  • chondroitin sulfate in water, adjusted to pH 9.0, is u ⁇ ed to elute the chondroitinase II protein, as a sharp peak (recovery 65%) and at a high purity of approximately 95%.
  • a 1% concentration of chondroitin sulfate is used.
  • a gradient of this solvent is also acceptable.
  • the chondroitin sulfate has an affinity for the chondroitinase II protein which is stronger than its affinity for the resin of the column, the chondroitin sulfate co-elutes with the protein.
  • Thi ⁇ ensures that only protein which recognizes chondroitin sulfate is eluted, which is desirable, but also means that an additional process step is neces ⁇ ary to ⁇ eparate the chondroitin ⁇ ulfate from the chondroitinase II protein.
  • the eluate is adjusted to a neutral pH and is loaded as i ⁇ onto an anion exchange resin-containing column, such as the Macro-PrepTM High Q resin.
  • the column is washed with a phosphate buffer.
  • the chondroitin sulfate binds to the column, while the chondroitinase II protein flows through in the unbound pool with greater than 95% recovery.
  • the protein i ⁇ pure, except for the presence of a single minor contaminant of approximately 37 kD.
  • the contaminant may be a breakdown product of the chondroitinase II protein.
  • This contaminant is effectively removed by a crytallization step.
  • the eluate from the anion exchange column is concentrated and the solution is maintained at a reduced temperature, such as 4°C, for several days to crystallize out the pure chondroitinase II protein.
  • the ⁇ upernatant contain ⁇ 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 washed with water.
  • the washed crystals are composed of the chondroitinase II protein at a purity of greater than 99%.
  • chondroitinase II protein 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 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 thi ⁇ invention.
  • Acid precipitation removes proteins that remain soluble; however, these proteins are removed anyway by the cation and anion exchange steps that follow (although smaller 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.
  • the additional acid precipitation and alkali dis ⁇ olution ⁇ tep ⁇ of the second embodiment mean that the second embodiment is more time consuming than the first embodiment.
  • 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 chondroitinase II enzyme at high yields.
  • the present invention further comprises DNA sequences which, by virtue of the redundancy 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 tho ⁇ e encoded by the DNA sequences set forth herein.
  • the invention contemplates those DNA sequences which are sufficiently duplicative of the sequence of SEQ ID NO:39 so as to permit hybridization therewith under standard high stringency Southern hybridization conditions, ⁇ uch a ⁇ 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 tho ⁇ e of the chondroitina ⁇ e II enzyme, but which are the biological equivalent to tho ⁇ e de ⁇ cribed for the enzyme (SEQ ID NO:40) .
  • amino acid ⁇ equence ⁇ may be said to be biologically equivalent to tho ⁇ e of the enzyme if their sequences differ only by minor deletion ⁇ from or con ⁇ ervative ⁇ ub ⁇ titutions to the enzyme sequence, such that the tertiary configurations of the sequences are essentially unchanged from tho ⁇ e of the enzyme.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be ⁇ ub ⁇ tituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine.
  • changes which result in sub ⁇ titution 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 a ⁇ changes based on similarities of residues in their hydropathic index, can also be expected to produce a biologically equivalent product.
  • one of ordinary skill in the art can ligate together the two pieces of DNA from the two depo ⁇ it ⁇ , for example, at the Hindlll ⁇ ite at nucleotide 3326, ⁇ o as to expres ⁇ both the chondroitinase I and chondroitinase II proteins under the control of the T7 promoter upstream of the coding sequence for chondroitinase I.
  • sample “B” is centrifuged and the cell pellet taken up with 7 ml of 0.05M glucose-0.025M Tris-HCl-0.01M EDTA (pH 8) containing 40 ⁇ g/ml of DNAa ⁇ e-free RNAa ⁇ e 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 Mannheim, Indianapolis, IN) is added to both samples to a final concentration of 100 ⁇ g/ml and incubation is continued overnight at 37°C.
  • proteinase K Boehringer Mannheim, Indianapolis, IN
  • sample ⁇ 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 shaking and finally, centrifugation to separate the two phase ⁇ .
  • the pelleted DNA is rinsed once with 70% (v/v) ethanol, dried under vacuum and then resuspended with 1 ml of TE (0.01M Tris-HCl-O.OOlM EDTA, pH 7.4).
  • Fragmentation of the genomic DNA to yield pieces of a size suitable for insertion into cosmid vectors is accom- pli ⁇ hed by partial digestion with the restriction endonuclease 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. vulgari ⁇ genomic DNA, 0.1M NaCl, 0.01M MgCl,, 0.01M Tri ⁇ -HCl (pH 7.5) and 80 unit ⁇ of the enzyme Sau3A.
  • the individual samples are heated to' 70°C and then 10 ⁇ l are removed for a size- di ⁇ tribution analysis on an agarose gel.
  • the sample obtained after five minutes of Sau3A digestion of preparation "A" and that obtained after 6 minutes with preparation "B" are chosen for further use.
  • an aliquot (4 ⁇ l, which is approximately equal to 2 ⁇ g) of the chosen partial digest is Iigated to the appropriate "left" and “right” arms of the cosmid vector DNA using approximately 1 ⁇ g and 2 ⁇ g of each, respectively, in 10 ⁇ l reactions containing 0.066M Tris-HCl (pH 7.4), 0.01M MgCl 2 , 0.001M ATP, and 400 units (as defined by the manufacturer (New England Biolabs, Beverly, MA)) of T4 DNA ligase. Incubation is carried out at 11°C overnight.
  • the "left" and "right” arms of the cosmids are DNA fragments which, when Iigated to an appropriately sized piece of P.
  • vulgaris DNA comprise a recombinant molecule of approximately 35-50 kb. Both arms contain "cos" site ⁇ which are recognized by the packaging enzymes in the next ⁇ tep. In addition, the ⁇ e arms carry the origin of replication and ampicillin-re ⁇ istance functions of pIBI24 (International Biochemical Inc., New Haven, CT) .
  • PDB 0.1M NaCl- 0.01M Tris-HCl (pH 7.9) -0.01M MgS0 4
  • Each tube of packaged DNA is, therefore, a gene bank of the P. vulgaris genome.
  • 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. vulgaris DNA)
  • the number of potential clones is quantitated 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.
  • 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 37°C.
  • the cells (1 ml) are then centrifuged, resuspended with PDB (0.2 ml) and 0.02 ml of the appropriate gene bank added. After adsorption for twenty minutes at 37°C, the sample ⁇ are diluted to 2 ml with 20-10-5 medium and grown at 37°C for 30 minute ⁇ . The culture is then spread on 20-10-5 plates containing 100 ⁇ g/ml of ampicillin and colonies scored after overnight incubation at 37°C. The results indicate that there are approximately 68,000 and 95,000 infectious particles (potential cosmid clone ⁇ ) present in the two sample ⁇ , designated PV1-GB and PV2- GB, corresponding to the "A" and "B" preparation of P. vulgaris genomic DNA, respectively.
  • P. vulgaris gene banks are prepared, as above, using two different cosmid vectors. These two cosmids differ from the above-mentioned vectors in that a kanamycin resi ⁇ tance determinant i ⁇ used in one ca ⁇ e 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.
  • Example 2 Example 2
  • PCR Polymera ⁇ e Chain Reaction
  • the oligonucleotides used must have sequences that are as close as possible to those of the target ⁇ equence -- the P. vulgari ⁇ chondroitinase I gene.
  • An approximation of that ⁇ equence can be derived from the limited available amino acid ⁇ equence data.
