CA2138948A1 - Molecular cloning of the genes responsible for collagenase production from clostridium histolyticum - Google Patents

Abstract

Genetically-engineered E. coli carrying vectors containing an insert that codes for non-native and native forms of Clostridium histolyticum collagenase. The recombinant DNA segments coding for collagenase are expressed efficiently in the transformed cells to yield enzymatically active and immunologically cross-reactive collagenase. Genetically-engineered collagenase is used for isolation of pancreatic islets, for isolation of endothelial cells, for the isolation of dispersed tumor cells, or for the treatment of "slipped disc".

Description

- WOg4/00~8~PCT/US93/0~9 MOLECULAR CLONING OF THE GENES
RESPONSIBLE FOR COLL~GENASE PRODUCTION
FROM C~OSTRIDIUM HISTOLYTICUM

BACKGROUND
This invention relates to the isolation and loning of genetic information coding for Clostridium _histolYticum collagenase and the expression of the genetic information in a suitable host. In particular, this invention is directed to the isolation and cloining of genetic information coding for forms of Clostridium histolyticum collagenase, including a form ha~ing a molecular weight higher than the products of translation determined by the native expression of the C. histol ~icum genomic coding sequence.

SUMMARY
In accordance with the present invention, a recombinant DNA segment is provided which codes for a polypeptide having the enzymatic activity and antigenicity of Clostridium histolyticium collagenase. This polypeptide, however, is distinguishable from native C.
histolyticu_ collagenases, that is, collagenases produced by the native expression of the C. histolyticum genome and subsequently purified from C. histolyticum. This polypeptide of the in~ention, referred to herein as a non-native form, has a higher molecular weight than the prcducts of translation determined by the native expresion of the C. histolyticum genomic coding sequence.
Th~ claimed recombinant DNA segment comprises promoter derived from C. histolyticum. This promoter .
' "`' W094/005g0 ~ 8 P~T/US93/059 operates independently and allows the claimed DNA segment to be transcribed under the control of said promoter to produce the claimed polypeptide without the functioning of a promoter external to the claimed recombinant DNA segment.
The claimed recombinant DNA segment is further capable of expressing native polypeptides with collagenase activity having molecular weights lower than the claimed non-native, high molecular weight polypeptide.
The invention further provides a vector comprising the claimed recombinant DNA segment and capable of transforming host cells to produce the claimed non-native polypeptide.
The inventors found that the expression of the claimed recombinant DNA segment in transformed host cells is determined by the strain of the host cell. Accordingly, different E. coli host cells transformed with the vector of the invention are provided. One strain of host cells produces the non-native polypeptide of the invention having collagenase activity and antigenicity. Greater than 50~ by weight of the total polypeptides produced ~y the host cells - 20 that have collagenase activity comprise the non-native high molecular weight polypeptide.
The invention further provides other E. coli host cells transformed with the vector of the invention. These host cells produce a polypeptide possessing collagenase activity and antigencity and having a molecular weight of 110,000. Greater than 50% of the polypeptides having collagenase activity produced by these cells comprises the 110 kd collagenase.
Another aspect of the invention involves substantially purified preparations of C. histol~ticum collagenase. One preparation comprises the non-native form of collagenase.
Another substantially purified preparation of the invention comprises collagenase having a molecular weight of 110 kd.
These forms of collagenase are derived from different - strains of E. coli host cells transformed with the vector of the invention.

- W094/00580 ~3~4~ pCT/U~93/0~9~
... . . ~. ~ -Further provides are methods for using the collagenase produced by the E. coli host cells genetically engineered according to the invention. These methods are suitale for such purposes as digesting connective tissue and releasing embedded cells, isolating dispersed pancreatic islets from :::
pancreatic tissue, isolating endothelial cells from blood vessels, and dissociating tumors for isolation of dispersed tumor cells. The method comprises two steps:
(1) incubating the tissue to be dispersed in a buffered solution containing the substantially purified genetically engineered collagenases of the present invention with shaking at about 25-39~ C to release and disperse the embedded cells; and (2) separating the dispersed cells from tissue debris. The step of separating the dispersed cells from tissue debris is typically performed by density gradient centrifugation.
The genetically engineered C. histolvticum collagenases of the present invention can also be used in a method for intradiscal treatment of herniation of nucleus pulposus ("slipped disc"). This method comprises the steps of: :
(1) preparing a sterile buffered ~olution containing the claimed substantially purified genetically engineered collagenase; and (2) injecting the sterile buffered solution containing the substantially purified claimed collagenase into the nucleus pulposus~
These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, appended claims, and the accompanying figure~.

W094/00580 21~8~ PCT/US93/059~

FIGURES ~;
: ~, Figure 1 outlines the cloning strategy for obtaining expression plasmid for intact 125 kd collagenase.
Figure 2 shows the expression of collagenase in E.
coli strain DH5a. `~
Figure 3 shows the construction of plasmids pCT6 and pC~7 for DNA sequencing of the collagenase gene.
Figure 4 shows various clones used to determine the partial DNA sequence of the gene encoding 125 kd collagenase. -Figure 5 is the transcriptional termination signal of the 125 kd collagenase gene. ~
Figure 6a is a Coomasie blue stained gel of 125 kd ~"
collagenase compared with co~mercially available ~ollagenases; Figure 6b is a Western blot of 125 kd collagenase compared with commercially available collagenases.
Figure 7 shows in lane 1 a Coomasie blue stained gel of culture media from E. coli strain DH5~ containing plasmid p70; lane 2 is a Coomasie blue stained gel of purified 12S kd collagenase from culture media.
Figure 8 is the restriction of plasmid pCT11.10.
Figure 9 is a Coomasie blue stained SDS-PAGE showing that IPTG does not induce the expression of recombinant collagenase from E. coli DH5~ carrying pCT11.10.
Figure 10 is a Western immunoblot comparison of recombinant collagenase produced from pRS21 and pCT8B. ~ -Figure lla is a Coomasie blue stained SDS-PAGE; llb is - -an immunoblot, both showing the intracellular localization of the recombinant collagenase produced in E. coli. `
Figure 12 shows the results of Coomasie blue staining and immunoblots of the purification and comparison of the ~ recombinant 110 kd collagenase to natively produced 110 kd i collagenase. ~-~

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--~ W094/~0580 2 ~ ~8 9~8 PCT/US93/059 DETAILED DESCRIPTION
The inventors have cloned a gene for Clostridium histolyticum into E. coli. The cloned gene, that is, the recombinant DNA ~egment of the invention, is capable of 5 expression as a polxpeptide product in E. coli. The product expressed in E. coli is detectable both in the form of protein immoreactive with anticollagenase antibody and in the form of assayable collagenase activity i.e. collagen digestion.
Depending on the E. coli host into which it was inserted, it was found that the same recombinant DNA
segment, having 4.9 kilobases, could be translated to yield several polypeptides having c~llagenase activity, as described below in Examples 3 and 11. E. coli strain DH5a, transformed with the claimed recombinant DNA segment according to the invention, produced, in particular, a polypeptide having collagenase activity and antigenicity, the polypeptide being distinguishable from native C.
histolyticum collagenases. The distinguishing feature of this non-native polypeptide was that it had a higher molecular weight than the products of translation determin d by the native expression of the C. histolYticum genomic coding sequence. C. histolyticum collagenases determined by native expression are those forms of collagenase directly produced by C. histolvticum without the introduction of genetic vectors carrying DNA segments coding for collagenase. As described in the Examples below, it was found that native collagenases of C.
histolyticum purchased from a variety of commercial sources do not have molecular weights as large as the non-native collagenase of the present invention.

W094/00580 X13~9~8 PCT/US93/059~

It was found, as detailed in Example 8, that the claimed recombinant DNA segment comprised a promoter derived from C. histolYticum. As described below, the 4.9 kb recombinant DNA fragment of the invention coding for collagenase comprised a Clostridium promtoer which functioned independently in E. coli.
The claimed recombinant DNA segment was found to express polypeptides having collagenase activity and molecular weights ower than the non-native collagenase of the invention. As described in the Examples, the relative `~ `
proportions of native and non-native collagenases expressed -;
from the claimed DNA segment vary according to the E. coli ~
host into which the claimed DNA segment is transformed via ~ `
the vector of the invention. ~
After DNA was isolated from C. histolvticum, a pRK290 ~-library containing clostridium DNA was constructed. ;~
(Example 1~ and the collagenase gene screened. ThP :.
inserted collagenase gene was characterized by restriction enzyme analysis (Example 2). Further characterization of the collagenase gene was carried out in Example 4, wherein the insert was placed in opposite insert orientations in plasmids pCT6 and pCT7, serially delted. 2551 base pairs were sequenced. A complete nucleotide sequence and inferred amino acid sequence is presented in Sequence ID ~`~
No. l. Further confirmation that the DNA sequence comprised the Clostridium collagenase gene is presented in Examples 5 and 6.
The expression of Clostridium collagenase genes in E.
coli requires transformation or transfection of the host E.
coli cells with a suitable plamid or other vector carrying ~`~
the Clostridium DNA and detection of the collagenase ~
produced by the transformed cells. Using standard ~-techni~ues to achieve transformation and collagenase detection as described in U.S.Application Serial No.
~ 07/498,919, the inventors in Examples 3, 7, 9, 10 and ll determined that the expresion of the claimed recombinant DNA segment in transformed host cells is determined by the - W094/00580 `2~38~8 P~T/US93/059~

