Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-09-09
2002-11-05
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S252300, C435S320100, C435S267000
Reexamination Certificate
active
06475764
ABSTRACT:
The present invention claims priority to the following applications: 1) EP 96 118 490.0, filed Nov. 19, 1996; 2) EP 97 101 085.5, filed Jan. 24, 1997; and 3) PCT/EP97/06002, filed Oct. 30, 1997.
The invention concerns a recombinant collagenase type I (CHC I) 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 collagenase types are known from Clostridium histolyticum (M. F. French et al., J. Protein Chemistry 11 (1992) 83-97). The isolation and purification of collagenases from Clostridium histolyticum is described for example in E. L. Angleton and H. E. van Wart, Biochemistry 27 (1988) 7413-7418 and 7406-7412.
Collagenases I were isolated and described by M. D. Bond and H. E. Van Wart, Biochemistry 23 (1984) 3077-3085 and 3085-3091 as well as by H. E. Van Wart, Biochemistry 24 (1985) 6520-6526. Accordingly the collagenases a (MW 68 kD), &bgr; (MW 115 kD), &ggr; (MW 79 kD) and &eegr; (MW 130 kD) are known as collagenases I. Collagenases I and collagenases II differ in their relative activity towards collagen and towards synthetic peptides. Collagenases I have higher activities towards collagen and gelatin and lower activities towards the short-chained peptides than collagenase II (Bond and Wart, Biochemistry 23 (1984) 3085-3091).
Further collagenases are described in the U.S. Pat. Nos. 5,418,157 and 5,177,011 with a molecular weight of 68 kD. WO 91/14447 also describes a collagenase with a molecular weight of 68 kD. A recombinant collagenase with a molecular weight of ca. 110 kD is described in WO 94/00580 and a sequence is stated for it. Nothing is stated in WO 94/00580 about its specificity and in particular whether it is a collagenase I or II. In addition to a collagenase with a molecular weight of 110 kD, a 125 kD collagenase is additionally mentioned which it is claimed can also be produced recombinantly. However, WO 94/00580 does not give more details about this collagenase.
The object of the present invention is to provide a highly active stable collagenase class I from Clostridium histolyticum (CHC I).
The object is achieved by a process for the production of a polypeptide which has the properties of a CHC I from Clostridium histolyticum, has a given amino acid composition and is obtainable by expression of an exogenous nucleic acid in prokaryotic or eukaryotic host cells and isolation of the desired polypeptide wherein the nucleic acid codes for a polypeptide having sequence ID NO:2 or a polypeptide which is extended N-terminally by one or several amino acids having the sequence ID NO:3.
It has surprisingly turned out that a CHC I according to the invention has a high collagenase class I activity and is very stable. A CHC I having the amino acid sequence SEQ ID NO:2 and a CHC I which is N-terminally extended by one or several amino acids having SEQ ID NO:3 are particularly preferred.
The CHC I according to the invention is a highly pure enzyme which can be produced in large amounts. The CHC I according to the invention is not contaminated by other clostridial enzymes and is free of toxins.
The CHC I according to the invention is especially suitable for isolating cells from tissues of mammals, preferably from human tissue, for an application in cell therapy (transplantation, immunotherapy) and for an application in gene therapy in tissues: e.g. pancreas, liver, bone, cartilage, skin, brain and nerve tissue, fat, muscle, heart, endothelium, kidney, solid tumours and for the purification of ulcers.
In addition this highly pure enzyme (preferably in a mixture with other-highly pure enzymes (such as collagenase II and neutral protease)) is particularly suitable for the isolation of cells whose surface molecules/markers (antigens) should not be changed. Preferred applications of this are for example the dissociation of solid tumours of all types in vitro (e.g. colon, breast etc.) for adoptive immunotherapy and for general diagnosis such as e.g. to detect rare cells. from tissues and solid tumours by means of specific surface markers/molecules.
The production of the recombinant CHC I can be carried out according to methods familiar to a person skilled in the art.
For this a DNA molecule is firstly produced which is capable of producing a protein which has the activity of CHC I. The DNA sequence is cloned into a vector which can be transferred into a host cell and can be replicated there. Such a vector contains promoter/operator elements which are necessary to express the DNA sequences in addition to the CHC I sequence. This vector which contains the CHC I sequence and the promoter/operator elements is transferred into a host cell which is able to express the DNA of CHC I. The host cell is cultured under conditions which are suitable for the amplification of the vector and CHC I is isolated. In this process suitable measures ensure that the protein can adopt an active tertiary structure in which it exhibits CHC I properties.
The nucleic acid sequence and protein sequence can be modified to the usual extent. 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 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.
Substitution or deletion of amino acids while retaining the basicity and the spatial structure of CHC I. It is advantageous to preserve 85% or more and preferably 90% or more of the original amino acid sequence.
A further subject matter of the invention is a polypeptide with the properties of a collagenase class I from Clostridium histolyticum with the amino acid sequence according to SEQ ID NO:2 or a polypeptide extended N-terminally by one or several amino acids of SEQ ID NO:3. A further subject matter of the invention is a nucleic acid coding for such a protein.
The protein is preferably produced recombinantly in microorganisms, in particular in prokaryotes and in this case in
E. coli.
Suitable expression vectors must contain a promoter which allows 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 (1977) 1056), trp promoter (Goeddel et al., Nuc. Acids Res. 8 (1980) 4057), &lgr;
PL
promoter (Shimatake et al., Nature 292 (1981) 128) and T5 promoter (U.S. Pat. No. 4,689,406). Synthetic promoters are also suitable such as for example the tac promoter (U.S. Pat. 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 the 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 DNA coding for the CHC I. 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 v
Ambrosius Dorothee
Burtscher Helmut
Hesse Friederike
Moore William W.
Roche Diagnostics Corporation
Roche Diagnostics GmbH
Saidha Tekchand
Waite Kenneth J.
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