  • the first approximation involves choosing an amino acid sequence that has the least degeneracy.
  • Thi ⁇ amino acid ⁇ equence could be encoded by any one of 512 different nucleotide ⁇ equence ⁇ , repre- ⁇ ented as 5' -CAY-TTY-GCN-CAR-AAY-AAY-CCN-3' (SEQ ID NO: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 will con ⁇ titute a nucleotide ⁇ equence that could encode the indicated amino acid ⁇ equence.
  • R stands for purine (A or G)
  • C or T pyrimidine
  • N indicates that any one of the four nucleotides (A T, G, or C) at this position will con ⁇ titute a nucleotide ⁇ equence that could encode the indicated amino acid ⁇ equence.
  • One po ⁇ sible approach would be to synthe ⁇ ize an
  • One of these pools is perfectly matched for the first eleven nucleotides (counting from the 3- end) , and, furthermore, within thi ⁇ pool of four oligonucleotides, one is a perfect match for the first fourteen nucleotide ⁇ .
  • Thi ⁇ 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 chondroitinase I gene.
  • a further aid in the design of oligonucleo ⁇ tides to be used in these PCR experiments is derived from the ob ⁇ ervation that the P. vulgaris 110 kD chodroitinase enzyme appear ⁇ to have a structure that leaves one particular region hypersensitive to proteolytic cleavage.
  • the result of this hydrolysi ⁇ i ⁇ that the normally approximately 110 kD protein i ⁇ split into two predominant species 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 re ⁇ ulting oligopeptides sequenced, affording still more information with which to design oligonucleotides for PCR.
  • This information from the "18 kD” and "90 kD” regions i ⁇ 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.
  • the nucleotide distance between the regions encoding the N-termini of the "110 kD" and "90 kD” entities can be predicted to be approximately 400-500 bp.
  • Two further set ⁇ of oligonucleotide pool ⁇ are then de ⁇ igned with one further con ⁇ ideration:
  • the fir ⁇ t eight oligonucleotides hybridize to one strand of the DNA and, during the in vitro DNA synthesis, they are extended toward the "90 kD" N-terminal coding sequence ⁇ . Consequently, the oligonucleotides corre ⁇ ponding to amino acid sequences from within the "18 kD" peptide and at the N-terminus of the "90 kD" peptide must be designed so that they anneal to the complementary DNA strand of the P. vulgaris genome, so that they extend, in vitro, toward the region encoding the N-terminus of the intact protein.
  • the oligonucleotides effectively "bracket" the region of the P. vulgaris chromosome that encodes the N-terminal region of the chondroitinase 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. vulgari ⁇ genomic DNA a ⁇ a template which will yield, potentially, microgram amounts of synthesized product which can be readily visualized, isolated and cloned.
  • 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 NO:2, amino acids 138-144) .
  • This heptapeptide is encoded by the following nucleotide sequences:
  • the complementary strand therefore, has the following sequences:
  • 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:
  • 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. vulgaris encoding chondroitina ⁇ e I.
  • oligonucleotide mixtures For a third ⁇ et of oligonucleotide mixtures, the following amino acid sequence, obtained as part of the N-terminal amino acid sequence of the "90 kD" peptide, is u ⁇ ed: Gly-Ala-Ly ⁇ -Val-A ⁇ p-Ser (SEQ ID NO:2, amino acid ⁇ 189-194) .
  • Thi ⁇ hexapeptide can be encoded by the following nucleotide sequences:
  • one base is deleted from the 5' end of oligonucleotides 17-24 in order to reduce the number of ⁇ equence permutations.
  • one oligonucleotide mixture ha ⁇ half of it ⁇ member ⁇ perfectly matched for the fir ⁇ t 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.
  • 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 obtained. This is resu ⁇ pended in 0.5 ml of water to yield a solution that contains approximately 50-60 pmoles of oligonucleotide per microliter. The remaining sample (oligonucleotide #20) contains 15
  • a typical 50 ⁇ l PCR reaction contains approximately 20 ng of P. vulgaris genomic DNA as template; 200 ⁇ M each of dATP, dGTP, dCTP, dTTP; 50mM KCl; lOmM Tris-HCl (pH 8.4); 1.5 mM MgCl 2 ; 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/Cetu ⁇ ThermalcyclerTM.
  • the instrument is programmed to denature the template DNA at 94°C for 1.25 minutes, anneal the oligonucleotide primers to the denatured template at 60°C or 62°C for one minute, and to extend these primers via DNA synthesis at 72°C for 2.25 minutes. Thirty such cycles are carried out in an experimental amplification.
  • the products are analyzed by running an aliquot on a 4% NuSieveTM (FMC Biochemicals, Rockland, ME) GTG gel containing approximately 0.5 ⁇ g/ml ethidium bromide using either Tris-borate or Tri ⁇ -acetate buffers at either full or half strength. These gels are usually run overnight at approximately lV/cm and photographed on a long wavelength UV transilluminator u ⁇ ing a red filter and Polaroid Type 57 film.
  • NuSieveTM FMC Biochemicals, Rockland, ME
  • PCR experiment ⁇ are run te ⁇ ting the pairwi ⁇ e combination ⁇ between oligonucleotide pool ⁇ #1-8 (derived from the "110 kD" amino-terminal ⁇ equence of chondroitina ⁇ e I) , pool ⁇ #9-16 (derived from a peptide sequence contained 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 are between oligonucleotide pools #4 and #18, and pools, #4 and #9,10,11, or 12.
  • oligonucletide pools #4 and #18 yield a product of approximately 500 bp as estimated relative to size standards (pBR322 digested with MSP-1 (New England Biolabs, Beverly, MA) ranging from 30 to 700 bp on NuSieveTM 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.
  • the larger product could be isolated from an agarose gel, diluted a thousand-fold, and then used as the template in a second PCR reaction employing oligonucleotide pools #4 and #9 as primers, which yield a product of approximately 350 bp. That is, the smaller PCR product is synthesized from the larger one in agreement with what would be expected if these ⁇ equence ⁇ were all derived from the P. vulgaris chondroitinase 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 i ⁇ then subjected to a "fill-in" reaction (11) to remove the extra, protruding adenine residue that Tag DNA polymerase tends to add to the 3'-end of DNA in a template- independent reaction (12) .
  • the isolated DNA is then treated with T. polynucleotide kinase to add a phosphate moiety to the 5' -ends of the PCR products to allow them to be joined to the vector DNA.
  • the PCR product is Iigated to pIBI24, a high copy vector containing a polylinker (IBI, New Haven, CT) , that is first sequentially digested with Pstl. "filled-in” and then treated with calf intestinal alkaline phosphatase (Boehringer- Mannheim) .
  • pIBI24 a high copy vector containing a polylinker
  • Pstl. "filled-in” and then treated with calf intestinal alkaline phosphatase (Boehringer- Mannheim) .
  • An eight residue oligopeptide derived from the DNA sequence (SEQ ID NO:2, amino acids 71-78) also matche ⁇ a previou ⁇ ly sequenced oligopeptide derived by a combination of trypsin digestion and cyanogen bromide treatment of the native protein.
  • the problems include: (1) the assumption that the protein being sequenced has not been processed at either end (not likely to be true, for example, with a secreted protein) , (2) the occasional lack of fidelity exhibited by Tag 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.
  • a total of approximately 260 ⁇ g of plasmid DNA is digested with Sail and the products separated by electrophoresis on a NuSieveTM GTG agaro ⁇ e gel.