strain of the host cell. Example 3 demonstrates that about 2% of the transformed E. coli soluble proteins was collagenase according to immunoblotting with anti-collagenase antibodies on Western blots and Coomasie blue staining of SDS-PAGE. ~`
It was found, as described in Example 8, that control of collagenase expression in E ! coli was under control of an independent promoter comprising the claimed DNA segment and derived fr~m Clostridium histolyticum.
The inventors produced and purified native and non-native collagenase from transformed E coli cells and from the culture medium in which these cells were grown, as described below in Example 10. Purification of the enzyme from cells involved sta~dard techn~ieus known in the art.
The inventors unexpectedly found large amounts of collagenase in the fermentation broth in which the transformed cells grew. Based on that observation, a strategy for purifying collagenase to o~tain substantially pure preparations of non-native collagenase and 110 kd collagenase were developed.
The collagen digestion activity of recombinant collagenase purifed from transformed cells was found to be ~i about equivalent to the activity of recombinant collagenase activity purified from cells. (Table 1 in Example lO~
The collagen digestion activity of the purified recombinant non-native ~ollagenase was compared to native forms of purified collagenases obtained commercially. (Table 2 in Example 11) the recombinant collagenase was 50% to 100%
higher in activity than native collagenases obtained from Worthington Chemical, Sigma Chemical, and CalBiochem.
The purified recombinant~ non-native or recombinant native llOkd collagenase produced by genetically engineered E. coli containing the claimed DNA segment can be used fo ~`
any application in which it is desired to digest collagen.
~ Particular applications for isolating or releasing cells from tissues include: (1) digesting connective tissue and releasing embedded cells without destroying cell membranes -.- W094/00580 z~3~ PCT/U~93/0~9~

and other essential features; (2) isolating endothelial - cells; (3) dissociating tumors; and (4) intradiscal treatment of herniation of the nucleus pulposus ("slipped disc"). As presented in Example 12, the purified llO kd recombinant collagenase of the invention, in combination with trypsin, was effective for isolating endothelial cells -' from human saphenous veins.
Depending on the targeted tissue and animal, the amounts o~ recombinant collagenase (SEQUENCE ID NO. 2), or ~` lO native class II collagenase, or neutral proteases can b~
varied to ob~ain dissociated tissue preparations using the method of the present invention. (G~H~Jo Wolters, I'An Analysis of the Role of Collagenase and Protease in the Enzymatic Dissociation of the Rat Pancreas for Islet Isolation," Diabetoloqia, 35:735-742 (1992)~
The type and amount of neutral proteases required in with the recombinant collagenase in the compositions and methods - of the present invention can be varied depending on the target tissue. Proteases other than crude or purifed native ~ollagenases can be employed in compositions comprising the recombinant collagenase (SEQUENCE ID NO. 2) to digest or dissociate tissues using the method of the present invention.

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--- W094~00~0 ~ ~3~9~8 EXAMPLES
The following examples are for illustxative purposes - only and are not to be construed as limiting the invention.

xample 1 Cloninq_and Screeninq of the Gene Encodinq For the Intact I25kd Colla~enase The orientation of the gene encoding for the intact 125 kd collagenase was determined in U.SO Application Serial No. 07/498,919. As described below in Examples 4 and 5, the termination of the gene was determined by sequencing the ~ene and found to be close to the BglII
site. As defined by the a~ove information, a 2.5 kb DNA
fragment extended from the first EcoRI site through the second EcoRI site to the BglII site. A 2.5 kb DNA fragment is not large enough to code for collagenase having a molecular weight of about 125 kd, which requires approximately 4.4 kb. Accordingly, a new library was prepared in an attempt to identify more collagenase gene sequence. This involved construction of a BglII li~rary.
An attempt was made to clone the BglII fragment directly from genomic DNA to small plasmids like pBluescript. The inventors identified a collagenase band at MW of 125 kd, but upon a second screening, involving two independent experiments, the molecular weight appeared to be Ç8 kd.
The inventors employed another plasmid with a low copy number (1-lO copies per cell), such as pRK290 (Haas, D.
Experientia, 39:11g9 (1983~). This resulted in the cloning of the entire collagenase gene. However, the difficulty o clonin~ the BglII fragment to smaller plasmids, such as ~ pUC8, remained. Nonetheless, the inventors achieved the construction of such a pUC8 plasmid incorporating the entire collagenase gene.

q, , WO94/~058Q ' Z ~ PCT/US~3/059 A. DNA isolation from_Clostridium~hlstolYticum Clostri ~ m histolYticum ATCC 21000 was obtained from the American Type Culture Collection. The paper tablet containing the bacteria was fir t ~oaked in TYE broth t15 g tryptone, 10 ~ yeast ex~ract, and 5 g NaCl per liter of culture medium) for 30 minutes at 4C with occasional shaking.
The cell suspension was streaked on a TYE agar plate and grown at 37C under anaerobic conditions. A single colony was picked and grown in 50 mL TYE broth and grown at 37-C under anaerobic conditions. The cells were collected by centrifugation and resuspended in TES (O.lM Tris, pH8.0, O.lmM EDTA, and 0.15M Na~1). Cells were partially lysed by , freezing and thawing the cell suspension four times. SDS
and pronase K were then added to final concentrations of 0.5% and 200 ~g/mL, respectively. The cell debris was removed by centrifugation at 10,000 rpm in a Beckman J2-21 centrifuge for 30 minutes at 4C. The supernatant was extracted three times with equal volumes of phenol-chloroform, and the DNA was precipitated with isopropanol. DNA concentration was measured by electrophoresing the DNA through an agarose gel and comparing fluorescence afte, the addition of ethidium bromide with the fluorescence of a known concentration standard. The average size of purified Clostridium DNA was measured by electrophoresis on a 0.6~ agarose gel and was 30 kb to 40 kb.
, ~ ~
B. Construction of a,,pRK290 library containinq clostridium DNA
Purified C. histolyticum chromosomal DNA was digested with r~striction enzyme BglII. After ethanol precipitation, the digested DNA was resuspended in TE
buffer (10 mM Tris, pH 7.4, 0.1 mM EDTA). Appropriate , ~ amounts of digested DNA were ligated with pRK290 that had ¦ 35 been previously cleaved with BglII and treated with alkaline phosphatase. The ligation was performed at 4C

W094~00580 213~ PCT/US~3/059~

for 16 hours. The ligated DNA was transformed into E. coli strain DH5~ as de~cribed. Transformed cells were plated on TYE-Tc (15 g tryptone, 10 g yeast extract, 5 g NaCl, and 15 mg of tetracycline per liter) plate and incubated at 35C
overnight.

C. Screenina_for Collaqenase Gene Approximately 1,000 colonies were obtained per plate.
About 10 plates were screened for intact collagenase gene.
Briefly, after 16 hours of incubation at 35C, a dried nitrocellulose paper (S&S) was overlayed ~n the petri dish to absorb th~ colonies. The nitrocellulose paper was carefully separated from the plates, transferred to a freshly prepared TYE-Tc agar plate, and incubated for another 4 hours at 35C. The original plates were stored as master plates at 4C. The nitrocellulose paper was carefully lifted from the plate, soaked in the O.lM Tris, pH 8.0, 0.2 M NaCl, and 5% skim milk, and shaken gently to remove the bacteria cells from the nitrocellulose paper.
The nitrocellulose paper was incubated with the rabbit anti-collagenase antibody (as detailed in Example 2 of U.S.
Application Serial No. 07/498,919) at a dilution of 1:1000 with 5% skim milk at room temperature for l hour, washed with 0.1 M Tris HCl buffer, p~i 8.0, containing 0.2 M NaCl and 1% Triton~ X-100, then incubated with protein A-horseradish peroxidase conjugate at 1:1000 dilution in PBS with 5% skim milk at room temperature for 1 hour. The washing procedure was repeated and the nitrocellulose paper developed with 4-chloro-1-naphthol and H202 in PBS as described in R.A. Young & R.W. Davis, "Efficient Isolation of Genes by Using Antibody Probes," Proc. Natl.
Acad. Sci. USA 80, 1194-1198 (1983).
The area of agarose on the plate corresponding to the location of a positive signal on the nitrocellulose was removed and resuspended in TYE-Tc broth. Serially diluted cell suspension to low cell densities (30-100 cells per plate) were replated on the TYE-Tc plates and a second , 11 ~

W094/00~80 21~3~ PCT/US93/0~9~

screening was performed to identify single colony. One colony designated P70 showed a strong signal and was collected and grswn in TYE-Tc broth. This colony was further characterized as below in Examples 2 and 3.

Restriction Enzyme Analysis of Inserted DNA
To characterize the organization of the Clostridium DNA in plasmid P70 more preciselyl P70 was subjected to further restriction enzyme analysis. DNA from plasmid P70 was prepared as described (Maniatis). Purified DNA was digested with BglII to release the cloned DNA insert. A
DNA fragment with apparent size of 4.9 kb was released from vector pRK290 by BglII digestion. p70 was further digested with a combination of BglII and EcoRI and compared to pBB1 and pRS21 digested with the ~same enzymes. All three plasmids released an identical 1.7kb EcoRI-BglII fra~ment.
Both P70 and pBBl releaesd an identical 0.8 kb EcoRI-EcoRI
fragment. These restriction enzyme analysis suggested that all three plasmids share common DNA fragment and P70 carries an extra piece of 2.4 kb BglII-EcoRI fragment.

