CA2144601C - Recombinant type ii collagenase from clostridium histolyticum and its use for isolating cells and groups of cells - Google Patents
Recombinant type ii collagenase from clostridium histolyticum and its use for isolating cells and groups of cells Download PDFInfo
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Abstract
Process for disintegrating cell tissue and releasing cells or groups of cells contained therein by incubation of the cell tissue with a class II collagenase from Clostridium histolyticum, which still has a considerable degree of collagenase activity after 12 freeze/thaw cycles and contains SEQ ID NO:2 or SEQ ID NO:10 in a characteristic partial region or it is coded in this region by SEQ ID NO:1, SEQ ID NO:9 or a sequence which codes for the same amino acid sequence within the scope of the degeneracy of the genetic code and is the product of a prokaryotic or eukaryotic expression of an exogenous DNA, until the cells or groups of cells are released to the desired extent and separating the cells or the groups of cells from the cell tissue fractions.
Description
Recombinant type II collagenase from Clostridium histolyticum and its use for isolating cells and groups of cells The invention concerns a recombinant type II collagenase from Clostridium histolyticum and its use for isolating cells and groups of cells.
Bacterial collagenases, e.g. from Clostridium histolyticum, are used to digest tissues and to isolate individual cells or groups of cells (e. g, islets) (islets: Sutton et al., Transplantation 42 (1986) 689 -691; liver: Quibel et al., Anal. Biochem. 154 (1986) 26 - 28; bones: Hefley et al., J. Bone Mineral Res. 2 (1987) 505 - 516; umbilical cord: Holzinger et al., Immunol. Lett. 35 (1993) 109 - 118. Two different types of collagenase are known from Clostridium histolyticum (M.F. French et al., J. Protein Chemistry 11 (1992) 83 -97). The production of class II collagenase from Clostridium histolyticum (CHC II) is described for example in E.L. Angleton and H.E. van Wart, Biochemistry 27 (1988) 7413 - 7418 and 7406 - 7412. However, it has turned out that CHC II is very sensitive to repeated freezing and thawing. The activity of CHC II almost completely disappears after 12 freeze/thaw cycles. (T. J.
Hefley, J. Bone and Minerals Res. 2 (1987) 505 - 516).
The object of the present invention is to provide a class II collagenase from Clostridium histolyticum which is stable when frozen.
Bacterial collagenases, e.g. from Clostridium histolyticum, are used to digest tissues and to isolate individual cells or groups of cells (e. g, islets) (islets: Sutton et al., Transplantation 42 (1986) 689 -691; liver: Quibel et al., Anal. Biochem. 154 (1986) 26 - 28; bones: Hefley et al., J. Bone Mineral Res. 2 (1987) 505 - 516; umbilical cord: Holzinger et al., Immunol. Lett. 35 (1993) 109 - 118. Two different types of collagenase are known from Clostridium histolyticum (M.F. French et al., J. Protein Chemistry 11 (1992) 83 -97). The production of class II collagenase from Clostridium histolyticum (CHC II) is described for example in E.L. Angleton and H.E. van Wart, Biochemistry 27 (1988) 7413 - 7418 and 7406 - 7412. However, it has turned out that CHC II is very sensitive to repeated freezing and thawing. The activity of CHC II almost completely disappears after 12 freeze/thaw cycles. (T. J.
Hefley, J. Bone and Minerals Res. 2 (1987) 505 - 516).
The object of the present invention is to provide a class II collagenase from Clostridium histolyticum which is stable when frozen.
The object is achieved by a polypeptide which has the properties of a class II collagenase from Clostridium histolyticum, still has a considerable collagenase activity after 12 freeze/thaw cycles and is obtainable by expression of an exogenous DNA sequence in prokaryotic or eukaryotic host cells and isolation of the desired polypeptide wherein the DNA sequence contains SEQ ID NO: 1 in the characteristic region for this collagenase or a sequence which codes for the same amino acid sequence within the scope of the degeneracy of the genetic code.
It surprisingly turned out that a CHC II which is the product of a recombinant production is much more stable to repeated freezing and thawing than CHC II isolated from natural sources and still has a considerable degree of collagenase activity after 12 freeze/thaw cycles.
The production of recombinant CHC II can be carried out according to methods familiar to a person skilled in the art.
For this a DNA sequence is firstly prepared which is able to produce a protein which has the activity of CHC II. By the term "DNA sequence" as used herein there is to be understood an isolated DNA molecule. The sequence coding for the protein can be determined with the aid of SEQ ID NO: 1 by determining the reading frame on the basis of a comparison with known peptide sequences of collagenase and the DNA sequence determined in this manner is converted into an amino acid sequence with the aid of the genetic code. The sequence coding for the active protein is ca. 2 to 3 times longer than SEQ ID NO: 1. The DNA sequence is cloned in a vector which can be transferred into a host cell and which can be replicated there. Such a vector also contains operator elements which are necessary for the expression of the DNA sequence in addition to the CHC II sequence.
This vector which contains the CHC II sequence and the operator elements is transferred into a vector which is able to express the DNA of CHC II. The host cell is cultured under conditions which are suitable for the amplification of the vector and CHC II is isolated. In this process suitable measures ensure that the protein can assume an active tertiary structure in which it exhibits CHC II properties.
The cloning and nucleotide sequence of CHC II is described in the post-published publication K. Yoshihara et al., J. Bacteriol. 176 (Nov. 1994) 6489 - 6494.
In this process it is not necessary that the expressed protein contains the exact CHC II amino acid sequence with the partial region SEQ ID NO: 2. Proteins are equally suitable which contain essentially the same sequence and have analogous properties.
The nucleic acid sequence of the protein can also be modified. Such modifications are for example:
- Modification of the nucleic acid in order to introduce various recognition sequences of restriction enzymes to facilitate the steps of ligation, cloning and mutagenesis.
- Modification of the nucleic acid to incorporate the preferred codons for the host cell.
- Extension of the nucleic acid by additional operator elements in order to optimize the expression in the host cell.
The invention in addition concerns a process for the production of a polypeptide which has the properties of a class II collagenase from Clostridium histolyticum and still has a considerable degree of collagenase activity after 12 freeze/thaw cycles by expression of an exogenous DNA sequence in prokaryotic or eukaryotic host cells and isolation of the desired polypeptide wherein the DNA sequence contains SEQ ID NO: 1 in the region characteristic for this collagenase or a sequence which codes for the same amino acid sequence within the scope of the degeneracy of the genetic code.
The expression of the protein is preferably carried out in microorganisms, in particular in prokaryotes and in this case in E. coli.
The expression vectors must contain a promoter which enables the expression of the protein in the host organism. Such promoters are known to a person skilled in the art and are for example the lac promoter (Chang et al., Nature 198 (1877) 1056), trp promoter (Goeddel et al., Nuc. Acids Res. 8 (1980) 4057), 7lpL promoter (Shimatake et al., Nature 292 (1981) 128) and T5 promoter (US patent No. 4,689,406). Synthetic promoters are also suitable such as for example the tac promoter (US Patent No. 4,551,433). Coupled promoter systems are also suitable such as the T7-RNA polymerase/promoter system (Studier et al., J. Mol. Biol. 189 (1986) 113).
Hybrid promoters composed of a bacteriophage promoter and the operator region of the microorganism (EP-A 0 267 851) are equally suitable. An effective ribosome binding site is necessary in addition to the promoter. In the case of E. coli this ribosome binding site is denoted Shine-Dalgarno (SD) sequence (Sambrook et al., "Expression of cloned genes in E. coli" in Molecular Cloning: A laboratory manual (1989) Cold Spring Harbor Laboratory Press, New York, USA).
In order to improve the expression it is possible to express the protein as a fusion protein. In this case a DNA sequence which codes for the N-terminal part of an endogenous bacterial protein or for another stable protein is usually fused to the 5' end of the sequence coding for CHC II. Examples of this are for example lacZ
(Phillips and Silhavy, Nature 344 (1990) 882 - 884), trpE (Yansura, Meth. Enzymol. 185 (1990) 161 - 166).
After expression of the vector, preferably a biologically functional plasmid or a viral vector, the fusion proteins are preferably cleaved with enzymes (e. g. factor Xa) (Nagai et al., Nature 309 (1984) 810).
Further examples of cleavage sites are the IgA protease cleavage site (WO 91/11520, EP-A 0 495 398) and the ubiquitin cleavage site (Miller et al., Bio/Technology 7 (1989) 698).
The proteins expressed in this manner in bacteria are isolated in the usual manner by lysing the bacteria and protein isolation.