  • the desired approximately 450 bp fragment is isolated using a QiaexTM extraction protocol.
  • the fragment is then denatured by heating at 95-100°C 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 ⁇ l reactions. Aliquots of the six P. vulgaris cosmid gene banks described in Example 1 above are used to infect the E.
  • coli strain ER1562 described above and a total of approximately 10,000 colonies are obtained on the appropriate selective plates. These colonies (on a total of 50 plates) are replica plated onto two nylon membranes 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 sulfate (SDS) and 0.5 M NaOH for 5-30 minute ⁇ each. The cells from the lysed colonies are neutralized by being placed on sheet ⁇ ⁇ aturated with 1 M Tris-HCl (pH 7.4) (twice) and then on paper saturated with 2X standard saline citrate prior to vacuum drying at 80°C. The DNA from the lysed colonies is then fixed to the membranes.
  • SDS sodium dodecyl sulfate
  • the filters are then washed by incubation of the filters at 42°C with agitation for 1-3 hours, using at least 10 ml/filter of 0.05 M Tris HCI, 0.5-1 M 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-hybrid!zed by incubation with a hybridization buffer at 65°C for 1-3 hour ⁇ .
  • the filters are then hybridized overnight at 65-68°C using the digoxigenin-labeled probe described above (0.5-50 ng/ml in a hybridization solution) .
  • the hybridized filters are wa ⁇ hed with SSC and SDS, re-blocked with a blocking reagent (Component #11 of DNA Labelling and Detection Kit, Nonradioactive, Boehringer Mannheim, Indianapolis, IN) and exposed to polyclonal sheep anti-digoxigenin Fab fragments conjugated to alkaline phosphatase.
  • a blocking reagent Component #11 of DNA Labelling and Detection Kit, Nonradioactive, Boehringer Mannheim, Indianapolis, IN
  • 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) .
  • BCIP bromo-chloro-indolyl-phosphate
  • 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.
  • the developed filters are used a ⁇ templates to guide the ⁇ election of a total of 117 clone ⁇ which are then picked to ⁇ elective 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.
  • 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. vulgaris 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 thi ⁇ nucleotide analogue in a PCR amplification.
  • thi ⁇ reaction the gel- purified product of a previous PCR amplification (that using P. vulgaris genomic DNA as template) is diluted 10,000-fold and serve ⁇ a ⁇ the template in a second PCR amplification.
  • Thi ⁇ latter reaction i ⁇ 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 #10 (see above) as the primers.
  • the normal complement of deoxyribonucleoside tripho ⁇ phate ⁇ i ⁇ replaced with a digoxigenin-dUTP labeling mixture from the manufacturer (Boehringer-Mannheim, Indianapoli ⁇ , IN) , which yields a final concentration of 100 ⁇ 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 de ⁇ cribed above.
  • the DNA (approximately 5 ⁇ l) is diluted into large (0.35 ml) volume ⁇ for dige ⁇ tion with the variou ⁇ re ⁇ triction enzyme ⁇ .
  • the DNA i ⁇ then concentrated by ethanol precipitation prior to fractionation on agaro ⁇ e gels and transfer to nylon membranes.
  • the data obtained in these experiments indicates that the chondroitinase 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 approximately 2800 bp, an EcoRV fragment of 5400 bp, and on large (equal to or greater than approximately lOkb) DNA fragments generated by Nsil. Bglll, Hindlll, and Stvl.
  • Two of these fragments are of special interest.
  • the first, a BstYI fragment of approximately 2800 bp, is observed in a number of cosmid clones, including those designated #2 and #45.
  • the DNA i ⁇ olated from these two cosmid clones i ⁇ designated LP 2 751 and LP 2 760.
  • LP 2 760 With LP 2 760, the approximately 2800 bp BstYI fragment is well separated from the other BstYI fragments and i ⁇ therefore more readily ⁇ ubcloned into another vector de ⁇ ignated pT660-3.
  • the plasmid designated pT660-3 is a derivative of pBR322 in which the DNA from a point immediately downstream of the promoter for tetracycline resistance (approximately bp 80) as far as the PvuII site (approximately bp 2070) is deleted and replaced with a BamHl linker.
  • the approximately 10 kb Nsil fragment (which hybridizes with the chondroitinase probe described above) is readily i ⁇ olated from a digest performed on LP 2 751. These two fragments are referred to as the "2800 bp BstYI" fragment and the "10 kb Nsil" 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 chondroitinase 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 (les ⁇ than 500 bp) compared to the predicted size of the intact gene (greater than 3000 bp) , thi ⁇ is not a trivial consideration.
  • the nucleotide sequence 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 site ⁇ , which ⁇ ugge ⁇ t ⁇ that the resulting fragments might be small enough for DNA sequencing.
  • the EcoRV sites are symmetrically placed within the 2800 bp BstYI fragment; each EcoRV site is approximately 1200 bp from one end, with the ⁇ pace between them equal to approximately 400 bp.
  • the ⁇ ubcloned fragment is digested asymmetrically by taking advantage of unique restriction sites present within the vector.
  • the "halves" of the 2800 bp B ⁇ tYI fragment are distinguished physically and, by Southern hybridi ⁇ zation, the "end" that contains the chondroitinase I N-terminal coding region is ascertained.
  • the appropriate piece which is a Hindlll-EcoRV fragment of approximately 1200 bp, is subcloned into both M13mpl8 and M13mpl9 vectors which are fir ⁇ t digested with both Hindlll and Smal and subsequently treated with calf intestinal alkaline phosphatase.
  • the DNA sequence derived from these subclone ⁇ reveal ⁇ a number of feature ⁇ that clearly establish the location of the chondroitinase I gene, as well as the direction in which it is read.
  • 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. vulgaris chondroitina ⁇ e I enzyme.
  • thi ⁇ sequence it is possible to discern a number of other features by their analogy to corresponding ⁇ equence motif ⁇ from previou ⁇ ly analyzed E. coli genes.
  • nucleotides 32-37 SEQ ID NO:l, nucleotide ⁇ 40-45
  • nucleotides 98-103 SEQ ID NO:l, nucleotide ⁇ 106-111
  • there i ⁇ an in-frame ATG initiation codon at nucleotide ⁇ 111-113 SEQ ID N0:1, nucleotide ⁇ 119-121) , which indicates that the P.
  • vulgaris chondroitinase I enzyme is synthe ⁇ ized with a 24 amino acid ⁇ ignal sequence which is, pre ⁇ umably, removed a ⁇ the protein i ⁇ transported across the inner membrane.
  • the second fragment that is subcloned (into a pIBI24 derivative that is fir ⁇ t modified to include an Nsil re ⁇ triction ⁇ ite in place of the P ⁇ tl ⁇ ite normally pre ⁇ ent in the polylinker of thi ⁇ vector) i ⁇ the approximately 10 kb Nsil fragment.
  • a double digestion with EcoRV and Hindlll 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 Hindlll. 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 chondroitinase probe, thereby serving to place one of the Hindlll site ⁇ . Since there i ⁇ a Hindlll ⁇ ite in the polylinker, it too can be placed, leaving the last Hindlll site to be placed by deduction.
  • Double digestion of the cloned approximately 10 kb Nsil fragment with EcoRV and EcoRl 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 EcoRl site ⁇ -- one in the polylinker and one in the cloned P. vulgaris DNA.
  • Southern hybridization reveals that the approximately 4.2 kb band in this double digest contains the chondroitinase I N-terminal coding sequence. Adding this information to the above data yields a preliminary restriction map for the subcloned approximately 10 kb Nsil fragment in pIBI24 ( Figure 1) .