Characterization of Collaqenase Produced in E. coli Strain DH5 To examine the size of immunoreactive protein, cells containing P70 were collected by centrifugation, resuspended in gel loading buffer, heated, and run on a 7.5~ SDS-Polyacrylamide gel as described in Laemmli, U.K., "Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4", Nature 227:680-685 (1970). The proteins were electroblotted to nitrocellulose paper and detected with the rabbit anticollagenase antibodies of U.S.
Application Serial No. 07~498,919 (W.H. Burnette, "Westerr.
Blotting: Electrophoretic Transfer of Proteins from SDS-~ polyacrylamide Gels to Unmodified Nitrocellulose and Radiographic Detection with Antibody and Radioiodinated Protein A" Anal. Biochem. 112. 195-203 (1981). The -~- W094/00580 213~ 8 PCT/US93~059~
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molecular weight of the collagenase from the P70 clone was determined by comparison with prestained protein standards. ;~
Immunoreactive protein bands with molecular weight of approximately 125 kd and downward were expressed from the P70 clone. Several bands with molecular weights between 68 and lO0 kd are similar to what we described in U.S.
Application Serial No. 07j498,919.
E. coli strain DH5~ was transformed with plasmids pUC8 or P70, respectively, ~nd grown in 50 ml of TYE broth in -250 ml flask. The cells were har~ested, resuspended in lOmM Tris, pH 7.4 and lmM P~5F, and sonicated. After ; -~
removing the cell debri by centrifugation, protein ~-concentrations were measured and the same amount of ~
prot~ins were loaded and run on a 7.5% SDS polyacrylamide ~-gel. ~-The expression of the recombinant DNA segment of the .. :--present invention is shown in Figure 2. Panel A is Coomassie blue-stainned SDS-PAGE. Lane 1, E. coli strain DH5~ carrying plasmid pUC8 and Lane 2, E. coli strain DH5~
carrying plasmid P70. Panel B is Coomassie blue-stainned (Lane 1) and immunoblotted tLane 2~ SDS-PAGE of cell extract prepared from E. coli strain carrying P70~ ~-In the cell extracts prepared from cells carrying plasmid P70 but not from cells carrying pUC8, a very strong Coomassie blue stained protein band was visualized between molecular weights 200 and 97 kilodaltons. Plasmid P70 --transformed E. coli strain DH5~ was grown in a 5 liter BioFloII Fermentor to produce collagenase. After cells were harvested by centrifugation, the cell paste was stored at -70C. Small amount of cells were futher processed to determine the purification condition. Sonicated and clarified supernatant was examined by Coomassiè blue stainning and immunoblotting with anti-collagenase antibodies and shown in the panel B of Figure 2. The ~ position of the Coomassie stainned band was the same position as the immunoreactive protein band. It therefore confirmed the identity of the Coomassie blue stainable band W~94/00580 PCT/US93/059~
2~3~
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that resid~d between 200 and 97 kd was collagenase. Under both growth conditions, it was estimated that about 2% of E. coli soluble protein was collagenase.

Characterization of the Collaqenase Gene A. DNA sequencing strategy.
A non-random DNA sequencing strategy published by ~ Henikoff, S. (Methods in~ Enzymolo~y, Unidirectional ,i Dig~stion With Exonuclease III in DNA Sequence Anallysis, 155:156 (1987)) with minor modification was used to sequence the 2.5 kb EcoRI-BglII DNA fragment encoded by pBBl. Plasmids pCT6 and pCT7 were constructed by inserting the 2.5 kb BamHI/BglII fragment into the Bam~I site of plasmid pBluescript SK(-) in the opposite orientation.
Plasmids pCT6 and pCT7 were constructed by inserting the 2.5 kb BamHI-BglII fragment from P42 to the BamHI site of plasmid pBluescript. Plasmids pCT6 and pCT7- represent opposite insert orientation as shown in Figure 3O To create unidirectional deletions, both plasmids were first digested with XhoI and KphI to create a recessed 3'-hydroxyl termini of double-stranded DNA and a protruding 3' termini at the other end. The recessed 3'-hydroxyl termini created by XhoI was susceptable to exonuclease III and was be removed stepwise while the protruding 3' termini created by KpnI remained intact. The digestion proceeds unidirectionally away from the cleavage site and into the - target DNA sequence. The degree of digestion was controlled by time. Aliquotes collected at different times were subjected to Sl nuclease digestion. The digested DNA
samples were analyzed by agarose gel electrophoresis to identify the samples containing DNA fragments with desired size. Klenow DNA polymerase was added to blunt both DNA
ends. T4 DNA ligase was added to recircularize the plasmid. Ligated DNAs were then transformed into E. coli strain DH5~.

~ W094/00580 2138948 PCT/US93/059~

After transformation, the cells were plated on TYE-Ap plates, and grown overni~ht at 35C To examine the deletion and prepare DNA for sequencing, the colonies were randomly picked and grown in 5 ml of TYE-Ap broth overnight. Cells were collected from overnight culture by centrifugation. The cell pellet was resuspended in Tri~
EDTA buffer and lysed by using the alkaline method as described in Maniatis. After ethanol precipitation, the DNA pellet was briefly dried and resuspended in TE (lOmM
Tris, pH 8.2, 0.1 mM EDTA). The size of deletion was examined by restriction enz~me analysis. Clones with appropriate deletions were identified and further treated with RNase A. After RNase digestion, the DNA was precipitated with 10 PEG and 1.25M NaCl. DNA pellets were washed with 100% ethanol and resuspended in TE buffer. DNA
prepared by this method was adequate for DNA sequencing using Sanger's dideoxy chain termination method (Sanger, F.
and A.R. Coulson, J. Molec. Biol. 94:441 (1975)). Clones used for DNA sequencing and the sequence information obtain~d are summarized in Figure 3. In some occasions, plasmid pCT12.8 was systematically deleted and subclones were used to determine part of the sequences. Plasmid pCT12~8 was constructed by inserting the 4.9 kb BglII
fragment of P70 into pBluescript (SK-) pretreated with BamHI.

B. DNA sequence and analysis A total of 2808 base pairs of the complete sequence is shown in (Sequence ID No. 1). The DNA sequence contains an unusually high proportion of A and T nucleotides (69%) and only 31% of G and C nucleotides. An open reading frame was identified from the first EcoRI site through the second EcoRI site and ends at a TAA termination codon located at base pair 2808. A deduced protein sequence containing 936 amino acids was identified and shown in Sequence ID No. 2.
This protein contains unusually high charged amino acids (30%) as compared to most other proteins.

W094/00~80 2~38948 PCT/US93/0~9~

A typical bacterial transcriptional termination signal ~Platt, T. and D.G. Bear in Gene Function in l~rokarYotes, J. Beckwith et al. editors, page 123, Cold Spring Harbor -~
Laboratory, Cold Spring Harbor, NY (1983)) was identified ~-ll base pairs downstream from the tran~lational termination signal (TAA) as sh~wn in Figure 5. This si~nal consists of a stem-loop structure followed by an A-T rich sequence.
This data suggested that the collagenase gene ends in the A-T rich area downstream of the stem loop structure.

EXA~PLE 5 ~;
pNA Seguence Comparison of Previous Clones `~
Although it is clear that all the previous clones -~
produced identical immunoreactive protein bands as judged by their mobilities on the immunoblots, it did not rule out the possibility of microheterogeneity. If microheterogeneity existed amon~ the clones obtained previously, it may have supported the notion proposed by Van Wart et al [REF~ that gene duplication and then mutation produced different collagenases.
To confirm their identities, two DNA sequence projects were carried out: 1). Comparison of sequences between clones with different inserts such as P6, P9, P41, P42, and P51 and 2) Comparison of sequences among different original isolates obtained previously, all apparently having the same insert size. DNA sequ nces flanked by multiple cloning sites were sequenced using the flanking universal primers. An average of 150 to 200 base pairs of Clostridium DNA sequence were obtained from both ends.
After comparing the sequences, no single base pair differences was identified. This result did not support gene duplication as the means responsible for obtaining differences in the same class of collagenase.