In a further embodiment it is possible to secrete the proteins as active proteins from the microorganisms. For this a fusion product is preferably used which is composed of a signal sequence which is suitable for the secretion of proteins in the host organisms used and the nucleic acid which codes for the protein. In this 21.4601 process the protein is either secreted into the medium (in the case of gram-positive bacteria) or into the periplasmatic space (in the case of gram-negative bacteria). It is expedient to place a cleavage site between the signal sequence and the sequence coding for CHC II which enables cleavage of the protein either during processing or in an additional step. Such signal sequences are derived for example from ompA (Ghrayeb et al. EMBO J. 3 (1984) 2437) or phoA (Oka et al., Proc.
Natl. Acad. Sci. USA 82 (1985) 7212).
The vectors in addition also contain terminators.
Terminators are DNA sequences which signal the end of a transcription process. They are usually characterized by two structural characteristics: an inverse repetitive G/C-rich region which can intramolecularly form a double helix and a number of U(or T) residues. Examples are the main terminator in the DNA of the phages fd (Beck and Zink, Gene 16 (1981) 35-58) and rrnB (Brosius et al., J.
Mol. Biol. 148 (1981) 107 - 127).
In addition the expression vectors usually contain a selectable marker in order to select the transformed cells. Such selectable markers are for example the resistance genes for ampicillin, chloroamphenicol, erythromycin, kanamycin, neomycin and tetracyclin (Davies et al., Ann. Rev. Microbiol. 32 (1978) 469).
Selectable markers which are also suitable are the genes for substances that are essential for the biosynthesis of substances necessary for the cell such as e.g.
histidine, tryptophan and leucine.
Numerous suitable bacterial vectors are known. Vectors have for example been described for the following _ 7 _ bacteria: Bacillus subtilis (Palva et al., Proc. Natl.
Acad. Sci. USA 79 (1982) 5582), E. coli (Arran et al., Gene 40 (1985) 183; Studier et al., J. Mol. Biol. 189 (1986) 113), Streptococcus cremoris (Powell et al., Appl. Environ. Microbiol. 54 (1988) 655), Streptococcus lividans and Streptomyces lividans (US patent No.
4,747,056).
Further genetic engineering methods for the construction and expression of suitable vectors are described in J.
Sambrook et al., Molecular Cloning: A laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, N.Y.
Apart from in prokaryotic microorganisms, recombinant CHC II can also be expressed in eukaryotes (such as for example CHO cells, yeast or insect cells). The yeast system or insect cells are preferred as the eukaryotic expression system. Expression in yeast can be achieved by means of three types of yeast vectors: integrating YIp (yeast integrating plasmids) vectors, replicating YRp (yeast replicon plasmids) vectors and episomal YEp (yeast episomal plasmids) vectors. Further details of this are for example described in S.M. Kingsman et al., Tibtech 5 (1987) 53 - 57).
The invention in addition concerns a process for disintegrating cell tissue and releasing cells or groups of cells contained therein by incubating the cell tissue with a class II collagenase from Clostridium histolyticum which still has a considerable degree of collagenase activity after 12 freeze/thaw cycles and contains SEQ ID NO: 2 in a characteristic partial region or it is coded in this region by SEQ ID NO: 1 or a ~~ ~~ s~~
-8_ sequence which codes for the same amino acid sequence within the scope of the degeneracy of the genetic code and is the product of a prokaryotic or eukaryotic expression of an exogenous DNA, until the cells or groups of cells have been released to the desired extent and separating the cells or groups of cells from the cell tissue fractions. The separation of the cells or groups of cells from the cell tissue fractions is preferably carried out by centrifugation using a density gradient.
Cells or groups of cells are usually isolated from tissues (e. g. pancreas, liver, skin, endothelium, umbilical cord, bones, tumour tissue) by incubating organs, organ parts or tissues with enzymes which disintegrate the surrounding extracellular connective tissue matrix (islets: Sutton et al., Transplantation 42 (1986) 689 - 691: liver: Quibel et al., Anal. Biochem.
154 (1986) 26 - 28; bones: Hefley et al., J. Bone Mineral Res. 2 (1987) 505 - 516; umbilical cord:
Holzinger et al., Immunol. Lett. 35 (1993) 109 - 118.
Tumour cells isolated in this manner may be used advantageously for tumour vaccination and/or for adoptive immunotherapy. Tissue disintegration can also be accomplished by perfusing the entire organ (Ricordi et al., Diabetes 37 (1988) 413 - 420) with an enzyme solution. Important factors in this process, in addition to the composition of the enzyme mixture, are the duration, the pH value and the temperature of the digestion as well as mechanical influence e.g. by shaking and addition of metal balls. Since extracellular connective tissue matrix often has a high proportion of collagen, collagenases and in particular type II
collagenase play a special role in this case (Wolters, Hormone and Metabolic Research 26 (1994), p. 80).
21 ~.4-~ ~ ~
_ g _ The process according to the invention is preferably used to isolate islets or islet cells from pancreatic tissue.
In addition the addition of further enzymes such as proteinases (e.g. neutral protease, cf example 5 or other metalloproteases; serine proteases such as trypsin, chymotrypsin, plasmin etc.; cysteine proteases:
aspartate proteases), elastases, hyaluronidases, lipases or other collagenases may be advantageous for the quality of the digestion.
The invention is elucidated in more detail by the following examples and sequence protocols.
The sequence protocols denote:
SEQ ID NO: 1: DNA fragment of CHC II
SEQ ID NO: 2: derived protein fragment SEQ ID NO: 3 - 8: primer sequences SEQ ID NO: 9: cDNA of CHC II
SEQ ID NO: 10: protein sequence of CHC II
SEQ ID NO: 11/12: primer Example 1 Purification of class II collagenase (CHC II) 1 g collagenase P (from Clostridium histolyticum, Boehringer Mannheim GmbH, Order No. 1213857) was dissolved in 20 ml H20 (MilliQ quality) and particulate components were separated by centrifugation. After precipitation with 60 % ammonium sulfate, the precipitate was taken up in 9.5 ml H20 and dialysed overnight at 4'C against 10 mM Tris-HC1, pH 7.5.
The dialysate was applied at room temperature to a Zn-chelate Sepharose*column (column volume CV = 50 ml, 2 cm diameter) at a flow rate of 1.5 CV (75 ml/H). Afterwards the column was washed with 10 mM Tris-HCl, pH 7.5 until the base line in the W chromatogram had been reached again. CHC II bound to the affinity matrix was eluted with a linear gradient (10 CV = 500 ml) up to 50 mM
Tris, pH 7.5, 50 mM imidazole. The active fractions (determined by cleavage of synthetic peptide substrates) were dialysed against 10 mM Tris, pH 8.0, 1 mM CaCl2 and stored at -20'C.
A strong increase in the bands in the region of > 85 kD
occurs in the SDS gel, further proteins (MW between 40 and 85 kD) as well as some smaller bands are recognizable.
The fractions containing CHC II were further separated within 30 minutes by means of a MonoQ*column (FPLC) in mM Tris-HC1, pH 8.0 using a linear NaCl gradient from 0 to 0.3 M NaCl in 10 mM Tris-HC1, pH 8Ø The peaks containing CHC II activity are further purified by reversed phase HPLC (mobile solvent A: 0.12 % TFA/H20;
mobile solvent B: 0.12 % TFA (trifluoroacetic acid)/
100 % acetonitrile). A total of 4 peaks are collected, evaporated to dryness and subjected to LysC digestion after carboxymethylation. The peptides were also separated by reversed phase HPLC on a C4*column using.a gradient~Qf 5 - 45 % acetonitrile/0.12 % TFA. The chromatograms of all digestions were identical with the exception of minor differences. They significantly * Trade-mark A
differed from LysC peptide maps of collagenase I
(purified by gel filtration on Superdex HiLoad S 200 or by repurification of the eluate from the Zn-chelate Sepharose). The~peptides were collected, evaporated to dryness and sequenced.
Example 2 Cloning collagenase II from Clostridium histolyticum The matching (degenerate) DNA sequence is derived from the peptide sequences determined according to example 1 after purification of CHC II. Sequences which exhibit an advantageous (lower) denaturation are used to construct a labelled DNA probe to screen gene banks e.g. via PCR.
2 peptides Coll-2-60 and Coll-2-51, are particularly suitable from which the following primers can be derived:
peptide Coll-2-60 (SEQ ID N0:3) primer Coll-2-60F (SEQ ID N0:4) primer Coll-2-60R (SEQ ID N0:5) peptide Coll-2-51 (SEQ ID N0:6) primer Coll-2-51F (SEQ ID N0:7) . .
primer Coll-2-51R (SEQ ID ~~T0:8) * Trade-mark A
Fragments of ca. 500 and ca. 750 by length are obtained after PCR with DNA isolated according to conventional methods from Clostridium histolyticum using primers 60F
and 51R.