  • This culture is then inoculated into fresh selective media either with or without isopropyl-beta-D-thiogalactopyrano ⁇ ide (IPTG) which is expected to increase the level of transcription from the lac promoter pre ⁇ ent in pIBI24.
  • IPTG isopropyl-beta-D-thiogalactopyrano ⁇ ide
  • the approximately 10 kb Nsil fragment, cloned into pIBI24, is digested with EcoRV (as described above) and Iigated together in the presence of EcoRI linkers.
  • the net result of this construction is the deletion of approximately 5 kb of P. vulgaris DNA from this subcloned piece of DNA and the simultaneous introduction of another EcoRI site into the molecule.
  • One hundred micrograms of thi ⁇ "EcoRV deletion" con ⁇ truction (LP 2 786) i ⁇ 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 ⁇ l TE described above.
  • One-third of this material is then Iigated 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.
  • coli strain MV1190 and 500 of the phage plaques obtained are picked into SM buffer (NaCl, 100 mM, MgS0 4 , 8 mM, Tris-HCl, pH 7.4, 50 mM and 0.01% gelatin) to serve as stock ⁇ for the infection of ⁇ mall (le ⁇ than or equal to 10 ml) cultures that are then used for the isolation of single stranded template DNA.
  • SM buffer NaCl, 100 mM, MgS0 4 , 8 mM, Tris-HCl, pH 7.4, 50 mM and 0.01% gelatin
  • DNA sequencing is carried out at elevated temperatures using Tag DNA polymerase and fluorescently-labeled oligonucleotide primer ⁇ .
  • the data are collected u ⁇ ing a Model 370A DNA ⁇ equencing ⁇ y ⁇ tem (Applied Biosysterns, Foster City, CA) .
  • Sequence editing, overlap determinations and derivation of a consensus sequence are performed using a collection of computer programs obtained from the
  • 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 Iigated into this site; it is not present in the P. vulgaris chromosome. This position actually is an EcoRV site in the cloned cosmid DNA.
  • the site-specific mutagenesis method employed is based on that of Kunkel (15) , using materials purcha ⁇ ed from Bio-Rad, Melville, N.Y.
  • This oligonucleotide serve ⁇ as a primer for T7 DNA polymerase which copies the entire recombinant molecule.
  • T4 DNA ligase is then used to seal the nick between the first residue of the mutagenic oligonucleotide and the last residue added in vitro.
  • the newly synthesized DNA (containing the desired base changes) therefore does not contain uracil, while the template DNA does. Transformation of a non-mutant (with respect to the dut and ung alleles) male E. coli strain yields phage progeny that are primarily derived from the mutagenized strand synthe ⁇ ized in vitro a ⁇ a re ⁇ ult of the inactivation of the uracil-containing template ⁇ trand.
  • resuspended plaques (aliquots of which had been used for DNA sequencing which established the N-terminal coding region of the chondroitinase 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 ung) .
  • Individual plaques are picked to 0.5 ml of phage dilution buffer (PDB) .
  • PDB phage dilution buffer
  • One picked plaque 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 55°C for 30 minutes and then stored at 4°C.
  • Single stranded 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 to modify the "ends" of thi ⁇ gene to allow it to be moved, precisely, into an appropriate high-level E. coli expression system.
  • the target vector chosen (pET9-A; see above) is one derived from genetic regulatory elements present in the bacteriophage T7. In this sytem, there i ⁇ a unique Ndel site (CATATG) that includes the translation initiation codon as well as a downstream Ba Hl site that, together, allow the direct, unidirectional, insertion of a gene encoding the protein that is to be expressed.
  • 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: 1) 5' -GCCAGCGTTTCTAAGGAGAAAAATAATGCCGATATT- TCGTTTTACTGC-3' (SEQ ID NO:l, nucleotides 94-141)
  • 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 nucleotide ⁇ , including the location of the initiation codon) :
  • the underlined GCC in line 3 corresponds to the codon for alanine which is the N-terminal amino acid for the mature, processed form of the P. vulgaris chondroitinase I.
  • oligonucleotide # 25 (5 O.D. units) is resuspended with 0.5 ml of TE
  • oligonucleotide # 26 (also 5 O.D. units) is resuspended in 0.65 ml TE to yield stock ⁇ that are approximately 20 nM, i.e., 20 pmole/ ⁇ l.
  • Template DNA (5 ⁇ l of the preparation de ⁇ scribed above) and pho ⁇ phorylated mutagenic primer (approximately 2 pmole) are annealed in a 20 ⁇ l volume containing 20 mM Tris-HCl (pH 7.4), 2 mM MgCl 2 , and 50 mM NaCl.
  • the sample is heated at 70°C for 45 minutes in a Perkin-Elmer/Cetus ThermalcyclerTM.
  • the sample is then gradually cooled from 70°C to 25°C over a 45 minute period.
  • the annealed mixture is placed on ice and the following components added: 2 ⁇ l of 10 X synthesis buffer (Bio-Rad) : 5mM each of dATP, dGTP, dCTP, dTTP; 10 mM ATP; lOO M Tris-HCl (pH 7.4); 50 mM MgCl 2 ; 20 mM dithiothreitol) , 2 ⁇ l of T4 DNA ligase (6 units) and 1 ⁇ l of T7 DNA polymerase (1 unit) . These reactions are incubated for 5 minutes each at 0°C (on ice), 11°C, 25°C, and finally for 30 minutes at 37°C.
  • 10 X synthesis buffer Bio-Rad
  • 5mM each of dATP, dGTP, dCTP, dTTP 10 mM ATP; lOO M Tris-HCl (pH 7.4)
  • 50 mM MgCl 2 20 mM di
  • Example 6 described the site- ⁇ pecific mutageneses that created an Ndel site immediately preceeding the signal sequence, as well as a second construction which placed the Ndel site adjacent to the triplet which codes for the N-terminal alanine found on the mature, processed P. vulgaris chondroitinase I gene.
  • the ATG sequence of the Ndel recognition site can function as the translation initiation codon for the protein (either with or without the signal sequence) .
  • the isolated replicative form is digested with Kpnl and Clal.
  • the Kpnl site is part of the M13mpl9 polylinker, while the Clal site is found approximately 490 bp from the end of the cloned fragment of the chondroitinase I gene.
  • the restriction digestion products obtained are fractionated on a 4% NuSieveTM GTG agarose gel run in 1/2 X Tris-Acetate buffer (TAE) . The appropriate approximately 500 bp band is extracted from the gel u ⁇ ing QiaexTM.
  • plasmid DNA (LP 2 786) carrying the chondroitinase I gene is also digested with Kpnl and Clal and then fractionated on a 0.8% agarose gel run in 1/2 X TAE.
  • the Kpnl site is part of the polylinker of pIBI24, while the Clal site corresponds to the one described above.
  • Clal site corresponds to the one described above.
  • the approximately 7 kb fragment containing the pIBI24 vector and the large fragment of the chondroitinase 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 phosphata ⁇ e, 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 encoding fragments are each Iigated to the approximately 7 kb fragment encompassing the remainder of the chondroitinase I gene and the pIBI24 vector.
  • the ligase reaction is 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 Ndel to verify the presence of this restriction site within the reconstructed DNA. In order to remove the (apparent) P.
  • the modified chondroitina ⁇ e I genes are isolated as approximately 4.5 kb Ndel-Nsil fragments and ⁇ ubcloned into a pBR322 variant in which the EcoRI ⁇ ite i ⁇ fir ⁇ t filled-in, then depho ⁇ phorylated, and finally a phosphorylated Nsil linker (New England Biolabs) inserted.