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romparison of the DNA Seguence of Clones Derived From P70 to Sequences from Cloness Obtai d Previously To examine the identities of P70 and other clones obtained previously, many clones were partially sequenced and compared. The 0.7 kb EcoRI and 1.8 kb EcoRI-BglII
- fragments fr~m clones P70 were subcloned into pUC13 or pBluescript. The DNA seguence flanked by multiple cloning sites was se~uenced using the flanking universal primers.
Flanking sequences of plasmids pBBl, pRS21, and P41 were determined and compared to the sequences obtained from subclones of P70. No differences was observed. This result suggested that the P70 shares the same DNA fragment as pBB1 and was located at the same place on the chromosome.
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Comparison of Collaaenase Produced from Subclone of P70 To the Collaqenase Produced from PRS21 To further characterize the identity between P70 and the previously described clones, the collagenase products produced from subclones apparently having the same inserts were compared. p70 was digested with BglII and EcoRI and ligated with pUC18 predigested with BamHI and EcoRI.
Ligated DNA was transformed to E. coli strain DH5~, plated on TYE-Ap plate, and incubated at 35 C overnight. A clone containing the 1.7kb EcoRI-BglII insert were and having the same insert orientation as pRS21 was identified and designated as pC~8B. E. coli strain DH5~ containing either plasmid pCT8B, pRS21, or pUC18 were grown in TYE-Ap broth overnight at 35 C. Cells were collected by centrifugation and disrupted with SDS-PAGE loading buffer. The release~
proteins were run on 7.5% SDS-polyacrylamide gel and electroblotted to nitrocellulose paper. The immunoreactive bands were detected as previously described and compared.
As shown in lane 1 of Figure 10, lysate produced from E.

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W094~00~80 ~13~94~ P~T~US93/05~

coli carrying pUC18 did not produce an immunoreactive collagenase band. However, both cell lysates produced from E. coli carrying pRS21 (lane 2~ or pCT8B (lane 3) ~howed identical immunoreactive protein bands with molecular weight of 68 kd, a sign that both are producing the same protein (Figure lO).

EXAMP~ 8 ~-Control of Collaaenase ~xpression in E. Coli BY Independent Promoter Derived~from .
Clostridium HistolYticum Plasmid pCTll.10 was constructed by cloning the 4.9 kb BglII fragment from P70 into plasmid pUC13 that was predigested with BamHI and is shown in Figure 8. The transcription direction of the collagenase gene is the same as the Lac promoter.
E. coli DH5~ carrying plasmid pCTll.10 was grown in 50 ml of TYE-Ap broth with or without lmM IPTG overnight at 32C with shaking (130rpm). The cells and supernatant were separated by centrifugation. The cells and supernatant were incubated with loading buffer at 95~C before running on 7.5% SDS polyacrylamide gel. Twenty ul and 80 ul .
equivalent to the original volume of supernatant and cells, respectively, were loaded per lane and shown in Figure 9. .
After Coomassie blue stainning, protein profiles of cell extracts are shown in lanes l ~with IPTG) and 2 (without IPTG) of Figure 9~ Protein profiles of culture media are shown in Lanes 3 (with IPTG) and 4 (without IPTG) of Figure 9.
With or without IPTG, both cell extracts and culture media did not show any expression differences of recombinant collagenase. IPTG, a potent lac promot~r ; inducer, did not increase the expression of collagenase, j which further confirmed that the 4.9 kb DNA fragment contained a Clostridium promoter that could function ! 35 independently in E. coli.

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Unexpectedly, a large amount of collagenase was discovered in the supernatant as shown in Lanes 3 and 4 of Figure 9. Since the equivalent volume of supernatant loaded (20 ul) was one fourth of the equivalent volume of cells 1oaded (80 ul~, it was estimated that 80% of collagenase resided in the supernatant, as judged from the stained gel. The acc~mulation of collagenase in the culturP media indicated that the cells excreted collagenase into the media from E. coli cellsO The e cells provide the advantage of a ~imple and cost effective method to produce and purify collagenase from culture media in which these -~
host cells comprising the recombinant gene segment of the present invention coding for collagenase have grown.

Intracellular Localization of ~Collaaenases Ex~ressed in . coli The intracellular localization of collagenase in different E. coli compartment was examined. E. coli strain ~
DH5~ containing plasmid P70 was grown in TYE-Tc broth at ;
35C for 5 hours with shaking. Cells were collected by centrifugation. Cell pellets were resuspended in 30 mM
Tris-HCl, pH 8.0, 20% sucrose buffer. Lysozyme (70 micrograms/ml) and EDTA (2mM) were added and incubated at 4C for 30 minutes to decompose the cell wall. The periplasmic fraction was separated from cytoplasmic and `~
membrane fractions (CM fraction) by centrifugation. The periplasmic fraction and CM fraction were examined on SDS-PAGE and detected by both Coomossie blue staining and immunoblot methods. As shown in Figure 11, panel A, a distinct protein band with MW of 125kd was seen in the - periplasmic fraction (lan~ 1) and barely seen in the cytoplasmic fraction (lane 2) on the Coomossie blue stainned gel. ~`~
~ Many immunoreactive bands were seen by the immunoblot as shown in Panel B of Figure 11. Most of the bands ; existed in the periplasmic region (lane l of Panel B), . ' ' 19 :':

., ~^l W094J00580 2~389'~a Pcr/us93~05944 ~ ..

which suggested that the protein represented by these bands can ~ecrete through the inner membrane and accumulate in the periplasmic space. Based on the amount loaded on the gel (th~ periplasmic fraction was loaded at half the eguivalent original volume as compared to the CM fraction), it is estimated that 80% of the 125 kd collagenase was lorat~d in the periplasmic region. Although the ratio of immunoreactive bands residing in the periplasmic and cytoplasmic regions (lane 2) are different among the immunoreacti~e bands, most of these bands apparently are prefera~ly located in the perplasmic region except for a protein band with MW of 68kd. The 68kd protein resides preferably in the cytoplasmic region, and not in the periplasmic region. This ~uggests that the 68 kd collagenase cannot be efficiently transported through the inner membrane. Although it was not clear that this 68 kd protein was identical to the 68 kd protein produced in pRS21, the 68 kd protein produced in pRS21 was not able to secrete into the periplasmic region. These data show that collagenase with MW of 125 kd produced in E. coli can utilize E. coli's secretion mechanism to transport through the inner membrane to accumulate in the periplasmic region.

EXAMP~E 1,O
Colla~enase Production and Purification From E. coli cells and culture medium.
A. Production of Collagenase from E. coli E. coli strain W3110 carrying plasmid P70 was grown in New Brunswick BioFlo III fermentor to produce collagenase. The fermentation media (FM, grams per liter of media) consists of: KH2PO4, 3~5; K2HPO4, 5.0;
- (NH4)2HPO4, 3.5; MgSO4.7H20, 3.5; Yeast Extract, and 5;
Tryptone, 5. 50% of glucose was autoclaved separately and lO ml per liter was added. lM stock solutions of CaCl2 and ZnCl2 were prepared and autoclaved separately and 20 ul of each per liter were,added. E. coli strain carrying P70 was grown in TYE-Tc overnight at 30C. The overnight culture , 20 W094/0~580 ~8~4~ PCT/US93/059~ ;~

was directly innoculated in the fermentor. After cell density reached OD600 equals to 16, the cells were harvested by centrifugation and separated from the supernatant.
;,~
B. Purification from Cells Cells were collected by centrifugation and resuspended -~
in 10mM Bis-Tris buffer, pH 6.5, with lmM PMSE. The cells were disrupted by sonication and debri were removed by centrifugation. Proteins were fractionated by sequential ammonium sulfate precipi~ation. Fractions containing collagenase were resuspended in 10mM Bis-Tris buffer (Buffer A) and dialyzed against the same buffer overnight - at 4C. Dialyzed samples were first fractionated by DE52 column chromatography. Fractions containing collagenase were pooled and dialyzed against buffer A. Dialyzed -~-samples were fractionated by Q Sepharose column chromatography. To obtain greater than 98% of purity, gel filtration may be required. Many immunoreactive collagenase bands co-existed in t~e starting material. ~ -Different molecular weight collagenase could be separated with anion exchange column chromatography to a certain extent. The experiment described below in ~xample 11 used the largest form of collagenase.
C. Purification from Media ~
Unexpectedly, a large amount of collagenase was -detected in the fermen~ation broth. To evaluate the ~`
possibility of collecting and purifying collagenase from culture media, a purification strategy was developed.
Briefly, after the cells were removed by centrifugation, the supernatant was concentrated and diafiltrated using hollow fiber with molecular weight cut-off of 30,000 , (Amicon). Concentrated supernatant was fractionated by ammonium sulfate differential precipitation. Fractions containing collagenase were collected, resuspended in Bis-Tris Buffer, pH6.5, and dialyzed against the same buffer.
Dialyzed sample was loaded onto the DE52 ~olumn (Whatman) :
: ' -:

W094/00580 2~3~9~ ~ PCT/US93/0~9~

and eluted with NaCl. Fractions containing the collagenase were collected and further purified on the Q Sepharose column (Pharmacia). Figure 7, lane 1 shows a Coomassie blue stained gel of culture media from E. coli strain W3110 carrying plasmid P70 (hane 1) and collagenase purified from this culture media (Lane 2). Collagen digestion activities of collagenase prepared from both cells and culture medium ~.
were compared. Collagenase prepared from medium had about two times the activity as that prepared from cells.
lQ D. Collagen Digestion Activity of Collagenase ::
Purified from Cells and Medium.
The collagen digestion activity of the recombinant collagenase of the present invention either purified from :~
cells or purified from culture medium was determined. The ;;:
activity was measured as described below in Example 11.
Table 1 below summarizes these activities. ~-:

Table 1. Collagenase Activity or Recombinant Collagenase Purified From Cells or Purified from Medium -:.
. ~ .............................. . ~ ~
Sources Collagen Digestion Units per ¦ ~.
mg Protein Collagenase Purified 16,000 from Cells ~:
. ..
Collagenase Purified 14,400 from Culture Medium _ The results of this table show that the collagenase activity purified from the medium is about equivalent to the activity of the recombinant collagenase purified from cells.