The 500 by fragment is labelled, for example also in a PCR reaction, with dig-dUTP (prepared according to WO 89/06698, EP-A 0 324 468), and serves to identify positive clones from a gene bank. The gene bank can be prepared according to generally known methods from Clostridium histolyticum DNA after digestion with restriction enzymes.
The 500 by fragment was sequenced and contains DNA from the sequence SEQ ID NO: 1.
The 750 by fragment can be fragmented into a ca. 600 by large and a 150 by large fragment using PstI. The 600 by fragment also contains SEQ ID NO: 1. pC2-600 is a vector which contains the 600 by fragment and was deposited under the number DSM 8996 on the 24.02.1994 at the DSM
("Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig).
The amino acid sequence has ca. 25 $ homology to a collagenase from Vibrio alginolyticus and is shown in SEQ ID N0:2. SEQ ID N0:2 is coded by the nucleotides 2 -487 from SEQ ID NO:1.
The missing parts of the coding region and of the untranslated regions of the gene can be amplified and sequenced by inverse PCR after digestion of the genomic DNA from Clostridium histolyticum using suitable restriction enzymes and primers derived from SEQ ID
NO:1. The coding region of the gene (ca. 3 kb) is contained in SEQ ID NO: 1.
The reading frame of SEQ ID NO: 9 can be unequivocally determined with the aid of peptide sequences found by protein sequencing and the sequence can be translated into the peptide sequence SEQ ID NO:10.
The ends of the coding region of nucleotide 688 to nucleotide 3792 were modified with the aid of two primers (SEQ ID NO: 11 and 12) and cloned into pUCBM 20 via XmaI and NotI. The resulting plasmid pC2-Exl/3-23 was deposited on the 09.12.1994 under the number DSM
9579 at the "Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig".
Example 3 Expression of class II aollagenase DNA fragments which contain the gene for CHC II or parts thereof are sequenced and their ends are modified in a suitable manner (e.g. via PCR) and they are used as a whole or in combination in an expression vector for E.
coli. The tac promoter is used as the promoter. However, it is also possible to use other - preferably well-regulatable - promoters.
A fragment of SEQ ID NO: 9 can be used for the expression which starts at position 688 or 757 and ends in the region of position 3792 - position 3900 which is brought under the control of one of the usual promoters.
As an alternative a fragment can be used which starts at position 847 and ends in the region of position 3792 -position 3900 which is firstly provided with the sequence for a heterologous signal peptide and is then placed behind the usual promoters. Those vectors with a high copy number are suitable as the vectors such as those of the pUC series. However, vectors with a low copy number derived from pACYC 177 or pACYC 184 can also be used when the selected host cell does not tolerate expression in high copy numbers. A range of 25 - 37°C is preferred as the growth temperature.
An E. coli strain which has been transformed with an expression plasmid is either cultured overnight at 30 to 37°C (e.g. in 5 ml culture) in minimum medium or LB
medium under antibiotic selection. The overnight culture is inoculated into a larger volume (e.g. 1 1) and allowed to grow further. If this volume is already used to obtain biomass, it can be induced at an OD550 of 0.2 to 2 with IPTG and the cells are allowed to grow further until there is no further increase in the OD and/or the enzyme activity that forms. At this time it is centrifuged and the biomass is used to purify collagenase.
If a significant proportion of the collagenase activity is present in the medium; the medium is also collected and collagenase is purified therefrom.
Example 4 Islet isolation from porcine pancreas The pancreas from a freshly slaughtered pig is prepared and cooled in ice-cold HBSS buffer (Gibco) until further processing. A Braunule is inserted into the ductus pancreaticus and fastened there, the water-tightness of the pancreas is tested using HBSS buffer. An enzyme solution in HBSS buffer + Ca2+ which contains purified recombinant type II collagenase alone or in combination with a purified native or recombinant neutral protease from Clostridium histolyticum is injected. The pancreas treated in this way is connected to the perfusion unit that also contains the above-mentioned enzyme solution (discontinuous perfusion). The digestion is carried out between 4°C - 37°C during a time period of 5 to 120 minutes. The pump is stopped after the time that is assumed to be optimal and the vessel containing the pancreas is carefully shaken for 3 to 20 minutes by hand. If necessary the prior addition of metal balls additionally facilitates the mechanical dissociation of the tissue and release of the islets from the surrounding exocrine tissue.
The digestion is stopped by addition of ice-cold HBSS/
% FCS (foetal calf serum) and the suspension is filtered through a sieve (mesh size 300 ~,m) in order to separate coarse particles. The islets present in the filtrate are centrifuged for 10 minutes at 100 g in 250 ml Nalgene round-bottom flasks. The supernatant is decanted and the pellet containing the islets is resuspended in 50 ml FCS.
The islets can be purified further by means of a density gradient made by hand. Firstly 7 ml islet suspension is added to 250 ml Nalgene round-bottom flasks. These are firstly overlayered with 93 ml of a Ficoll~ solution (cp = 1.077 g/cm3), and then with 50 ml medium (RPMI
1640). These gradients are centrifuged for 10 minutes at '" 214460 1 100 g in a swing-out rotor. Fractions of 10 ml are collected, the size, purity and yield of the islets stained with dithizone is determined microscopically in every fraction~or with the aid of image analysis.
Example 5 Isolation of neutral protease Collagenase P is dissolved in 5 mM HEPES buffer, pH 7.5, 1 mM CaCl2 (c = 50 mg/ml) and centrifuged. An aliquot (10 mg) is injected onto a MonoQ*column (Pharmacia FPLC) and eluted with a CaCl2 gradient (up to 150 mM in 20 min.). The fraction having the highest specific casinolytic activity (determined by means of resorufin-labelled casein, Boehringer Mannheim GmbH) contained a protein with a MW of 30 - 35 kDa (SDS gel). The peptides which formed after carboxymethylation and LysC digestion were separated by reversed phase HPLC on a Cg*column, concentrated to dryness and sequenced. As an alternative the protein fractions with a high caseinolytic activity were separated on a SDS gel, transferred onto PVDF*
membranes (Millipore) (Western blot; transfer buffer 50 mM Tris, 50 mM boric acid, pH 8.3) and the relevant bands were cut out after staining with amido black.
After LysC digestion of the immobilized protein, the peptides were detached with HCOOH as described above, separated and sequenced.
.
* Trade-mark A, SEQUENCE LISTING
NUMBER OF SEQUENCES: 12 (2) INFORMATION FOR SEQ ID NO: 1:
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NO: 1:
214-4~~I
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(A) LENGTH: 162 amino acids (B) TYPE: amino~acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Asp Xaa Tyr Glu Asn Leu Thr Val Xaa Xaa Val Ala Asp Asp Tyr Leu Val Arg His Ala Tyr Lys Asn Pro Asn Glu Ile Tyr Ser Glu Ile Ser Glu Val Ala Lys Leu Lys Asp Ala Lys Ser Glu Val Lys Lys Ser Gln Tyr Phe Ser Thr Phe Thr Leu Arg Gly Ser Tyr Thr Gly Gly Val Ser Lys Gly Lys Leu Glu Asp Gln Lys Ala Met Asn Lys Phe Ile Asp Asp Ser Leu Lys Lys Leu Asp Thr Tyr Ser Trp Ser Gly Tyr Lys Thr Leu Thr Ala Tyr Phe Thr Asn Tyr Lys Val Asp Ser Ser Asn Lys''?Val Thr Tyr Asp Val Val Phe His Gly Tyr Leu Pro Xaa Xaa Gly Asp Ser Arg ~1~.4601 Asn Ser Leu Pro Tyr Gly Lys Thr Asn Gly Thr Tyr Lys Gly Thr Glu Lys Glu Lys Ile Lys Phe Ser Ser Glu Gly Ser Xaa Asp Pro Asp Gly Lys Ile (2) INFORMATION FOR SEQ ID NO: 3:
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Tyr Gln Asp His Met Gln Glu (2) INFORMATION FOR SEQ ID NO: 9:
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(A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3979 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: double (D) TOPOLOGY: linear lii} MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: sig-peptide (B) LOCATION:688..846 (ix) FEATURE:
(A) NAME/KEY: sig-peptide (B) LOCATION:757..846 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:847..3792 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CCTAATATTC TCTTATATAC TTAATTAAAT ATTAATAAAA AATTAATGA,A CAGGTATATC 590 ,...