  • the sequence of the linker used (TGCATGCATGCA) to place the Nsil site (ATGCAT) into pBR322 also includes an SphI site (GCATGC) .
  • pla ⁇ mid ⁇ (repre ⁇ enting two clones each with the signal sequenc retained [LP 2 861 and LP 2 863] and two with the signal ⁇ equence deleted [LP 2 865 and LP 2 867] ) containing the approximately 4500 bp Ndel-Nsil segment ⁇ including the chondroitina ⁇ e I gene are first digested with SphI, the end ⁇ "filled-in” with the "Klenow" fragment (11) of the E.
  • Ndel-BamHl fragment which contains the chondroitinase I gene. Seventeen clones (eight with and nine without the signal sequence) yield the desired fragment which is extracted from the agarose gel with QiaexTM. These approximately 3.4 kb Ndel-Bam-HI chondroitina ⁇ e I gene-containing fragments (both with and without the signal sequence) are then used to construct a high-level expres ⁇ ion system.
  • chondroitina ⁇ e I gene fragments (both with and without the signal sequence) is Iigated to the expression vector fragment.
  • the resulting recombinant DNA mixture is used to transform the E. coli K-12 host, HMS174 (Novagen) .
  • Kanamycin- resistant colonies obtained 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 expres ⁇ ion host BL21(DE3)/pLysS (10).
  • T7 lysozyme is expres ⁇ ed at a relatively low level in this construction and serves as an inhibitor of the T7 RNA Polymerase (16) , thereby minimizing the basal-level expres ⁇ ion of the gene to be overexpressed.
  • 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 30°C 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 containing M9 salts (17) supplemented with 20 g/1 tryptone, 10 g/1 yeast extract, and 10 g/1 dextrose in addition to the same level of kanamycin and chloramphenicol.
  • the culture is grown at 30°C to an appropriate density (a value of 1 at A 600 ) and then chondroitinase I expres ⁇ ion is induced by the addition 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 as ⁇ ay. The frozen pellets are thawed, resuspended in buffer and sonicated. A value of 56 unit ⁇ /ml i ⁇ obtained
  • the bacterial cells are fir ⁇ t recovered from the medium and resuspended in buffer.
  • the cell suspension is then homogenized to lyse the bacterial cells.
  • a charged particulate such as 50 ppm Bioacryl (Toso Haas, Philadelphia, PA) , is added to remove DNA, aggregates and debris from the homogenization step.
  • the solution is brought to 40% saturation of ammonium sulfate to precipitate out undesired proteins.
  • the chondroitina ⁇ e I remain ⁇ in ⁇ olution.
  • the ⁇ olution is then filtered using a 0.22 micron SP240 filter (Amicon, Beverly, MA) , and the retentate is washed using nine volumes of 40% ammonium ⁇ ulfate solution to recover most of the enzyme.
  • the 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 containing chondroitinase I is subjected to cation exchange chromatography using a CellufineTM cellulo ⁇ e ⁇ ulfate column (Chi ⁇ o Corporation, di ⁇ tributed by Amicon) .
  • a CellufineTM cellulo ⁇ e ⁇ ulfate column Cho ⁇ o Corporation, di ⁇ tributed by Amicon
  • pH 7.2 20 mM ⁇ odium pho ⁇ phate
  • more than 98% of the chondroitina ⁇ e I binds to the column.
  • the native chondroitinase I is then eluted from the column using a 0 to 250 mM sodium chloride gradient, in 20 mM sodium phosphate buffer.
  • chondroitinase I i ⁇ obtained at a purity of 90-97% as measured by SDS-PAGE scanning (see above) .
  • the yield of the native protein is only 25-35%, determined as described above. This method also results in the cleavage of the approximately 110 kD chondroitinase I protein into a 90 kD and an 18 kD fragment. Nonetheless, the two fragments remain non-ionically bound and exhibit chondroitinase I activity.
  • the host cells which express the recombinant chondroitinase I enzyme are homogenized to lyse the cells. This releases the enzyme into the supernatant.
  • the supernatant is first subjected to diafiltration to remove salts and other small molecules.
  • a suitable filter is a spiral wound 30 kD filter made by Amicon (Beverly, MA) .
  • 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- containing column.
  • a strong, high capacity anion exchange resin- containing column An example of such a resin is the Macro-PrepTM High Q resin (Bio-Rad, Melville, N.Y.) .
  • Other strong, high capacity anion exchange 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.
  • the eluate from the anion exchange column i ⁇ directly loaded to a cation exchange resin- containing column.
  • cation exchange resin- containing column examples include the S- Sepharo ⁇ eTM (Pharmacia, Pi ⁇ cataway, N.J.) and the Macro-PrepTM High S (Bio-Rad) .
  • S- Sepharo ⁇ eTM Pulcoa, Pi ⁇ cataway, N.J.
  • Macro-PrepTM High S Bio-Rad
  • Each of the ⁇ e two resin-containing columns has S0 3 " ligands bound thereto in order to facilitate the exchange of cations.
  • Other cation exchange 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.
  • Any salt which increase ⁇ 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 a ⁇ 0 to 250 mM sodium chloride is acceptable, as i ⁇ a step elution using 200 mM sodium chloride.
  • the purity of the protein is measured by scanning 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 i ⁇ scanned using the procedure described above.
  • two additional ⁇ tep ⁇ 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 4.5, to precipitate out the desired enzyme.
  • the pellet is obtained 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 i ⁇ then subjected to the diafiltration and subsequent step ⁇ of the fir ⁇ t embodiment of this invention.
  • Acid precipitation removes proteins that remain soluble; however, these proteins are removed anyway by the cation and anion exchange ⁇ tep ⁇ 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.
  • the additional acid precipitation and alkali dissolution steps of the second embodiment mean that the ⁇ econd embodiment is more time consuming than the first embodiment.
  • 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.
  • 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 represent ⁇ molecular weight standards.
  • the approach taken in the case of the chondroitinase II gene is to modify the naturally- occurring ATG initiation codon to embed it within an Ndel site. Thi ⁇ re ⁇ ults in a construction in which the signal peptide is retained, 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 bases are upstream of the coding region.
  • the method used for this ⁇ ite-specific alteration is that described above for the expres ⁇ ion of the chondroitina ⁇ e I gene and i ⁇ based on the work of Kunkel (15) using the Muta-GeneTM In Vitro Mutagenesis Kit Version 2 (Bio-Rad, Melville, N.Y.).
  • 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 ung alleles.
  • dut duTPase
  • ung uracil-N-glyco ⁇ yla ⁇ e
  • T7 DNA polymerase which copies the entire recombinant molecule.
  • T4 DNA ligase is then used to seal the nick between the fir ⁇ t residue of the mutagenic oligonucleotide and the last residue added in vitro.
  • the newly synthe ⁇ ized DNA (containing the de ⁇ ired 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 ung and dut alleles) male E. coli ⁇ train yields phage progeny that are primarily derived from the mutagenized strand synthesized in vitro as a re ⁇ ult of the inactivation of the uracil-containing template strand.
  • the fragment to be cloned for the mutagenesis is a Muni-EcoRI fragment that span ⁇ the region between nucleotide ⁇ 2943 to 3980 (SEQ ID NOS:l and 39) .
  • the DNA dige ⁇ ted to obtain this fragment is designated LP 2 783.