SlJBSTITUTE SHEET

~ W094~00580 2~3~948 PCT/U~93/05~

Comparisons of Recombinant Collagenase and Native Colla~enase Produced by C. histoly~icum A. Size .
The identity of recombinant collagenase was compared to the native collagenase produced by C. histolYticum.
- Both unpurified and purified native forms of collagenase were purchased from Sigma, Boehringer Mannheim Products ;~-(Collagenase A, ~CHSA), Collagenase B (CHSB), Collagenase :
D (CHSD)), Worthington, and CalbioChem. Different sources of collagenase were run on the SDS-PAGE to examine :
their molecular weight. A typical Coomossie hlue stained gel and immunoblot is shown in Figure 6. Figure ~ show ;~:
comparisons between recombinant collagenase (RCL) and native collagenase; panel A is a Coomassie blue stained gel; panel B is a Western blot of SDS-PAGE.
The lane assigments for Figure 6 are as follows~
Panel A: Lane 1, 0.5 ug of Lot 17 RCL; Lane 2, 2.5 ug of Lot 17 RCL; Lane 3, 2.5 ug of CHSD tBMB); Lane 4, ~;
10 ug of CHSD; Lane 5, 2.5 ug of CHSB; Lane 6, 10 ug of CHSB; Lane7, 2.5 ug of CHSA; and Lane 8, 10 -~
ug of CHSA. ~`
Panel B: Lane 1, 0.01 ug of Lot 17 RCL; Lane 2, 0.05 ug of Lot 17 RCL; Lane 3, 0.2 ug of CHSD; Lane 4, 0.2 ug of CHSB; and Lane 5, 0.5 ug of CHSA. `~
Purified recombinant collagenase of the present - .
invention was compared with commercially available collagensaes obtained from Boehringer Mannheim Co., catalog ~ ;
numbers CHSA, CHSB, and CHSD. Different amounts of proteins were run on the 7.5% SDS polyacrylamide gel. The -`~.
collagenases on the Western blots were detected with rabbit anti-collagenase antibodies. ~reparation of the anti- :.
collagenase antibodies was described in U.S. Serial No. :~.
07/498,919.
~ Although a lot of proteins can be visualized by :
Coomossie blue staining, more than 50% of the stainable ~
bands can not be detected by collagenase specific ~:
`

W094/00580 Z ~ ~ 9~8 PCT/US93/~59~

antibodies. The largest form of recombinant collagenase clearly shows a larger size than any of the native forms of collagenase obtained from Boehringer ~annheim. The identical result was obtained in comparing the recombinant collagenase of the present invention to the native collagenases obtained from Calbiochem, Worthington, and Sigma.
! A form of recombinant collagenase similar to the s- largest collagenase (110kd) of native products could be produced quantitatively and consistently in a special ~.
coli host (Strain JM105).
B. Activity The collagen digestion activities of c:ommercially available purified native collagenase from different vendors were compared to the purified recombinant collagenase having molecular weight of 125 kd. Collagen digestion activity was measured according to Mendl, I, et al, J.Clin Invest. 32:1323 (~953) with modifications. The major modifications are l). to reduce the amount of enzyme used down to 0.2 or 0.4 ug and 2). to increase the substrate (Type 1 collagen) concentration to lO mg/ml. The collagenase activity is defined as the following: One unit of collagenase activity equals to one umole of L-leucine equivalents released from collagen after 5 hours of incubation at 37C. The enzyme activities of different purified collagenases are shown in Table 2 below.

., ., . ,,, ,,, . -., --- W~94~00580 . 2~3~ PCT/US93/059~

Table 2. Comparisons of Collagen Digestion Activities among Different Purified Collagenases , . ~ ~ ::
Sources Collagen Digestion Units per mg Protein I .
Trigen 14,400 _ . , . . . _ :
Worthington 6,~00-8,000 ~-. _ .
Sigma 8,00~ 9,00 CalBiochem 11,500 -~

The collagen digestion acti~ity of the recombinant collagenase was about 25% to 100% higher than that of the native form of purified collagenases. The result clearly suggest that the recombinant collagenase of the present invention was superior to native c~llagenase. ~ ~

EXAMPLE 12 ~- -Endothelial Cell Isolation With Recombinant Collaqenase ~-Plus Trypsin Purified recombinant collagenase of the present invention produced in E. coli was used to harvest endothelial cells from human saphenous veins. A human saphenous vein was divided in half and perfused one half with 0.1% type II collagenase (Worthington), 0.5% BSA, and PBS/CMF and the other half with 0.048% recombinant collagenase in the same buffer with or without 0.01%
trypsin. Recombinant collagenase alone did not release endothelial cells from the vein. By combining RCL and trypsin, the yield of endothelial cells doubled as compared to the crude collagenase. These results demonstrated that more endothelial cells can be isolated by combining recombinant collagenase of the present invention with a -- constant concentration of trypsin. These results further suggested that it was possible to replace native collagenase with recombinant collagenase of the present invention for cell isolation from different tissues.

SUBSTITlJTE SHEET

W094tOOS80 ~3~ 8 PCr/US93/059~

Rat Islet Isolation Usinq ~næyme Mixture Comprisinq Recombi~ant Colla~enase A composition comprising recomblnant collagenase (SEQUENCE ID NO. 2), native class II collagenase, and neutral proteases was developed to disperse pancreatic tissue to release islets.
Purified recombinant collagenase having a molecul2r weight of about 110,000 daltons (SEQUENCE ID N0. 2) and produced in E. coli was used to harvest rat islets from rat pancreas according to a method of the present invention.
Native class I and II collagenases and neutral protease were prepared according to G.H.J. Wolters, "An Analysis of the Role of Collagenase and Protease in the Enz~matic Dissociation of the Rat Pancreas for Islet Isolation,"
Diabetologia, 35:735-742 (1992).
Rat pancxeas digestion was carried out as in Wolters (1992). A mixture comprising recombinant collagenase (SEQUENCE ID N0. 2), native class II collagenase and neutral protease produced the same rat islet yield as compared to using the crude collagenase preparation or a combination of purified native collagenase and neutral protease. This mixture comprised per 10 ml of RRH buffer (Wolters, 1992) 2 mg of class I recombinant collagenase (SEQUENCE ID N0. 2), 0.8 mg class II native collagenase, and 100 units of neutral protease. Similar digestion time required to obtain the same islet yields were also observed.
These results demonstrated that a composition which comprised the substantially purified recombinant collagenase (SEQUENCE ID N0. 2) dispersed pancreatic tissue to release islets from other pancreatic components.
Furthermore, it was observed that fewer islets each having a larger islet mass per islet resulted when pancreases were ~ digested using a composition comprising recombinant collagenase (SEQUENCE ID N0. 2) and other proteases as compared to other combinations described in the Wolters, W094/00580 ~3~ PCT/US93/059~

1992 reference. It appeared that digesting pancreases .~ -using the method of the present invention resulted in less damage to the integrity of islets as aompared to usiny enzyme compositions which did not comprise the recombinant collagenase (SEQUENCE ID NO. 2) of the pxesen~ i~vention. ~
It is known that larger animals require different lots :
of crude collagenase preparations to obtain satisfactory islets from pancreas digestion. Accordingly, the amounts of recombinant collagenase (SEQUENCE ID NO. 2), or native `:-~
class ~I ~ollagenase, or neutral proteases can be varied to --;
obtain pancreatic islet preparation from pancreas digestions of other animals. Protocols for determining the concentrations of proteolytic enzymes useful for tissue digestions are exemplified in Wolters, 1992. Compositions comprising the highly purified recombinant collagenase (SEQUENCE ID NO. 2) can be used in the method of the present invention for islet isolation from humans. ~:~

Although the present invention has been described in considerable detail with regard to certain preferred versions thereof, other versions are possible. Therefore, the spirit and scope of the amended claims should not be -:
l~mited to the descriptions of the preferred versions herein.

, .

WO g4/0~580 PCr~VSg3~05944 ~ ~
213~ 8 -SEQUENCE LISTING ~ -(1) GENERAL INFORMATION:

(i) APPLICANT: Lin et al., Hun-Chi (ii) TITLE OF INVENTION: Molecular cloning of the genes 5 responsible for collagena6e product ~`~

(iii) NUMBER OF SE~UENCES: 3 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Harris Brotman (B) STREET: 401 B. St Ste 1700 (C) CITY: San Die~o jD3 STATE: CA
(E) COUNTRY: USA
(F) ZIP: 92101-4297 ~

15 (v~ COMPUTER READABLE FORM: ~:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible :. :
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 20(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE-(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Brotman, Harris F.