4~
~i4~so1 - 25 - ~ ~ 1 (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 981 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Val Gln Asn Glu Ser Lys Arg Tyr Thr Val Ser Tyr Leu Lys Thr Leu Asn Tyr Tyr Asp Leu Val Asp Leu Leu Ala Lys Thr Glu Ile Glu Asn Leu Pro Asp Leu Phe Gln Tyr Ser Ser Asp Ala Lys Glu Phe Tyr Gly Asn Lys Thr Arg Met Asn Phe Ile Met Asp Glu Ile Gly Arg Arg Ala Ser Gln Tyr Thr Glu Ile Asp His Lys Gly Ile Pro Thr Leu Val Glu Val Val Arg Ala Gly Phe Tyr Leu Gly Phe His Asn Lys Glu Leu Asn Glu Ile Asn Lys Arg Ser Phe Lys Glu Arg Val Ile Pro 5er Ile Leu Ala Ile Gln Lys Asn Pro Asn Phe Lys Leu Gly Thr Glu Val Gln Asp Lys Ile Val Ser Ala Thr Gly Leu Leu Ala Gly Asn Glu Thr Ser Pro Ala Glu Val Val Asn Asn Phe Thr Pro Ile Leu Gln Asp Cys Ile Lys Asn Met Asp Arg Tyr Ala Leu Asp Asp Leu Lys Ser Lys Ala Leu Phe Asn Val Leu Ala Ala Pro Thr Tyr Asp Val Thr Glu Tyr Leu Arg Ala Thr Lys Glu Lys Pro Glu Asn Thr Pro Trp Tyr Gly Lys Ile Asp Gly Phe Ile Asn Glu Val Lys Lys Leu Ala Leu Tyr Gly Lys Ile Asn Asp Lys Asn Ser Trp Ile Ile Asp Asn Gly Ile Tyr His Ile Ala Pro Leu Gly Lys Leu His Ser Asn Asn Lys Ile Gly Ile Glu Thr Leu Thr Glu Val Met Lys Val Tyr Pro Tyr Leu Ser Met Gln His Leu Gln Ser Ala Asp Gln Ile Lys Arg His Tyr Asp Ser Lys Asp Ala Glu Gly Asn Lys 2?5 280 285 Ile Pro Leu Asp Lys Phe Lys Lys Glu Gly Lys Glu Lys Tyr Cys Pro Lys Thr Tyr Thr Phe Asp Asp Gly Lys Val Ile Ile Lys Ala Gly Ala Arg Val Glu Glu Glu Lys Val Lys Arg Leu Tyr Trp Ala Ser Lys Glu Val Asn Ser Gln Phe Phe Arg Val Tyr Gly Ile Asp Asn Pro Leu Glu Glu Gly Asn Pro Asp Asp Ile Leu Thr Met Xaa Ile Tyr Asn Ser Pro Glu Glu Tyr Lys Leu Asn Ser Val Leu Xaa Gly Tyr Asp Thr Asn Asn Gly Gly Met Tyr Ile Glu Pro Glu Gly Thr Phe Phe Thr Tyr Glu Arg Glu Ala Gln Glu Ser Thr Tyr Thr Leu Xaa Glu Leu Phe Arg His Glu Tyr Thr His Tyr Leu Gln Gly Arg Tyr Ala Val Pro Gly Gln Trp Gly Arg Pro Lys Leu Tyr Asp Asn Asp Arg Leu Thr Trp Tyr Glu Glu Gly Gly Ala Glu Leu Phe Ala Gly Ser Thr Arg Thr Ser Gly Ile Leu Pro Arg Lys Ser Ile Val Ser Asn Ile His Asn Thr Thr Arg Asn Asn Arg Tyr Lys Leu Ser Asp Thr Val His Ser Lys Tyr Gly Ala Ser Phe Glu Phe Tyr Asn Tyr Ala Cys Met Phe Met Asp Tyr Met Tyr Asn Lys Asp Met Gly Ile Leu Asn Lys Leu Asn Asp Leu Ala Lys Asn Asn Asp Val Asp Gly Tyr Asp Asn Tyr Ile Arg Asp Leu Ser Ser Asn His Ala Leu Asn Asp Lys Tyr Gln Asp His Met Gln Glu Arg Ile Asp Asn Tyr Glu Asn Leu Thr Val Pro Phe Val Ala Asp Asp Tyr Leu Val Arg His Ala Tyr Lys Asn Pro Asn Glu Ile Tyr Ser Glu Ile Ser Glu Val Ala Lys Leu Lys Asp Ala Lys Ser Glu Val Lys Lys Ser Gln Tyr Phe Ser Thr Phe Thr Leu Arg Gly Ser Tyr Thr Gly Gly Val Ser Lys Gly Lys Leu Glu Asp Gln Lys Ala Met Asn Lys Phe Ile Asp Asp Ser Leu Lys Lys 2~446~1 Leu Asp Thr Tyr Ser Trp Ser Gly Tyr Lys Thr Leu Thr Ala Tyr Phe Thr Asn Tyr Lys Val Asp Ser Ser Asn Xaa Val Thr Tyr Asp Val Val Phe His Gly Tyr Leu Pro Asn Glu Gly Asp Ser Lys Asn Ser Leu Pro Tyr Gly Lys Thr Asn Gly Thr Tyr Lys Gly Thr Glu Lys Glu Lys Ile Lys Phe Ser Ser Glu Gly Ser Phe Asp Pro Asp Gly Lys Ile Val Ser Tyr Glu Trp Asp Phe Gly Asp Gly Asn Lys Ser Asn Glu Glu Asn Pro Glu His Ser Tyr Asp Lys Val Gly Thr Tyr Thr Val Lys Leu Lys Val Thr Asp Asp Lys Gly Glu Ser Ser Val Ser Thr Thr Thr Ala Glu Ile Arg Asp Leu Ser Glu Asn Lys Leu Pro Val Ile Tyr Met His Val Pro Thr Ser Gly Ala Leu Asn Gln Lys Val Val Phe Tyr Gly Lys Gly Thr Tyr Asp Pro Asp Gly Ser Ile Ala Gly Tyr Gln Trp Asp Phe Gly Asp ..r~
2~.4~~~I
Gly Ser Asp Phe Ser Ser Glu Gln Asn Pro Ser His Val Tyr Thr Lys Lys Gly Glu Tyr Thr Val Thr Leu Arg Val Met Asp Ser Ser Gly Gln Met Ser Glu Lys Thr Met Lys Ile Lys Ile Thr Asp Pro Val Tyr Pro Ile Gly Thr Glu Lys Glu Pro Asn Asn Ser Lys Glu Thr Ala Ser Gly Pro Ile Val Pro Gly Ile Pro Val Ser Gly Thr Ile Glu Asn Thr Ser Asp Gln Asp Tyr Phe Tyr Xaa Asp Val Ile Thr Pro Gly Glu Val Lys Ile Asp Ile Asn Lys Leu Gly Tyr Gly Gly Ala Thr Trp Val Val Tyr Asp Glu Asn Asn Asn Ala Val Ser Tyr Ala Thr Asp Asp Gly Gln Asn Leu Ser Gly Lys Phe Lys Ala Asp Lys Pro Gly Arg Tyr Tyr Ile His Leu Tyr Met Phe Asn Gly Ser Tyr Met Pro Tyr Arg Ile Asn Ile Glu Gly Ser Val Gly Arg 21~-4~~~
(2) INFORMATION FOR SEQ ID NO: il:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
It surprisingly turned out that a CHC II which is the product of a recombinant production is much more stable to repeated freezing and thawing than CHC II isolated from natural sources and still has a considerable degree of collagenase activity after 12 freeze/thaw cycles.
The production of recombinant CHC II can be carried out according to methods familiar to a person skilled in the art.
For this a DNA sequence is firstly prepared which is able to produce a protein which has the activity of CHC II. By the term "DNA sequence" as used herein there is to be understood an isolated DNA molecule. The sequence coding for the protein can be determined with the aid of SEQ ID NO: 1 by determining the reading frame on the basis of a comparison with known peptide sequences of collagenase and the DNA sequence determined in this manner is converted into an amino acid sequence with the aid of the genetic code. The sequence coding for the active protein is ca. 2 to 3 times longer than SEQ ID NO: 1. The DNA sequence is cloned in a vector which can be transferred into a host cell and which can be replicated there. Such a vector also contains operator elements which are necessary for the expression of the DNA sequence in addition to the CHC II sequence.
This vector which contains the CHC II sequence and the operator elements is transferred into a vector which is able to express the DNA of CHC II. The host cell is cultured under conditions which are suitable for the amplification of the vector and CHC II is isolated. In this process suitable measures ensure that the protein can assume an active tertiary structure in which it exhibits CHC II properties.
The cloning and nucleotide sequence of CHC II is described in the post-published publication K. Yoshihara et al., J. Bacteriol. 176 (Nov. 1994) 6489 - 6494.