  • Thi ⁇ plasmid is constructed in the same way as LP 2 786 (described in Example 4) , except that a Hindlll linker is inserted into the EcoRV deletion of LP 2 776 rather than the EcoRI linker.
  • the four ba ⁇ e overhang produced by Muni digestion can be Iigated to an EcoRI site, but the re ⁇ ulting recombinant ⁇ equence cannot be dige ⁇ ted by either enzyme.
  • the EcoRI dige ⁇ ted LP 2 941 i ⁇ also dephosphorylated with calf inte ⁇ tinal alkaline pho ⁇ phatase (Boehringer Mannheim, Indianapolis IN) prior to gel purification and use.
  • the Iigated DNA mixture is used to infect the male E. coli strain MV1190 and the plaques obtained 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 viru ⁇ . The ⁇ upernatant ⁇ , which contain the corresponding phage particles, are ⁇ tored under refrigeration until needed. The orientation of the cloned fragment is determined by digestion of the replicative form DNA and Hindlll. because there is one site within the polylinker and a second, aymmetrically placed site (SEQ ID NOS:l and 39, nucleotides 3326-3331) within the above Muni-EcoRI fragment.
  • the corresponding phage-containing 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 phage-containing supernatant using QiaexTM column ⁇ and material ⁇ and methods recommended by the manufacturer (Qiagen, Chatsworth, CA) and finally re ⁇ uspended in a volume of 0.01 ml.
  • the recombinant phage are grown on CJ236 (dut " ung " ) for two rounds in order to maximize the accumulation of uracil residues in the template and strand prior to the actual site- ⁇ pecific mutagenesis.
  • the mutagenic oligonucleotide used is obtained from Bio-Synthesis (Denton, TX) and has the following sequence:
  • This sequence differs from the corresponding region of SEQ ID NOS:l and 39 in that an AT sequence (base pairs 3235 and 3236) is replaced by a CA sequence which creates the desired Ndel sequence (CATATG) at the start of the presumed leader sequence for the chondroitinase II gene.
  • AT sequence base pairs 3235 and 3236
  • CA sequence which creates the desired Ndel sequence (CATATG) at the start of the presumed leader sequence for the chondroitinase II gene.
  • One optical density unit of this oligonucleotide is dissolved in 0.46 ml. of TE 7.4 (0.01M TrisHCl, pH 7.8-0.001M EDTA, pH 8.0), yielding an oligonucleotide concentration of approximately 6 pmol/ ⁇ l.
  • Three hundred picomole ⁇ of this oligonucleotide are phosphorylated in a 0.1 ml reaction containing 0.05 M TrisHCl, pH 7.8, 0.01 M MgCl 2 , 0.02M dithiothreitol, 0.001 M ATP, 25 ⁇ g/ml bovine serum albumin and 100 units of T4 polynucleotide kinase (New England Biolab ⁇ ) at 37°C for 30 minute ⁇ , followed by incubation at 75° for 20 minute ⁇ to inactivate the enzyme.
  • the pho ⁇ phorylated oligonucleotide i ⁇ then ⁇ tored frozen at -20° at a concentration of approximately 3 pmoles/ ⁇ l.
  • 1 ⁇ l (3 pmole) of the mutagenic oligonucleotide is mixed with 6 ⁇ l of the single-stranded DNA prepared above in a 10 ⁇ l volume of 0.02 M TrisHCl, pH 7.4, 0.002 M MgCl 2 , 0.05 M NaCl.
  • the oligonucleotide is annealed to this template by fir ⁇ t incubating the ⁇ ample at 70°C for 5 minute ⁇ and then cooling thi ⁇ ⁇ ample at 25°C over a 45 minute period in a DNA Thermal CyclerTM (Perkin-Elmer Cetus/Norwalk, CT) .
  • the sample is maintained at 25°C for another 5 minute ⁇ before being cooled to 20°C and finally transferred to an ice bath.
  • the annealed primer is then extended after the addition of 1 ⁇ l of 10X synthesi ⁇ buffer (Bio-Rad; containing 0.005 M of each of the dNTP' ⁇ , 0.01 M ATP, 0.1 M Tri ⁇ HCl, pH 7.4, 0.05 M MgCl 2 , 0.02 M DTT).
  • 10X synthesi ⁇ buffer Bio-Rad; containing 0.005 M of each of the dNTP' ⁇ , 0.01 M ATP, 0.1 M Tri ⁇ HCl, pH 7.4, 0.05 M MgCl 2 , 0.02 M DTT.
  • T4 DNA ligase 3 units/ ⁇ l Bio-Rad
  • T7 DNA polymerase 0.5 units/ ⁇ l Bio-Rad.
  • the in vitro DNA synthesis is carried out on ice for 5 minutes, at 11°C for ten minute ⁇ , and at 37°C for 30 minute ⁇ prior to transfer to ice. This ⁇ ample i ⁇ used directly to transform the male E.
  • coli host MV1190 (dut* ung*) and the resulting plaques, containing the site-specifically mutagenized phage, are obtained, picked and eluted as described above. Aliquots of these phage stock ⁇ 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 QiaexTM columns and methods recommended by the manufacturer (Qiagen, Chatsworth CA) . The DNA isolated is resuspended in 0.1 ml of TE 7.4.
  • the four sample ⁇ are then combined and the DNA extracted from the gel using a QiaexTM resin and buffer ⁇ according to the manufacturer' s recommendations (Qiagen, Chat ⁇ worth 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. vulgaris gene for the chondroitinase II gene is then subcloned into the plasmid pNEB193 (New England Biolabs, Beverly MA) between the (dephosphorylated) unique Ndel and EcoRI sites present in this plasmid.
  • the DNA sample from one of the positive clones is designated m#15-5712. This sample represents the modified N- terminal region that is to be joined to the C-terminal coding region for the chondroitinase II gene, which is described in Example 12.
  • the DNA sequence contained in SEQ ID NO: 39 indicates that chondroitinase II is encoded by a region that is downstream of that for chondroitinase I. This information is derived from a portion of a 10 kilobase Nsil fragment of P. vulgaris that is ⁇ ubcloned originally from a co ⁇ mid clone designated LP 2 751.
  • the combination of the DNA sequencing and the restriction map in Figure 1 reveal ⁇ that the chondroitinase II coding region initiates to the "left" of the EcoRI site that lies within the P. vulgaris derived DNA and proceeds toward the Nsil site at the "right” end of the fragment depicted in Figure 1. Therefore, this re ⁇ triction map ⁇ hould be expanded to the "right” to find a ⁇ uitable fragment that will include the C-terminal coding region for the chondroitina ⁇ e II gene.
  • digestion ⁇ are carried u ⁇ ing the restriction enzymes Afllll, Clal, EcoRV, and Hindlll each of which has been noted by Applicants to yield eight to ten fragments upon digestion of the original cosmid clone designated LP 2 751.
  • the recombinant molecule carrying the subcloned approximately 10 kb Nsil fragment (LP 2 770) and the individually gel-purified approximately 20 kb EcoRI and approximately 10 kb EcoRI fragments are digested with each of the ⁇ e enzyme ⁇ to yield pattern ⁇ of fragments that are compared.
  • the ⁇ e digestions reveal that the approximately 20 kb EcoRI and the LP 2 770 pattern ⁇ have a number of fragments in common.
  • the second deletion removes the region between the Hindlll site at the other end of the polylinker and the (now unique) PvuII site, maintaining the Hindlll site, while removing the PvuII site.