(ix) TELECOM~UNICATION INFORMATION:
(A) TELEPHONE: (619) 699-3630 (B) TELEFAX: (619) 236-1048 28 `

' W O 94/005~0 ~38~8 PCT/US93/05944 (2) INFORMATION FOR SEQ ID NO~

(i) SEQUENCE CHARACTERISTICS~
(A~ LENGT~: 2817 base pairs (B) TYPE: nucleic acid (C) ST~ANDEDNESS: single (D) TOPOLOGY: linear -(ii) MOLECULE TYPE: DNA (genomic) ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1.... 2808 ~ ~
~ ~, :::
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Met Lys ~ly Ile Glu Thr Phe Thr Glu Val Leu Arg Ala Gly Phe Tyr :~ "
TTa5GGG TAC TAT AAT GAT GGT TTA TCT TAT TTA AAT GAT AGA ~AC TTC 96 Leu Gly Tyr Tyr Asn Asp Gly Leu Ser Tyr Leu Asn Asp Arg Asn Phe ::~

CAA GAT AAA TGT ATA CCT GCA ATG ATT GCA ATT CAA AAA AAT CCT AAC 144 :
Gln Asp Lys Cys Ile Pro Ala Met Ile Ala Ile Gln Lys Asn Pro Asn Phe Lys Leu Gly Thr Ala Val Gln Asp Glu Val Ile Thr Ser Leu Gly 50 55 60 :~

Ll'8 WO 94/~0580 PCI`/US93/05944 - :~

Lys Leu Ile Gly Asn Ala Ser Ala Asn Ala Glu Val Val Asn Asn Cys Val5Pro Val Leu Lys Gln Phe Arg GlU Asn Leu Asn Gln Tyr Ala Pro GAT TAC GTT AAA GGA ACA GCT GTA AAT GAA TTA ATT AAA GGT ATT GAA336 :
Asp Tyr Val Lys Gly Thr Ala Val Asn Glu Leu Ile Lys Gly Ile Glu Tll~GAT ~TT TCT GGT GCT GCA TAT GAA AAA GAT GTT AAG ACA ATG CCT 384 Ph~ Asp Phe Ser Gly Ala Ala Tyr Glu Lys Asp Val Lys Thr Met Pro ~-TGG TAT GGA AAA ATT GAT CCA TTT ATA AAT GAA CTT AAG GCC l`TA GGT432 Trp Tyr Gly Lys Ile Asp Pro Phe Ile Asn Glu Leu Lys Ala Leu Gly Leu Tyr Gly Asn Ile Thr Ser Ala Thr Glu Trp Ala Ser Asp Val Gly Il~OTyr Tyr Leu Ser Lys Phe Gly Leu Tyr Ser Thr Asn Arg Asn Asp Ile Val Gln Ser Leu Glu Lys Ala Val Asp Met Tyr Lys Tyr Gly Lys :.. :' :
AT~5GCC TTT GTA GCA ATG GAG AGA ATA ACT TGG GAT TAT GAT GGG ATT624 ~
Ile Ala Phe Val Ala Met Glu Arg Ile Thr Trp Asp Tyr Asp Gly Ile :--~:~
-"~; ':' -- W0 94/00580 ~3~948 PCl/U~i93/05~

Gly Ser Asn Gly Lys Lys Val Asp His Asp Lys Phe Leu ~sp Asp Ala .

GAA AAA CAT TAT CTG CCA AAG ACA TAT ACT TTT GAT AAT GGA ACC TTT 720 ~ .-Glu5Lys His Tyr ~eu Pro Lys Thr Tyr Thr Phe ~sp Asn Gly Thr Phe - -225 230 235 240 ~.

Ile Ile Arg Ala Gly Asp Lys Val Ser Glu &lu Lys Ile Lys Arg Leu Tyr Trp Ala Ser Arg Glu Val Lys Ser Gln Phe His Arg Val Val Gly 260 265 270 : :

Asn Asp Lys Ala Leu Glu Val Gly Asn Ala Asp Asp Val Leu Thr Met 15 275 ~80 285 ~ -AAA ATA TTT AAT AGC CCA GAA GAA TAT AAA TTT AAT ACC AAT ATA AAT 912 ~
Lys Ile Phe Asn Ser Pro Glu Glu Tyr Lys Phe Asn Thr Asn Ile Asn ;:.
29~ 295 300 :~

GGT GTA AGT ACT GAT AAT GGT GGT CTA TAT ATA GAA CCA AGA GGG ACT 960 :~ -Gl~OVal Ser Thr Asp Asn Gly Gly Leu Tyr Ile Glu Pro Arg Gly Thr :~

Phe Tyr Thr Tyr Glu Arg Thr Pro Gln Gln Ser Ile Phe Ser Leu Glu .:
325 330 335 ~:

GAa5TTG TTT AGA CAT GAA TAT ACT CAC TAT TTA CAA GCG AGA TAT CTT 1056 ~
Glu Leu Phe Arg His Glu Tyr Thr His Tyr Leu Gln Ala Arg Tyr Leu . :

;. ' ~.: ~'., ;;;" ` ~

W O 94/00580 2138~48 PCT/USg3/0~944 Val Asp Gly Leu Trp Gly Gln Gly Pro Phe Tyr Glu Lys Asn Arg Leu ACT TGG TTT GAT GAA GGT ACA GCT GAA TTC TTT GCA GGA TCT ACC CGT 1152.
Thr5Trp Phe Asp Glu Gly Thr Ala Glu Phe Phe Ala Gly Ser Thr Arg 370 375 380 . : `

Thr Ser Gly Val Leu Pro Arg Ly~ Ser Ile Leu Gly Tyr Leu Ala Lys GATOAAA GTA GAT CAT AGA TAC TCA TTA AAG AAG ACT C~T AAT TCA GGG 1248 Asp Lys Yal Asp His Arg Tyr Ser Leu ~ys Lys Thr Leu Asn Ser Gly TAT GAT GAC AGT GAT TGG ATG TTC TAT AAT TAT GGA TTT GCA GTT GCA 1296::
Tyr Asp Asp Ser Asp Trp Met Phe Tyr Asn Tyr Gly Phe Ala Val Ala CAT TAC CTA TAT GAA AAA GAT ATG CCT ACA m ATT AAG ATG AAT AAA 1344 His Tyr Leu Tyr Glu Lys Asp Met Pro Thr Phe Ile Lys Met Asn Lys ~
435 440 445 ~.
: -GCT ATA TTG AAT ACA GAT GTG AAA TCT TAT GAT GAA ATA ATA AAA AAA 1392 .
ALaOIle Leu Asn Thr Asp Val Lys Ser Tyr Asp Glu Ile Ile Lys Lys 4S0 455 460 ~-~

Leu Ser Asp Asp Ala Asn Lys Asn Thr Glu Tyr Gln Asn His Ile Gln .:

GA~5TTA GTA GAT AAA TAT CAA GGA GCT GGA CTA CCT CTA GTA TCA GAT 1488~
Glu Leu Val Asp Lys Tyr Gln Gly Ala Gly Leu Pro Leu Val Ser Asp ~ :
485 490 495 ~:~

~.

32 ~ ~:

_W O 94/00580 -~13~ pcT/uss3/o~s44 GAT TAC TTA AAA GAT CAT GGA TAT AAG AAA GCA TCT GAA GTA TAT TCT 1536 ~:
Asp Tyr Leu Lys Asp His Gly Tyr Lys Lys Ala Ser Glu Val Tyr Ser :~

GAA ATT TCA AAA GCT GCT TCT CTT ACA AAC ACT AGT GTA AC~ GCA GAA 1584 Glu5Ile Ser Lys Ala Ala Ser Leu Thr Asn Thr Ser Val Thr Ala Glu 515 520 S25 ~
': :.'.
AAA TCT CAA TAC TTT AAC ACA TTC ACT TTA AGA GGA ACT TAT ACA GGT 1632 :
Lys Ser Gln Tyr Phe Asn Thr Phe Thr Leu Arg Gly Thr Tyr Thr Gly 530 535 540 ~:

GAaoAcT TCT AAA GGT GAA TTT AAA GAT TGG GAT GAA ATG AGT AAA AAA 1680 Glu Thr Sex Lys Gly Glu Phe Lys Asp Trp ~sp Glu Me~ Ser Lys Lys Leu Asp Gly Thr Leu Glu Ser Leu Ala Lys Asn Ser Trp Ser Gly Tyr ~`.`, ' ~
AAA ACC TTA ACA GCA TAC TTT ACG AAT TAT AGA GTT ACA AGC GAT AAT 1776Lys Thr Leu Thr Ala Tyr Phe Thr Asn Tyr Arg Val Thr Ser Asp Asn :~

AAA GTT CAA TAT GAT GTA GTT TTC CAT GGG GTT TTA ACA GAT AAT GGG 1824Ly~OVal Gln Tyr Asp Val Val Phe His Gly Val Leu Thr Asp Asn Gly ~::
595 600 605 ~;

GAT ATT AGT AAC AAT AAG GCT CCA ATA GCA AAG GTA ACT GGA CCA AGC 1872 ~.
Asp Ile Ser Asn Asn Lys Ala Pro Ile Ala Lys Val Thr Gly Pro Ser :
610 615 620 ;~

AC~5GGT GCT GTA GGA AGA AAT ATT GAA TTT AGT GGA AAA GAT AGT AAA 1920 ~ -Thr Gly Ala Val Gly Arg Asn Ile Glu Phe Ser Gly Lys Asp Ser Lys 33 `

" "' ~

W O 94/00~80 2~g PCT/US~3/0~944 ~ ~

Asp ~lu Asp Gly Lys Ile Val Ser Tyr Asp Trp Asp Phe Gly Asp Gly 645 65~ 655 Ala5Thr Ser Arg Gly Lys Asn Ser Val His Ala Tyr Lys Lys Ala Gly 660 665 670 . :

ACA TAT AAT GTT ACA ~TA AAA GTA ACT GAC GAT AAG GGT GCA ACA GCT 206 ~hr Tyr Asn Val Thr Leu Lys Val Thr Asp Asp Lys Gly Ala Thr Ala ~:

Thr Glu Ser Phe Thr Ile Glu Ile Lys Asn Glu Asp Thr Thr Thr Pro 6~0 695 700 :~

ATA ACT AAA GAA ATG GAA CCT AAT GAT GAT ATA AAA GAG GCT AAT GGT 2160 -~
Ile Thr Lys Glu Met Glu Pro Asn Asp Asp Ile Lys Glu Ala Asn Gly ..
70~5 710 715 720 Pro Ile Val Glu Gly Val Thr Val Lys Gly Asp Leu Asn Gly Ser Asp 725 730 735 .