In this process it is not necessary that the expressed protein contains the exact CHC II amino acid sequence with the partial region SEQ ID NO: 2. Proteins are equally suitable which contain essentially the same sequence and have analogous properties.
The nucleic acid sequence of the protein can also be modified. Such modifications are for example:
- Modification of the nucleic acid in order to introduce various recognition sequences of restriction enzymes to facilitate the steps of ligation, cloning and mutagenesis.
- Modification of the nucleic acid to incorporate the preferred codons for the host cell.
- Extension of the nucleic acid by additional operator elements in order to optimize the expression in the host cell.
The invention in addition concerns a process for the production of a polypeptide which has the properties of a class II collagenase from Clostridium histolyticum and still has a considerable degree of collagenase activity after 12 freeze/thaw cycles by expression of an exogenous DNA sequence in prokaryotic or eukaryotic host cells and isolation of the desired polypeptide wherein the DNA sequence contains SEQ ID NO: 1 in the region characteristic for this collagenase or a sequence which codes for the same amino acid sequence within the scope of the degeneracy of the genetic code.
The expression of the protein is preferably carried out in microorganisms, in particular in prokaryotes and in this case in E. coli.
The expression vectors must contain a promoter which enables the expression of the protein in the host organism. Such promoters are known to a person skilled in the art and are for example the lac promoter (Chang et al., Nature 198 (1877) 1056), trp promoter (Goeddel et al., Nuc. Acids Res. 8 (1980) 4057), 7lpL promoter (Shimatake et al., Nature 292 (1981) 128) and T5 promoter (US patent No. 4,689,406). Synthetic promoters are also suitable such as for example the tac promoter (US Patent No. 4,551,433). Coupled promoter systems are also suitable such as the T7-RNA polymerase/promoter system (Studier et al., J. Mol. Biol. 189 (1986) 113).
Hybrid promoters composed of a bacteriophage promoter and the operator region of the microorganism (EP-A 0 267 851) are equally suitable. An effective ribosome binding site is necessary in addition to the promoter. In the case of E. coli this ribosome binding site is denoted Shine-Dalgarno (SD) sequence (Sambrook et al., "Expression of cloned genes in E. coli" in Molecular Cloning: A laboratory manual (1989) Cold Spring Harbor Laboratory Press, New York, USA).
In order to improve the expression it is possible to express the protein as a fusion protein. In this case a DNA sequence which codes for the N-terminal part of an endogenous bacterial protein or for another stable protein is usually fused to the 5' end of the sequence coding for CHC II. Examples of this are for example lacZ
(Phillips and Silhavy, Nature 344 (1990) 882 - 884), trpE (Yansura, Meth. Enzymol. 185 (1990) 161 - 166).
After expression of the vector, preferably a biologically functional plasmid or a viral vector, the fusion proteins are preferably cleaved with enzymes (e. g. factor Xa) (Nagai et al., Nature 309 (1984) 810).
Further examples of cleavage sites are the IgA protease cleavage site (WO 91/11520, EP-A 0 495 398) and the ubiquitin cleavage site (Miller et al., Bio/Technology 7 (1989) 698).
The proteins expressed in this manner in bacteria are isolated in the usual manner by lysing the bacteria and protein isolation.
In a further embodiment it is possible to secrete the proteins as active proteins from the microorganisms. For this a fusion product is preferably used which is composed of a signal sequence which is suitable for the secretion of proteins in the host organisms used and the nucleic acid which codes for the protein. In this 21.4601 process the protein is either secreted into the medium (in the case of gram-positive bacteria) or into the periplasmatic space (in the case of gram-negative bacteria). It is expedient to place a cleavage site between the signal sequence and the sequence coding for CHC II which enables cleavage of the protein either during processing or in an additional step. Such signal sequences are derived for example from ompA (Ghrayeb et al. EMBO J. 3 (1984) 2437) or phoA (Oka et al., Proc.
Natl. Acad. Sci. USA 82 (1985) 7212).
The vectors in addition also contain terminators.
Terminators are DNA sequences which signal the end of a transcription process. They are usually characterized by two structural characteristics: an inverse repetitive G/C-rich region which can intramolecularly form a double helix and a number of U(or T) residues. Examples are the main terminator in the DNA of the phages fd (Beck and Zink, Gene 16 (1981) 35-58) and rrnB (Brosius et al., J.
Mol. Biol. 148 (1981) 107 - 127).
In addition the expression vectors usually contain a selectable marker in order to select the transformed cells. Such selectable markers are for example the resistance genes for ampicillin, chloroamphenicol, erythromycin, kanamycin, neomycin and tetracyclin (Davies et al., Ann. Rev. Microbiol. 32 (1978) 469).
Selectable markers which are also suitable are the genes for substances that are essential for the biosynthesis of substances necessary for the cell such as e.g.
histidine, tryptophan and leucine.
Numerous suitable bacterial vectors are known. Vectors have for example been described for the following _ 7 _ bacteria: Bacillus subtilis (Palva et al., Proc. Natl.
Acad. Sci. USA 79 (1982) 5582), E. coli (Arran et al., Gene 40 (1985) 183; Studier et al., J. Mol. Biol. 189 (1986) 113), Streptococcus cremoris (Powell et al., Appl. Environ. Microbiol. 54 (1988) 655), Streptococcus lividans and Streptomyces lividans (US patent No.
4,747,056).
Further genetic engineering methods for the construction and expression of suitable vectors are described in J.
Sambrook et al., Molecular Cloning: A laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, N.Y.
Apart from in prokaryotic microorganisms, recombinant CHC II can also be expressed in eukaryotes (such as for example CHO cells, yeast or insect cells). The yeast system or insect cells are preferred as the eukaryotic expression system. Expression in yeast can be achieved by means of three types of yeast vectors: integrating YIp (yeast integrating plasmids) vectors, replicating YRp (yeast replicon plasmids) vectors and episomal YEp (yeast episomal plasmids) vectors. Further details of this are for example described in S.M. Kingsman et al., Tibtech 5 (1987) 53 - 57).
The invention in addition concerns a process for disintegrating cell tissue and releasing cells or groups of cells contained therein by incubating the cell tissue with a class II collagenase from Clostridium histolyticum which still has a considerable degree of collagenase activity after 12 freeze/thaw cycles and contains SEQ ID NO: 2 in a characteristic partial region or it is coded in this region by SEQ ID NO: 1 or a ~~ ~~ s~~
-8_ sequence which codes for the same amino acid sequence within the scope of the degeneracy of the genetic code and is the product of a prokaryotic or eukaryotic expression of an exogenous DNA, until the cells or groups of cells have been released to the desired extent and separating the cells or groups of cells from the cell tissue fractions. The separation of the cells or groups of cells from the cell tissue fractions is preferably carried out by centrifugation using a density gradient.
Cells or groups of cells are usually isolated from tissues (e. g. pancreas, liver, skin, endothelium, umbilical cord, bones, tumour tissue) by incubating organs, organ parts or tissues with enzymes which disintegrate the surrounding extracellular connective tissue matrix (islets: Sutton et al., Transplantation 42 (1986) 689 - 691: liver: Quibel et al., Anal. Biochem.
154 (1986) 26 - 28; bones: Hefley et al., J. Bone Mineral Res. 2 (1987) 505 - 516; umbilical cord:
Holzinger et al., Immunol. Lett. 35 (1993) 109 - 118.
Tumour cells isolated in this manner may be used advantageously for tumour vaccination and/or for adoptive immunotherapy. Tissue disintegration can also be accomplished by perfusing the entire organ (Ricordi et al., Diabetes 37 (1988) 413 - 420) with an enzyme solution. Important factors in this process, in addition to the composition of the enzyme mixture, are the duration, the pH value and the temperature of the digestion as well as mechanical influence e.g. by shaking and addition of metal balls. Since extracellular connective tissue matrix often has a high proportion of collagen, collagenases and in particular type II
collagenase play a special role in this case (Wolters, Hormone and Metabolic Research 26 (1994), p. 80).
21 ~.4-~ ~ ~
_ g _ The process according to the invention is preferably used to isolate islets or islet cells from pancreatic tissue.
In addition the addition of further enzymes such as proteinases (e.g. neutral protease, cf example 5 or other metalloproteases; serine proteases such as trypsin, chymotrypsin, plasmin etc.; cysteine proteases:
aspartate proteases), elastases, hyaluronidases, lipases or other collagenases may be advantageous for the quality of the digestion.
The invention is elucidated in more detail by the following examples and sequence protocols.