  • the recombinant DNA molecule carrying the subcloned approximately 10 kb EcoRI fragment in the vector lacpo ⁇ pNEB193 is de ⁇ ignated LP 2 1263.
  • the orientation of the 112 kD C-terminal coding region within LP 2 1263 i ⁇ determined by re ⁇ triction enzyme mapping.
  • the re ⁇ ult ⁇ indicate that thi ⁇ region is positioned so as to proceed from the EcoRI site (defined as the "left" end) toward the Hindlll site at the other end of the polylinker.
  • Thi ⁇ con ⁇ truction also "places" a BamHl site (present in the polylinker) downstream of the coding region for the chondroitinase II gene.
  • This recombinant DNA molecule which carries the chondroitinase II gene from the EcoRI site to (and presumably just beyond) the termination codon for this gene has been designated m#25-5712.
  • DNA sequence analysi ⁇ i ⁇ initiated on the approximately 10 kb EcoRI fragment derived from LP 2 1263 and is completed after the assembly of the intact gene for chondroitinase II.
  • the materials and methods for the DNA sequencing of thi ⁇ fragment are essentially the same a ⁇ tho ⁇ e u ⁇ ed for the approximately 4 kb fragment containing the gene for chondroitina ⁇ e 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 a ⁇ by partial dige ⁇ tion with the re ⁇ triction enzymes Sau3A or Msel.
  • the ⁇ e piece ⁇ are then eventually cloned into M13 derived vector ⁇ and the single- stranded recombinant molecules sequenced using the standard protocols described above. Finally, with the two set of sequence data available, an approximately 300 base-pair Bell fragment is identified that is predicted to contain the EcoRI site that is the junction between the two P. vulgaris fragments of approximately 20 kb and approximately 10 kb obtained by dige ⁇ tion with EcoRI. This small fragment is sequenced in both directions to verify the nucleotide sequence through this junction point used in the con ⁇ truction ⁇ described below.
  • the molecule designated m#25-5712 is digested with EcoRI and BamHl, This releases a DNA fragment of approximately 2.6 kb.
  • the construction designated m#15-5712 is digested with EcoRI and BamHl and then dephosphorylated prior to purification by gel electrophore ⁇ is.
  • the latter molecule therefore carries the N-terminal coding region of the chondroitinase II gene from the ATG initiation codon (now present as part of an Ndel site from the site- specific mutagenesis) to the EcoRI site.
  • the coding region of the chondroitinase II gene includes nucleotides 3238-6276 of the SEQ ID NO: 39, which encodes 1013 amino acids (SEQ ID NO:40) .
  • nucleotide ⁇ 3238-3306 encode the 23 amino acid ⁇ ignal peptide (SEQ ID NO:40, amino acid ⁇ 1-23)
  • nucleotide ⁇ 3307-6276 encode the mature 990 amino acid chondroitina ⁇ e II protein (SEQ ID NO:40, amino acid ⁇ 24-1013) .
  • restriction analysis with Sau3AI reveals a multiplicity of site ⁇ , including tho ⁇ e at SEQ ID NO:39, nucleotide ⁇ 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 chondroitina ⁇ e II gene in ⁇ erted into pET9A) obtained in thi ⁇ experiment i ⁇ grown in large ⁇ cale (0.5 liter) and the expre ⁇ ion system containing the chondroitinase II gene isolated and designated LP 2 1359.
  • the re ⁇ ulting strain is designated TD112 and is used for large-scale fermentation and isolation of the chondroitinase II enzyme. A fermentation at a 10 liter scale carried out with this E.
  • coli strain containing the plasmid expressing the chondroitinase II protein provide ⁇ a maximum chondroitina ⁇ e II titer of approximately 0.3 mg/ml, which i ⁇ approximately 25 time ⁇ that of the approximately 0.012 mg/ml obtained from the native P. vulgari ⁇ fermentation proce ⁇ for chondroitina ⁇ e II.
  • the initial part of this method is the same as that used for the recombinant chondroitinase I enzyme.
  • the host cells which express the recombinant chondroitinase II enzyme are homogenized to lyse the cells. This releases the enzyme into the supernatant.
  • the supernatant is first subjected to diafiltration to remove salts and other small molecules.
  • a ⁇ uitable filter is a spiral wound 30 kD filter made by Amicon (Beverly, MA) .
  • Amicon Billerly, MA
  • this ⁇ tep only removes the free, but not the bound form of the negatively charged molecules.
  • the bound form of the ⁇ e charged species is removed by pas ⁇ ing the supernatant (see the SDS-PAGE gel depicted in Figure 5, lane 1) through a strong, high capacity anion exchange resin- containing column.
  • An example of such a resin is the Macro-PrepTM High Q re ⁇ in (Bio-Rad, Melville, N.Y.) .
  • Other ⁇ trong, high capacity anion exchange column ⁇ are al ⁇ o suitable.
  • the negatively charged molecules bind to the column, while the enzyme passes through the column with approximately 90% recovery of the enzyme. It is also found that some unrelated, undesirable proteins also bind to the column.
  • the method diverges from that used for the chondroitinase I protein.
  • a specific elution using a solution containing chondroitin sulfate is used.
  • a 1% concentration of chondroitin sulfate is used; however, a gradient of this solvent is also acceptable.
  • the specific chondroitin sulfate solution is preferred to the non-specific salt solution becau ⁇ e the recombinant chondroitina ⁇ e II protein i ⁇ expressed at levels approximately several-fold lower than the recombinant chondroitinase I protein; therefore, a more powerful and selective solution is necessary in order to obtain a final chondroitinase II product of a purity equivalent to that obtained for the ' chondroitinase I protein.
  • the cation exchange column i ⁇ next washed with a phosphate buffer, pH 7.0, to elute unbound proteins, followed by washing 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 chondroitinase II protein using the ⁇ ub ⁇ trate, chondroitin ⁇ ulfate.
  • a phosphate buffer pH 7.0
  • borate buffer pH 8.5
  • chondroitin sulfate a 1% solution of chondroitin sulfate in water, adjusted to pH 9.0, is used to elute the chondroitinase II protein, as a sharp peak (recovery 65%) and at a high purity of approximately 95% ( Figure 5, lane 3) .
  • the chondroitin sulfate has an affinity for the chondroitinase II protein which is stronger than its affinity for the resin of the column, and therefore the chondroitin sulfate co- elutes with the protein.
  • Thi ⁇ ensures that only protein which recognizes chondroitin sulfate is eluted, which is desirable, but also means that an additional proce ⁇ ⁇ tep is neces ⁇ ary to separate the chondroitin sulfate from the chondroitinase II protein.
  • the eluate is adju ⁇ ted to pH 7.0 and i ⁇ loaded a ⁇ is onto an anion exchange resin-containing column, such as the Macro- PrepTM High Q resin.
  • the column is washed with a 20 mM phosphate buffer, pH 6.8.
  • the chondroitin sulfate binds to the column, while the chondroitina ⁇ e II protein flow ⁇ through in the unbound pool with greater than 95% recovery.
  • the protein i ⁇ pure, except for the pre ⁇ ence of a ⁇ ingle minor contaminant of approximately 37 kD ( Figure 5, lane ⁇ 4 and 6) .
  • the contaminant may be a breakdown product of the chondroitinase II protein.
  • This contaminant is effectively removed by a crytallization step.
  • the eluate from the anion exchange column is concentrated to 15 mg/ml protein using an Amicon stirred cell with a 30 kD cutoff.
  • the solution is maintained at 4°C for ⁇ everal days to crystallize out the pure chondroitinase II protein.