GAT GCT GAT ACC TTC TAT TTT GAT GTA AAA GAA GAT GGT GAT GTT ACA 2256 -~
AspOAla Asp Thr Phe Tyr Phe Asp Val Lys Glu Asp Gly Asp Val Thr `~:~
740 745 750 ` :`

ATT GAA CTT CCT TAT TCA GGG TCA TCT AAT TTC ACA TGG TTA GTT TAT 2304 .~. .
Ile Glu Leu Pro Tyr 5er Gly Ser Ser Asn Phe Thr Trp Leu Val Tyr ~-755 760 765 - ~.:
~'"
AAa5GAG GGA GAC GAT CAA AAT CAT ATT GCA AGT GGT ATA GAT AAG AAT 2352 Lys Glu Gly Asp Asp Gln Asn His Ile Ala Ser Gly Ile Asp Lys Asn :
770 775 780 .-.,:

34 ~ `~

- WO 94/00~80 PCI'/US93/05944 2~a3~4s Asn Ser Lys Val Gly Thr Phe Lys Ala Thr Lys Gly Arg His Tyr Val 785 790 795 800 ::

Phe5Ile Tyr Lys His Asp ~er Ala Ser Asn Ile Ser Tyr Ser Leu Asn ~ .
ATA AAA GGA TTA G&T AAC GAG AAA TTG AAG GAA AAA GAA AAT AAT GAT 2496 Ile Lys Gly Leu Gly Asn Glu Lys Leu Lys Glu Lys &lu Asn Asn Asp TCTOTCT GAT AAA GCT ACA GTT ATA CCA AAT TTC AAT ACC ACT ATG CAA 2544 :::~
Ser Ser Asp Lys Ala Thr Val Ile Pro Asn Phe Asn Thr Thr Met Gln GGT TCA CTT TTA GGT GAT GAT TCA AGA GAT TAT TAT TCT TTT GAG GTT 2592 ~--Gly Ser Leu Leu Gly Asp Asp Ser Arg Asp Tyr Tyr Ser Phe Glu Val 15850 855 860 .

Lys Glu Glu Gly Glu Val Asn Ile Glu Leu Asp Lys Lys Asp Glu Phe ~ -865 870 875 880 ~-:

Gl~OVal Thr Trp Thr Leu His Pro Glu Ser Asn Ile Asn Asp Arg Ile ~:

Thr Tyr Gly Gln Val Asp Gly Asn Lys Val Ser Asn Lys Val Lys Leu :~

AGa5ccA GGA AAA TAT TAT CTA CTT GTT TAT AAA TAC TCA GGA TCA GGA 2784 Arg Pro Gly Lys Tyr Tyr Leu Leu Val Tyr Lys Tyr Ser Gly Ser Gly :~
915 920 925 :~

~13~ 8 WO 94/00~80 PCr/US93/05944 Asn Tyr Glu Leu Arg Val Asn Lys :

(2) INFORMATION FOR SEQ ID NO:2:

5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 936 a~ino acids (B) TYPE: amino acid tD) TOPOL~GY: linear (ii) MOLECULE TYPE: protein : :

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

Met Lys Gly Ile Glu Thr Phe Thr Glu Val Leu Arg Ala Gly Phe Tyr S 10 15 "~

Leu Gly Tyr Tyr Asn Asp Gly Leu Ser Tyr Leu Asn Asp Arg Asn Phe .~- -20 25 3Q --.

G1~5Asp Lys Cys Ile Pro Ala Met Ile Ala Ile Gln Lys Asn Pro Asn . . .~
Phe Lys Leu Gly Thr Ala Val Gln Asp Glu Val Ile Thr Ser Leu Gly :~

:. .: :.
Lys Leu Ile Gly Asn Ala Ser Ala Asn Ala Glu Val Val Asn Asn Cys ;~
6~0 70 75 80 ..
Val Pro Val Leu Lys Gln Phe Arg Glu Asn Lel~ Asn Gln Tyr Ala Pro ~ ~
85 90 95 . ~.

Asp Tyr Val Lys Gly Thr Ala Val Asn Glu Leu Ile Lys Gly Ile Glu ~:
100 105 110 ~:~

_. W O 94/00580 ~ :2~948 PCT/US93~0~944 Phe Asp Phe Ser Gly Ala Ala Tyr Glu Lys Asp Val Lys Thr Met Pro Trp Tyr Gly Lys Ile Asp Pro Phe Ile Asn Glu Leu Lys Ala Leu Gly Leu5Tyr Gly Asn Ile Thr Ser Ala Thr Glu Trp Ala Ser Asp Val Gly Ile Tyr Tyr Leu Ser Lys Phe Gly Leu Tyr Ser Thr Asn Arg Asn Asp Ile Val Gln S~r Leu Glu Lys Ala Val Asp Met Tyr Lys Tyr Gly Lys Ile Ala Phe Val Ala Met Glu Arg Ile Thr Trp Asp Tyr Asp Gly Ile Gly Ser Asn Gly Lys Lys Val Asp His Asp Lys Phe Leu Asp Asp Ala Gl1l5Lys His Tyr Leu Pro Lys Thr Tyr Thr Phe Asp Asn Gly Thr Phe Ile Ile Arg Ala Gly Asp Lys Val Ser Glu Glu Lys Ile Lys Arg Leu ' 245 250 255 - Tyr Trp Ala Ser Arg Glu Val Lys Ser Gln Phe His Arg Val Val Gly Asn Asp Lys Ala Leu Glu Val Gly Asn Ala Asp Asp Val Leu Thr Met Lys Ile Phe Asn Ser Pro Glu Glu Tyr Lys Phe Asn Thr Asn Ile Asn 21;~ 8 W 0 94~00580 PCT/USg3/05944 ~ : :

Gly Val Ser Thr Asp Asn Gly Gly Leu Tyr Ile Glu Pro Arg Gly Thr 305 310 315 320 ~:

Phe Tyr Thr Tyr Glu Arg Thr Pro Gln Gln Ser Ile Phe Ser L~u Glu 325 330 335 ~:

Glu5Leu Phe Arg His Glu Tyr Thr His Tyr Leu Gln Ala Arg Tyr Leu -340 345 350 ~ ~

Val Asp Gly Leu Trp Gly Gln Gly Pro ~he Tyr Glu Lys Asn Arg Leu :~;
355 360 365 ';

Thr Trp Phe Asp Glu Gly Thr Ala Glu Phe Phe Ala Gly Ser Thr Arg Thr Ser Gly Val Leu Pro Arg Lys Ser Ile Leu Gly Tyr Leu Ala Lys Asp Lys Val Asp His Arg Tyr Ser Leu Lys Lys Thr Leu Asn Ser Gly 405 410 415 `~ ".
. .
Ty~5Asp Asp Ser Asp Trp Met Phe Tyr Asn Tyr Gly Phe Ala Val Ala ~ :
420 425 430 `~

His Tyr Leu Tyr Glu Lys Asp Met Pro Thr Phe Ile Lys Met Asn Lys 435 440 445 -~

Ala Ile Leu Asn Thr Asp Val Lys Ser Tyr Asp Glu Ile Ile Lys Lys Leu Ser Asp Asp Ala Asn Lys Asn Thr Glu Tyr Gln Asn His Ile Gln :-465 470 475 480 :

Glu Leu Val Asp Lys Tyr ~ln Gly Ala Gly Leu Pro Leu Val Ser Asp 485 490 4g5 .

-WO 94~00~80 ~ 48 PCI/US93/05944 ~ ~:
, . ~ .
Asp Tyr Leu Lys Asp His Gly Tyr Lys Lys Ala Ser Glu Val Tyr Ser 500 505 510 ~:

Glu Ile Ser Lys Ala Ala Ser Leu Thr Asn Thr Ser Val Thr Ala Glu 515 520 525 ; :

Lys5Ser Gln Tyr Phe Asn Thr Phe Thr Leu Arg Gly Thr Tyr Thr Gly 530 535 540 ~-Glu Thr Ser Lys Gly Glu Phe Lys Asp Trp Asp Glu Met Ser Lys Lys Leu Asp Gly Thr Leu Glu Ser Leu Ala Lys Asn Ser Trp Ser Gly Tyr Lys Thr Leu Thr Ala Tyr Phe Thr A~n Tyr Arg Val Thr Ser Asp Asn 580 585 590 ; .~.