The sequence protocols denote:
SEQ ID NO: 1: DNA fragment of CHC II
SEQ ID NO: 2: derived protein fragment SEQ ID NO: 3 - 8: primer sequences SEQ ID NO: 9: cDNA of CHC II
SEQ ID NO: 10: protein sequence of CHC II
SEQ ID NO: 11/12: primer Example 1 Purification of class II collagenase (CHC II) 1 g collagenase P (from Clostridium histolyticum, Boehringer Mannheim GmbH, Order No. 1213857) was dissolved in 20 ml H20 (MilliQ quality) and particulate components were separated by centrifugation. After precipitation with 60 % ammonium sulfate, the precipitate was taken up in 9.5 ml H20 and dialysed overnight at 4'C against 10 mM Tris-HC1, pH 7.5.
The dialysate was applied at room temperature to a Zn-chelate Sepharose*column (column volume CV = 50 ml, 2 cm diameter) at a flow rate of 1.5 CV (75 ml/H). Afterwards the column was washed with 10 mM Tris-HCl, pH 7.5 until the base line in the W chromatogram had been reached again. CHC II bound to the affinity matrix was eluted with a linear gradient (10 CV = 500 ml) up to 50 mM
Tris, pH 7.5, 50 mM imidazole. The active fractions (determined by cleavage of synthetic peptide substrates) were dialysed against 10 mM Tris, pH 8.0, 1 mM CaCl2 and stored at -20'C.
A strong increase in the bands in the region of > 85 kD
occurs in the SDS gel, further proteins (MW between 40 and 85 kD) as well as some smaller bands are recognizable.
The fractions containing CHC II were further separated within 30 minutes by means of a MonoQ*column (FPLC) in mM Tris-HC1, pH 8.0 using a linear NaCl gradient from 0 to 0.3 M NaCl in 10 mM Tris-HC1, pH 8Ø The peaks containing CHC II activity are further purified by reversed phase HPLC (mobile solvent A: 0.12 % TFA/H20;
mobile solvent B: 0.12 % TFA (trifluoroacetic acid)/
100 % acetonitrile). A total of 4 peaks are collected, evaporated to dryness and subjected to LysC digestion after carboxymethylation. The peptides were also separated by reversed phase HPLC on a C4*column using.a gradient~Qf 5 - 45 % acetonitrile/0.12 % TFA. The chromatograms of all digestions were identical with the exception of minor differences. They significantly * Trade-mark A
differed from LysC peptide maps of collagenase I
(purified by gel filtration on Superdex HiLoad S 200 or by repurification of the eluate from the Zn-chelate Sepharose). The~peptides were collected, evaporated to dryness and sequenced.
Example 2 Cloning collagenase II from Clostridium histolyticum The matching (degenerate) DNA sequence is derived from the peptide sequences determined according to example 1 after purification of CHC II. Sequences which exhibit an advantageous (lower) denaturation are used to construct a labelled DNA probe to screen gene banks e.g. via PCR.
2 peptides Coll-2-60 and Coll-2-51, are particularly suitable from which the following primers can be derived:
peptide Coll-2-60 (SEQ ID N0:3) primer Coll-2-60F (SEQ ID N0:4) primer Coll-2-60R (SEQ ID N0:5) peptide Coll-2-51 (SEQ ID N0:6) primer Coll-2-51F (SEQ ID N0:7) . .
primer Coll-2-51R (SEQ ID ~~T0:8) * Trade-mark A
Fragments of ca. 500 and ca. 750 by length are obtained after PCR with DNA isolated according to conventional methods from Clostridium histolyticum using primers 60F
and 51R.
The 500 by fragment is labelled, for example also in a PCR reaction, with dig-dUTP (prepared according to WO 89/06698, EP-A 0 324 468), and serves to identify positive clones from a gene bank. The gene bank can be prepared according to generally known methods from Clostridium histolyticum DNA after digestion with restriction enzymes.
The 500 by fragment was sequenced and contains DNA from the sequence SEQ ID NO: 1.
The 750 by fragment can be fragmented into a ca. 600 by large and a 150 by large fragment using PstI. The 600 by fragment also contains SEQ ID NO: 1. pC2-600 is a vector which contains the 600 by fragment and was deposited under the number DSM 8996 on the 24.02.1994 at the DSM
("Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig).
The amino acid sequence has ca. 25 $ homology to a collagenase from Vibrio alginolyticus and is shown in SEQ ID N0:2. SEQ ID N0:2 is coded by the nucleotides 2 -487 from SEQ ID NO:1.
The missing parts of the coding region and of the untranslated regions of the gene can be amplified and sequenced by inverse PCR after digestion of the genomic DNA from Clostridium histolyticum using suitable restriction enzymes and primers derived from SEQ ID
NO:1. The coding region of the gene (ca. 3 kb) is contained in SEQ ID NO: 1.
The reading frame of SEQ ID NO: 9 can be unequivocally determined with the aid of peptide sequences found by protein sequencing and the sequence can be translated into the peptide sequence SEQ ID NO:10.
The ends of the coding region of nucleotide 688 to nucleotide 3792 were modified with the aid of two primers (SEQ ID NO: 11 and 12) and cloned into pUCBM 20 via XmaI and NotI. The resulting plasmid pC2-Exl/3-23 was deposited on the 09.12.1994 under the number DSM
9579 at the "Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig".
Example 3 Expression of class II aollagenase DNA fragments which contain the gene for CHC II or parts thereof are sequenced and their ends are modified in a suitable manner (e.g. via PCR) and they are used as a whole or in combination in an expression vector for E.
coli. The tac promoter is used as the promoter. However, it is also possible to use other - preferably well-regulatable - promoters.
A fragment of SEQ ID NO: 9 can be used for the expression which starts at position 688 or 757 and ends in the region of position 3792 - position 3900 which is brought under the control of one of the usual promoters.
As an alternative a fragment can be used which starts at position 847 and ends in the region of position 3792 -position 3900 which is firstly provided with the sequence for a heterologous signal peptide and is then placed behind the usual promoters. Those vectors with a high copy number are suitable as the vectors such as those of the pUC series. However, vectors with a low copy number derived from pACYC 177 or pACYC 184 can also be used when the selected host cell does not tolerate expression in high copy numbers. A range of 25 - 37°C is preferred as the growth temperature.
An E. coli strain which has been transformed with an expression plasmid is either cultured overnight at 30 to 37°C (e.g. in 5 ml culture) in minimum medium or LB
medium under antibiotic selection. The overnight culture is inoculated into a larger volume (e.g. 1 1) and allowed to grow further. If this volume is already used to obtain biomass, it can be induced at an OD550 of 0.2 to 2 with IPTG and the cells are allowed to grow further until there is no further increase in the OD and/or the enzyme activity that forms. At this time it is centrifuged and the biomass is used to purify collagenase.
If a significant proportion of the collagenase activity is present in the medium; the medium is also collected and collagenase is purified therefrom.
Example 4 Islet isolation from porcine pancreas The pancreas from a freshly slaughtered pig is prepared and cooled in ice-cold HBSS buffer (Gibco) until further processing. A Braunule is inserted into the ductus pancreaticus and fastened there, the water-tightness of the pancreas is tested using HBSS buffer. An enzyme solution in HBSS buffer + Ca2+ which contains purified recombinant type II collagenase alone or in combination with a purified native or recombinant neutral protease from Clostridium histolyticum is injected. The pancreas treated in this way is connected to the perfusion unit that also contains the above-mentioned enzyme solution (discontinuous perfusion). The digestion is carried out between 4°C - 37°C during a time period of 5 to 120 minutes. The pump is stopped after the time that is assumed to be optimal and the vessel containing the pancreas is carefully shaken for 3 to 20 minutes by hand. If necessary the prior addition of metal balls additionally facilitates the mechanical dissociation of the tissue and release of the islets from the surrounding exocrine tissue.
The digestion is stopped by addition of ice-cold HBSS/
% FCS (foetal calf serum) and the suspension is filtered through a sieve (mesh size 300 ~,m) in order to separate coarse particles. The islets present in the filtrate are centrifuged for 10 minutes at 100 g in 250 ml Nalgene round-bottom flasks. The supernatant is decanted and the pellet containing the islets is resuspended in 50 ml FCS.
The islets can be purified further by means of a density gradient made by hand. Firstly 7 ml islet suspension is added to 250 ml Nalgene round-bottom flasks. These are firstly overlayered with 93 ml of a Ficoll~ solution (cp = 1.077 g/cm3), and then with 50 ml medium (RPMI
1640). These gradients are centrifuged for 10 minutes at '" 214460 1 100 g in a swing-out rotor. Fractions of 10 ml are collected, the size, purity and yield of the islets stained with dithizone is determined microscopically in every fraction~or with the aid of image analysis.