  • the supernatant contains the 37 kD contaminant ( Figure 5, lane 7) .
  • Centrifugation causes the crystal ⁇ to form a pellet, while the ⁇ upernatant with the 37 kD contaminant i ⁇ removed by pipetting, and the cry ⁇ tal ⁇ wa ⁇ hed twice with water.
  • two additional step ⁇ are inserted in the method for purifying the chondroitinase II enzyme before the diafiltration step of the fir ⁇ t embodiment.
  • the supernatant i ⁇ 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 obtained by centrifugation at 5,000 x g for 20 minutes.
  • the pellet is then dissolved in an alkali solution, such as 20-30 mM
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • CAC AAC GTA AAG CCA CAA CTA CCT GTA ACA CCT GAA AAT TTA GCG GCC 886 His Asn Val Lys Pro Gin Leu Pro Val Thr Pro Glu Asn Leu Ala Ala 245 250 255
  • GGC AGA CAT CTG ATC ACT GAT AAA CAA ATC ATT ATT TAT CAA CCA GAG 1078 Gly Arg His Leu He Thr Asp Lys Gin He He He Tyr Gin Pro Glu 305 310 315 320
  • GGT AGC AAT ATA AAT AGT AGT GAT AAA AAT AAA AAT GTT GAA ACG ACC 2470 Gly Ser Asn He Asn Ser Ser Asp Lys Asn Lys Asn Val Glu Thr Thr 770 775 780
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • GATCGATCAC AGCACTCGCC CCAAAGATGC CAGTTATGAG TATATGGTCT TTTTAGATGC 2760
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANT -SENSE NO
  • GGAATTCCAT CACTCAATCA TTAAATTTAG GCACAACGAT GGGCTATCAG CGTTATGACA 60
  • AATTTAATGA AGGACGCATT GGTTTCACTG TTAGCCAGCG TTTCTAAGGA GAAAAATAAT 120
  • GAT AAA CAA CTA TTT GAT AAT TAT GTT ATT TTA GGT AAT TAC ACG ACA 1141 Asp Lys Gin Leu Phe Asp Asn Tyr Val He Leu Gly Asn Tyr Thr Thr 305 310 315
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:10: CACTTCGCNC AAAATAACCC 20
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES

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Abstract

L'invention se rapporte à la séquence d'ADN codant le constituant protéique majeur de chondroitinase ABC, appelé 'chondroitinase I', provenant de Proteus vulgaris (P. vulgaris), cette séquence étant contenue dans le fragment de Nsi illustré par la figure. L'invention se rapporte également à la séquence d'ADN codant un second constituant protéique de chondroitinase ABC, appelé 'chondroitinase II', provenant de P. vulgaris, au clonage et à l'expression des gènes contenant ces séquences d'ADN, aux séquences d'aminoacide de la chondroitinase recombinante I et II, et à des procédés d'isolation et de purification de la chondroitinase recombinante I ou II. Ces procédés permettent d'obtenir des rendements et une pureté significativement plus élevés que ceux obtenus par l'adaptation, aux enzymes recombinantes, du procédé précédemment utilisé pour isoler et purifier la chondroitinase I native provenant de P. vulgaris.
PCT/US1994/004495 1993-04-23 1994-04-22 CLONAGE ET EXPRESSION DE GENES DE CHONDROITINASE I ET II A PARTIR DE $i(P. VULGARIS) WO1994025567A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP6524437A JPH09500011A (ja) 1993-04-23 1994-04-22 Ｐ．ブルガリスからのコンドロイチナーゼｉ及びｉｉ遺伝子のクローニング及び発現
AU68183/94A AU697156B2 (en) 1993-04-23 1994-04-22 Cloning and expression of the chondroitinase I and II genes from (p. vulgaris)
EP94916561A EP0702715A4 (fr) 1993-04-23 1994-04-22 CLONAGE ET EXPRESSION DE GENES DE CHONDROITINASE I ET II A PARTIR DE $i(P. VULGARIS)

Applications Claiming Priority (4)

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US5220693A 1993-04-23 1993-04-23
US5361593A 1993-04-23 1993-04-23
US08/053,615 1993-04-23
US08/052,206 1993-04-23

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WO1994025567A1 true WO1994025567A1 (fr) 1994-11-10

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JP (1) JPH09500011A (fr)
AU (1) AU697156B2 (fr)
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US5716617A (en) * 1994-04-22 1998-02-10 American Cyanamid Company Compositions of proteus vulgaris chondroitinase I and chondroitinase II
US5855883A (en) * 1994-04-22 1999-01-05 American Cyanamid Company Method of disinsertion of vitreous body from neural retina of the eye with Proteus vulgaris chondroitinases I and II
EP0756636A1 (fr) * 1994-04-22 1997-02-05 American Cyanamid Company Chondroitinases i et ii, leurs procedes de preparation et leur utilisation
US5741692A (en) * 1994-04-22 1998-04-21 American Cyanamid Company Protein vulgaris chondroitinase II
EP0756636A4 (fr) * 1994-04-22 1997-12-17 American Cyanamid Co Chondroitinases i et ii, leurs procedes de preparation et leur utilisation
WO1996040897A3 (fr) * 1995-06-07 1997-04-24 American Cyanamid Co Chrondroitinase i et chrondroitinase ii produisant des mutants du p.vulgaris
WO1996040938A1 (fr) * 1995-06-07 1996-12-19 American Cyanamid Company Production de chondroitinase dans les souches de proteus vulgaris recombinees
US5888798A (en) * 1995-06-07 1999-03-30 American Cyanamid Company Chondroitinase I and chondroitinase II producing mutants of P. vulgaris
WO1996040897A2 (fr) * 1995-06-07 1996-12-19 American Cyanamid Company Chrondroitinase i et chrondroitinase ii produisant des mutants du p.vulgaris
US8183350B2 (en) 2002-05-04 2012-05-22 Acorda Therapeutics, Inc. Compositions and methods for promoting neuronal outgrowth
US9956273B2 (en) 2002-05-04 2018-05-01 Acorda Therapeutics, Inc. Compositions and methods for promoting neuronal outgrowth
US9468671B2 (en) 2002-05-04 2016-10-18 Acorda Therapeutics, Inc. Compositions and methods for promoting neuronal outgrowth
US8785606B2 (en) 2002-05-04 2014-07-22 Acorda Therapeutics, Inc. Compositions and methods for promoting neuronal outgrowth
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AU2009251124B2 (en) * 2003-05-16 2012-12-06 Acorda Therapeutics, Inc. Proteoglycan degrading mutants for treatment of CNS
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US9528102B2 (en) 2003-05-16 2016-12-27 Acorda Therapeutics, Inc. Proteoglycan degrading mutants for treatment of CNS
US7959914B2 (en) 2003-05-16 2011-06-14 Acorda Therapeutics, Inc. Methods of reducing extravasation of inflammatory cells
US7968089B2 (en) 2003-05-16 2011-06-28 Acorda Therapeutics, Inc. Proteoglycan degrading mutants for the treatment of CNS
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JP2014236729A (ja) * 2003-05-16 2014-12-18 アコーダ セラピューティクス、インク． Ｃｎｓ治療用のプロテオグリカン分解変異体
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AU697156B2 (en) 1998-10-01
CA2161125A1 (fr) 1994-11-10
EP0702715A1 (fr) 1996-03-27
JPH09500011A (ja) 1997-01-07
EP0702715A4 (fr) 2000-01-19
AU6818394A (en) 1994-11-21

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