Lys Val Gln Tyr Asp Val Val Phe His Gly Val Leu Thr Asp Asn Gly :-As~5Ile Ser Asn Asn Lys Ala Pro Ile Ala Lys Val Thr Gly Pro Ser : ... :-Thr Gly Ala Val Gly Arg Asn Ile Glu Phe Sex Gly Lys Asp Ser Lys Asp Glu Asp Gly Lys Ile Val Ser Tyr Asp Trp Asp Phe Gly Asp Gly Ala Thr Ser Arg Gly Lys Asn Ser Val His Ala Tyr Lys Lys Ala Gly Thr Tyr Asn Val Thr Leu Lys Val Thr Asp Asp Lys Gly Ala Thr Ala 2~3f~ 8 Thr Glu Ser Phe Thr Ile Glu Ile Lys Asn Glu Asp Thr Thr Thr Pro :

Il~ Thr Lys Glu Met Glu Pro Asn Asp Asp Ile Lys Glu Ala Asn ~ly ~:

ProbIle Val Glu Gly Val Thr Yal Lys Gly Asp Leu Asn Gly Ser Asp Asp Ala Asp Thr Phe Tyr Phe Asp Val Lys Glu Asp Gly Asp Val Thr ~.:

. .. ;,.-.
Ile Glu Leu Pro Tyr Ser Gly Ser Ser Asn Phe Thr Trp Leu Val Tyr ::~
10 755 760 765 ~:.

Lys Glu Gly Asp Asp Gln Asn His Ile Ala Ser Gly Ile Asp Lys Asn -770 775 780 ~ .:

Asn Ser Lys Val Gly Thr Phe Lys Ala Thr Lys Gly Arg His Tyr Val Ph~5Ile Tyr Lys His Asp Ser Ala Ser Asn Ile Ser Tyr Ser Leu Asn 805 810 815 - ~
-: ' "
Ile Lys Gly Leu Gly Asn Glu Lys Leu Lys Glu Lys Glu Asn Asn Asp ~ -~
820 825 830 :~

Ser Ser Asp Lys Ala Thr Val Ile Pro Asn Phe Asn Thr Thr Met Gln Gly Ser Leu Leu Gly Asp Asp Ser Arg Asp Tyr Tyr Ser Phe Glu Val : .
850 855 860 :

Lys Glu Glu Gly Glu Val Asn Ile Glu Leu Asp Lys Lys Asp Glu Phe qo .. .

.- W O 94/00~80 2~3~g48 PCT/US93/05944 Gly Val Thr Trp Thr Leu His Pro Glu Sér Asn Ile Asn Asp Arg Ile ' `'~' -' ':
Thr Tyr Gly Gln Val Asp Gly Asn Lys Val Ser Asn L~s Val Lys Leu 900 ~05 910 Arg5Pro Gly Lys Tyr Tyr Leu Leu Val Tyr Lys Tyr Ser Gly Ser Gly ,',''`` . '~".
Asn Tyr Glu Leu Arg Val Asn Lys ~30 935 :`

(2) INFORMATION FOR SEQ ID NO:3~
'. ,' ' ' 10 (i) SEQUENCE CHARACTERISTICS: ~.
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid -: ~
(C) STRANDEDNESS: single - .
(D) TOPOLOGY: lin~ar 15(ii) MOLECULE TYPE: DNA (genomic) ~

(xi) SEQUENCE DE5CRIPTION: SEQ ID NO:3: ~ :
: :~
AAATAATTTA TCTTATAAAA AAGAGTGTGC CTAATACATG GCACACTCTT TTTATTTATT 60 ~.

Claims (26)

What is claimed is:

1. A recombinant DNA segment comprising DNA derived from Clostridium histolyticum coding for a polypeptide having the enzymatic activity of C. histolyticum collagenase, the polypeptide being distinguisable from the products of translation determined by the native expression of the C. histolyticum genomic coding sequence by having a higher molecular weight than the products of translation determined by the native expression of the C. histolyticum genomic coding sequence.

2. The DNA segment of claim 1 capable of being transcribed to yield mRNA, the mRNA capable of being translated to yield the polypeptide of claim 1.

3. The DNA segment of claim 1 capable of being transcribed to yield mRNA, the mRNA being capable of being translated to yield the polypeptide of claim 1 displaying the antigenicity of C. histolyticum collagenase.

4. The DNA segment of claim 1 wherein said DNA
segment comprises a promoter derived from C. histolyticum such that said DNA segment when contained in the genome of a host cell can be transcribed under the control of said promoter and without the functioning of a promoter external to said DNA segment.

5. The DNA segment of claim 1 wherein the C.
histolyticum structural gene is capable of further expressing polypeptides having collagenase activity, said polypeptides having molecular weights lower than the polypeptide of claim 1.

6. A vector comprising the DNA segment of claim 1 incorporated into a plasmid capable of transforming host cells, said plasmid selected from the group of plasmids consisting of those with both a drug resistance marker and a replication of origin.

7. The vector of claim 6 wherein said host cell is selected from the group consisting of E. coli, Bacillus subtilis, Clostridium histolyticum, and yeast.

8. E. coli host cells transformed with the vector of claim 6 and producing a polypeptide having collagenase activity and having the antigenicity of C. histolyticum collagenase, said polypeptide being distinguisable from the products of translation determined by the native expression of the C. histolyticum genomic coding sequence by having a higher molecular weight than the products of translation determined by the native expression of the C. histolyticum genomic coding sequence.

9. E. coli host cells transformed with the vector of claim 6 and producing polypeptides having collagenase activity wherein greater than about 50% by weight of the total polypeptides produced which have collagenase activity is comprised of a polypeptide having a higher molecular weight than the products of translation determined by the native expression of the C. histolyticum genomic coding sequenc.

10. E. coli host cells transformed with the vector of claim 6 and producing a polypeptide having a molecular weight of about 110,000 daltons having collagenase activity, wherein said polypeptide of molecular weight of about 110,000 daltons comprises greater than about 50% by weight of the total polypeptides produced that have collagenase activity.

11. A substantially purified preparation of C.
histolyticum collagenase, said collagenase having a higher molecular weight than the products of translation determined by the native expression of the C. histolyticum genomic coding sequence.

12. The preparation of claim 10 purified from E. coli transformed with a vector comprising Clostridium DNA.

13. A substantially purified preparation of C.
histolyticum collagenase, said collagenase having a molecular weight of about 110,000 daltons and prepared from E. coli transformed with a vector comprising Clostridium DNA.

14. A composition comprising a substantially purified preparation of C. histolyticum collagenase, said collagenase having a higher molecular weight than the products of translation determined by the native expression of the C. histolyticum genomic coding sequence.

15. A composition comprising a substantially purified preparation of C. histolyticum collagenase, said collagenase having a molecular weight of about 110,000 daltons and prepared from E. coli transformed with a vector containing Clostridium DNA.

16. A method for digesting connective tissue and releasing embedded cells without destroying cell membranes and other essential structures comprising the steps of:
(a) incubating tissue in a buffered solution comprising said substantially purified collagenase of claim 11 with shaking at about 25-39° C to release and disperse the embedded cells; and (b) separating the dispersed cells from tissue debris.

17. The method of claim 16 wherein the step of separating the dispersed cells from tissue debris is performed by density gradient centrifugation.

18. The method of claim 16 using a substantially purified preparation of C. histolyticum collagenase, said collagenase having a molecular weight of about 110,000 daltons and prepared from E. coli transformed with a vector comprising Clostridium DNA.

19. A method for isolating dispersed pancreatic islets comprising the steps of:
(a) incubating pancreatic tissue in a buffered solution comprising the substantially purified collagenase of claim 11 with shaking at about 25-39° C to release and disperse the pancreatic islets; and (b) separating the dispersed pancreatic islets from tissue debris.

20. The method of claim 19 wherein the step of separating the dispersed pancreatic islets is performed by density gradient centrifugation.

21. The method of claim 19 using a substantially purified preparation of C. histolyticum collagenase, said collagenase having a molecular weight of about 110,000 daltons and prepared from E. coli transformed with a vector comprising Clostridium DNA.

22. A method for dissociating tumors comprising the steps of:
(a) incubating tumor tissue in a buffered solution comprising the substantially purified collagenase of claim 11 with shaking at about 25-39° C to dissociate and disperse the tumor cells; and (b) separating the dispersed tumor cells from tissue debris.

23. The method of claim 22 wherein the step of separating the dispersed tumor cells is performed by density gradient centrifugation.

24. The method of claim 22 using a substantially purified preparation of C. histolyticum collagenase, said collagenase having a molecular weight of about 110,000 daltons and prepared from E. coli transformed with a vector comprising Clostridium DNA.

25. A method for intradiscal treatment of herniation of nucleus pulposus comprising the steps of:
(a) preparing a sterile buffered solution comprising the substantially purifed collagenase of claim 11; and (b) injecting the sterile buffered solution comprising the substantially purified collagenase into the nucleus pulposus.

26. The method of claim 25 using a substantially purified preparation of C. histolyticum collagenase, said collagenase having a molecular weight of about 110,000 daltons and prepared from E. coli transformed with a vector comprising Clostridium DNA.