Example 5 Isolation of neutral protease Collagenase P is dissolved in 5 mM HEPES buffer, pH 7.5, 1 mM CaCl2 (c = 50 mg/ml) and centrifuged. An aliquot (10 mg) is injected onto a MonoQ*column (Pharmacia FPLC) and eluted with a CaCl2 gradient (up to 150 mM in 20 min.). The fraction having the highest specific casinolytic activity (determined by means of resorufin-labelled casein, Boehringer Mannheim GmbH) contained a protein with a MW of 30 - 35 kDa (SDS gel). The peptides which formed after carboxymethylation and LysC digestion were separated by reversed phase HPLC on a Cg*column, concentrated to dryness and sequenced. As an alternative the protein fractions with a high caseinolytic activity were separated on a SDS gel, transferred onto PVDF*
membranes (Millipore) (Western blot; transfer buffer 50 mM Tris, 50 mM boric acid, pH 8.3) and the relevant bands were cut out after staining with amido black.
After LysC digestion of the immobilized protein, the peptides were detached with HCOOH as described above, separated and sequenced.
.
* Trade-mark A, SEQUENCE LISTING
NUMBER OF SEQUENCES: 12 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 487 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO: 1:
214-4~~I
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 162 amino acids (B) TYPE: amino~acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Asp Xaa Tyr Glu Asn Leu Thr Val Xaa Xaa Val Ala Asp Asp Tyr Leu Val Arg His Ala Tyr Lys Asn Pro Asn Glu Ile Tyr Ser Glu Ile Ser Glu Val Ala Lys Leu Lys Asp Ala Lys Ser Glu Val Lys Lys Ser Gln Tyr Phe Ser Thr Phe Thr Leu Arg Gly Ser Tyr Thr Gly Gly Val Ser Lys Gly Lys Leu Glu Asp Gln Lys Ala Met Asn Lys Phe Ile Asp Asp Ser Leu Lys Lys Leu Asp Thr Tyr Ser Trp Ser Gly Tyr Lys Thr Leu Thr Ala Tyr Phe Thr Asn Tyr Lys Val Asp Ser Ser Asn Lys''?Val Thr Tyr Asp Val Val Phe His Gly Tyr Leu Pro Xaa Xaa Gly Asp Ser Arg ~1~.4601 Asn Ser Leu Pro Tyr Gly Lys Thr Asn Gly Thr Tyr Lys Gly Thr Glu Lys Glu Lys Ile Lys Phe Ser Ser Glu Gly Ser Xaa Asp Pro Asp Gly Lys Ile (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Tyr Gln Asp His Met Gln Glu (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
o - 21~460I
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Tyr Glx Trp Asp Phe Gly Asp (2) INFORMATION FOR SEQ ID N0: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear {ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3979 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: double (D) TOPOLOGY: linear lii} MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: sig-peptide (B) LOCATION:688..846 (ix) FEATURE:
(A) NAME/KEY: sig-peptide (B) LOCATION:757..846 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:847..3792 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CCTAATATTC TCTTATATAC TTAATTAAAT ATTAATAAAA AATTAATGA,A CAGGTATATC 590 ,...
4~
~i4~so1 - 25 - ~ ~ 1 (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 981 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Val Gln Asn Glu Ser Lys Arg Tyr Thr Val Ser Tyr Leu Lys Thr Leu Asn Tyr Tyr Asp Leu Val Asp Leu Leu Ala Lys Thr Glu Ile Glu Asn Leu Pro Asp Leu Phe Gln Tyr Ser Ser Asp Ala Lys Glu Phe Tyr Gly Asn Lys Thr Arg Met Asn Phe Ile Met Asp Glu Ile Gly Arg Arg Ala Ser Gln Tyr Thr Glu Ile Asp His Lys Gly Ile Pro Thr Leu Val Glu Val Val Arg Ala Gly Phe Tyr Leu Gly Phe His Asn Lys Glu Leu Asn Glu Ile Asn Lys Arg Ser Phe Lys Glu Arg Val Ile Pro 5er Ile Leu Ala Ile Gln Lys Asn Pro Asn Phe Lys Leu Gly Thr Glu Val Gln Asp Lys Ile Val Ser Ala Thr Gly Leu Leu Ala Gly Asn Glu Thr Ser Pro Ala Glu Val Val Asn Asn Phe Thr Pro Ile Leu Gln Asp Cys Ile Lys Asn Met Asp Arg Tyr Ala Leu Asp Asp Leu Lys Ser Lys Ala Leu Phe Asn Val Leu Ala Ala Pro Thr Tyr Asp Val Thr Glu Tyr Leu Arg Ala Thr Lys Glu Lys Pro Glu Asn Thr Pro Trp Tyr Gly Lys Ile Asp Gly Phe Ile Asn Glu Val Lys Lys Leu Ala Leu Tyr Gly Lys Ile Asn Asp Lys Asn Ser Trp Ile Ile Asp Asn Gly Ile Tyr His Ile Ala Pro Leu Gly Lys Leu His Ser Asn Asn Lys Ile Gly Ile Glu Thr Leu Thr Glu Val Met Lys Val Tyr Pro Tyr Leu Ser Met Gln His Leu Gln Ser Ala Asp Gln Ile Lys Arg His Tyr Asp Ser Lys Asp Ala Glu Gly Asn Lys 2?5 280 285 Ile Pro Leu Asp Lys Phe Lys Lys Glu Gly Lys Glu Lys Tyr Cys Pro Lys Thr Tyr Thr Phe Asp Asp Gly Lys Val Ile Ile Lys Ala Gly Ala Arg Val Glu Glu Glu Lys Val Lys Arg Leu Tyr Trp Ala Ser Lys Glu Val Asn Ser Gln Phe Phe Arg Val Tyr Gly Ile Asp Asn Pro Leu Glu Glu Gly Asn Pro Asp Asp Ile Leu Thr Met Xaa Ile Tyr Asn Ser Pro Glu Glu Tyr Lys Leu Asn Ser Val Leu Xaa Gly Tyr Asp Thr Asn Asn Gly Gly Met Tyr Ile Glu Pro Glu Gly Thr Phe Phe Thr Tyr Glu Arg Glu Ala Gln Glu Ser Thr Tyr Thr Leu Xaa Glu Leu Phe Arg His Glu Tyr Thr His Tyr Leu Gln Gly Arg Tyr Ala Val Pro Gly Gln Trp Gly Arg Pro Lys Leu Tyr Asp Asn Asp Arg Leu Thr Trp Tyr Glu Glu Gly Gly Ala Glu Leu Phe Ala Gly Ser Thr Arg Thr Ser Gly Ile Leu Pro Arg Lys Ser Ile Val Ser Asn Ile His Asn Thr Thr Arg Asn Asn Arg Tyr Lys Leu Ser Asp Thr Val His Ser Lys Tyr Gly Ala Ser Phe Glu Phe Tyr Asn Tyr Ala Cys Met Phe Met Asp Tyr Met Tyr Asn Lys Asp Met Gly Ile Leu Asn Lys Leu Asn Asp Leu Ala Lys Asn Asn Asp Val Asp Gly Tyr Asp Asn Tyr Ile Arg Asp Leu Ser Ser Asn His Ala Leu Asn Asp Lys Tyr Gln Asp His Met Gln Glu Arg Ile Asp Asn Tyr Glu Asn Leu Thr Val Pro Phe Val Ala Asp Asp Tyr Leu Val Arg His Ala Tyr Lys Asn Pro Asn Glu Ile Tyr Ser Glu Ile Ser Glu Val Ala Lys Leu Lys Asp Ala Lys Ser Glu Val Lys Lys Ser Gln Tyr Phe Ser Thr Phe Thr Leu Arg Gly Ser Tyr Thr Gly Gly Val Ser Lys Gly Lys Leu Glu Asp Gln Lys Ala Met Asn Lys Phe Ile Asp Asp Ser Leu Lys Lys 2~446~1 Leu Asp Thr Tyr Ser Trp Ser Gly Tyr Lys Thr Leu Thr Ala Tyr Phe Thr Asn Tyr Lys Val Asp Ser Ser Asn Xaa Val Thr Tyr Asp Val Val Phe His Gly Tyr Leu Pro Asn Glu Gly Asp Ser Lys Asn Ser Leu Pro Tyr Gly Lys Thr Asn Gly Thr Tyr Lys Gly Thr Glu Lys Glu Lys Ile Lys Phe Ser Ser Glu Gly Ser Phe Asp Pro Asp Gly Lys Ile Val Ser Tyr Glu Trp Asp Phe Gly Asp Gly Asn Lys Ser Asn Glu Glu Asn Pro Glu His Ser Tyr Asp Lys Val Gly Thr Tyr Thr Val Lys Leu Lys Val Thr Asp Asp Lys Gly Glu Ser Ser Val Ser Thr Thr Thr Ala Glu Ile Arg Asp Leu Ser Glu Asn Lys Leu Pro Val Ile Tyr Met His Val Pro Thr Ser Gly Ala Leu Asn Gln Lys Val Val Phe Tyr Gly Lys Gly Thr Tyr Asp Pro Asp Gly Ser Ile Ala Gly Tyr Gln Trp Asp Phe Gly Asp ..r~
2~.4~~~I
Gly Ser Asp Phe Ser Ser Glu Gln Asn Pro Ser His Val Tyr Thr Lys Lys Gly Glu Tyr Thr Val Thr Leu Arg Val Met Asp Ser Ser Gly Gln Met Ser Glu Lys Thr Met Lys Ile Lys Ile Thr Asp Pro Val Tyr Pro Ile Gly Thr Glu Lys Glu Pro Asn Asn Ser Lys Glu Thr Ala Ser Gly Pro Ile Val Pro Gly Ile Pro Val Ser Gly Thr Ile Glu Asn Thr Ser Asp Gln Asp Tyr Phe Tyr Xaa Asp Val Ile Thr Pro Gly Glu Val Lys Ile Asp Ile Asn Lys Leu Gly Tyr Gly Gly Ala Thr Trp Val Val Tyr Asp Glu Asn Asn Asn Ala Val Ser Tyr Ala Thr Asp Asp Gly Gln Asn Leu Ser Gly Lys Phe Lys Ala Asp Lys Pro Gly Arg Tyr Tyr Ile His Leu Tyr Met Phe Asn Gly Ser Tyr Met Pro Tyr Arg Ile Asn Ile Glu Gly Ser Val Gly Arg 21~-4~~~
(2) INFORMATION FOR SEQ ID NO: il:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Claims (13)
1. A process for disintegrating cell tissue and releasing cells or groups of cells contained therein, said process comprising the steps of:
- incubating the cell tissue with a recombinant class II collagenase from Clostridium histolyticum until the cells or groups of cells are released to the desired degree, said recombinant class II collagenase still retaining a collagenase activity after 12 freeze/thaw cycles and being produced by a prokaryotic or eukaryotic expression of an exogenous DNA, said exogenous DNA being a nucleic acid sequence of SEQ ID NO:1 or a nucleic acid sequence which codes for an amino acid sequence of SEQ ID NO:2; and - separating the cells or groups of cells from the cell tissue fractions by means of a density gradient centrifugation.
- incubating the cell tissue with a recombinant class II collagenase from Clostridium histolyticum until the cells or groups of cells are released to the desired degree, said recombinant class II collagenase still retaining a collagenase activity after 12 freeze/thaw cycles and being produced by a prokaryotic or eukaryotic expression of an exogenous DNA, said exogenous DNA being a nucleic acid sequence of SEQ ID NO:1 or a nucleic acid sequence which codes for an amino acid sequence of SEQ ID NO:2; and - separating the cells or groups of cells from the cell tissue fractions by means of a density gradient centrifugation.
2. A process for disintegrating cell tissue and releasing cells or groups of cells contained therein, said process comprising the steps of:
- incubating the cell tissue with a recombinant class II collagenase from Clostridium histolyticum until the cells or groups of cells are released to the desired degree, said recombinant class II collagenase still retaining a collagenase activity after 12 freeze/thaw cycles and being produced by a prokaryotic or eukaryotic expression of an exogenous DNA, said exogenous DNA being a nucleic acid sequence of SEQ ID NO:9 or a nucleic acid sequence which codes for an amino acid sequence of SEQ ID NO:10; and - separating the cells or groups of cells from the cell tissue fractions by means of a density gradient centrifugation.
- incubating the cell tissue with a recombinant class II collagenase from Clostridium histolyticum until the cells or groups of cells are released to the desired degree, said recombinant class II collagenase still retaining a collagenase activity after 12 freeze/thaw cycles and being produced by a prokaryotic or eukaryotic expression of an exogenous DNA, said exogenous DNA being a nucleic acid sequence of SEQ ID NO:9 or a nucleic acid sequence which codes for an amino acid sequence of SEQ ID NO:10; and - separating the cells or groups of cells from the cell tissue fractions by means of a density gradient centrifugation.
3. Process as claimed in claim 1 or 2, wherein the cell tissue is pancreatic tissue, the isolated cells are islet cells and the groups of cells are islets.
4. Process as claimed in claim 1 or 2, wherein the cell tissue is selected from the group consisting of liver, skin, umbilical cord, endothelial, bone, fat, heart, muscle, kidney, uterus, ovarian, placental or lung tissue.
5. Process for the production of a polypeptide, which has the properties of a class II collagenase from Clostridium histolyticum and which still retains collagenase activity after 12 freeze-thaw cycles, by expression of an exogenous DNA sequence in prokaryotic or eukaryotic host cells and isolation of the desired polypeptide, wherein the exogenous DNA sequence comprises a nucleic acid sequence of SEQ ID NO: 1 or a sequence substantially similar to SEQ ID NO:1 and which codes for amino acid sequence of SEQ ID NO:2.
6. Process as claimed in claim 5, wherein the DNA sequence comprises sequence SEQ ID NO:1.
7. Process as claimed in claim 5 or 6, wherein the host cells are E. coli cells.
8. Process as claimed in claim 5 or 6, wherein the host cells are yeast cells or insect cells.
9. A DNA sequence which comprises SEQ ID NO:1 and which codes for a polypeptide with collagenase activity.
10. DNA sequence SEQ ID NO:1.
11. DNA sequence SEQ ID NO:9.
12. A biologically functional plasmid or a viral DNA vector which comprises a DNA sequence as claimed in claims 9 to 11.
13. Prokaryotic or eukaryotic host cell which is stably transformed or transfected with a DNA vector as claimed in claim 12.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4408940A DE4408940A1 (en) | 1994-03-16 | 1994-03-16 | Recombinant type II collagenase from Clostridium histolyticum and its use for the isolation of cells and cell assemblies |
| DEP4408940.6 | 1994-03-16 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2144601A1 CA2144601A1 (en) | 1995-09-17 |
| CA2144601C true CA2144601C (en) | 2002-05-14 |
Family
ID=6512956
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002144601A Expired - Fee Related CA2144601C (en) | 1994-03-16 | 1995-03-14 | Recombinant type ii collagenase from clostridium histolyticum and its use for isolating cells and groups of cells |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0677586A1 (en) |
| JP (1) | JP2634782B2 (en) |
| CA (1) | CA2144601C (en) |
| DE (1) | DE4408940A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2272244A1 (en) * | 1996-11-19 | 1998-05-28 | Roche Diagnostics Gmbh | Recombinant collagenase type i from clostridium histolyticum and its use for isolating cells and groups of cells |
| AU9253898A (en) * | 1997-09-26 | 1999-04-23 | Novo Nordisk A/S | Use of proteases in passaging of adherent animal or human cell cultures |
| DE10331171A1 (en) | 2003-07-09 | 2005-01-27 | Nordmark Arzneimittel Gmbh & Co. Kg | Neutral protease (NP) and product using neutral tissue release tissue protease and method for its preparation |
| US8323642B2 (en) * | 2006-12-13 | 2012-12-04 | Depuy Mitek, Inc. | Tissue fusion method using collagenase for repair of soft tissue |
| US10071143B1 (en) | 2007-05-03 | 2018-09-11 | The Research Foundation For The State University Of New York | Methods for non-surgical treatment of carpal tunnel syndrome |
| WO2014066622A2 (en) | 2012-10-24 | 2014-05-01 | The Research Foundation For The State University Of New York | Use of collagenase to treat glaucoma |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5177017A (en) * | 1990-03-22 | 1993-01-05 | Trigen, Inc. | Molecular cloning of the genes responsible for collagenase production from Clostridium histolyticum |
| WO1994000580A1 (en) * | 1992-06-22 | 1994-01-06 | Trigen, Inc. | Molecular cloning of the genes reponsible for collagenase production from clostridium histolyticum |
-
1994
- 1994-03-16 DE DE4408940A patent/DE4408940A1/en not_active Withdrawn
-
1995
- 1995-03-03 EP EP95103015A patent/EP0677586A1/en not_active Withdrawn
- 1995-03-14 CA CA002144601A patent/CA2144601C/en not_active Expired - Fee Related
- 1995-03-15 JP JP7055676A patent/JP2634782B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH07313159A (en) | 1995-12-05 |
| JP2634782B2 (en) | 1997-07-30 |
| CA2144601A1 (en) | 1995-09-17 |
| DE4408940A1 (en) | 1995-10-05 |
| EP0677586A1 (en) | 1995-10-18 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |