Clostridium histolyticum enzymes and methods for the use thereof
View Patent ↗The invention relates to recombinant nucleic acid and polypeptides encoding collagenase I and collagenase II, methods for the preparation thereof and methods for the use thereof. The invention also encompasses methods related to releasing a composition comprising collagenase prior to therapeutic administration.
1. A recombinant nucleic acid molecule comprising;
a) a polynucleotide having the nucleic acid sequence of SEQ ID NO: 1 or the complement of SEQ ID NO: 1, and
b) a heterologous regulatory sequence operably linked to the polynucleotide.
2. The recombinant nucleic acid molecule of claim 1 , wherein the heterologous regulatory sequence is a heterologous promoter operably linked to the nucleic acid sequence of SEQ ID NO: 1 or to the complement of SEQ ID NO: 1.
3. A vector comprising the nucleic acid molecule of claim 1 .
4. The vector of claim 3 , wherein the vector is a plasmid.
5. A recombinant host cell comprising the vector of claim 3 .
6. The recombinant host cell of claim 5 , wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, an insect cell, a plant cell and a mammalian cell.
7. The recombinant host cell of claim 6 , wherein the host cell is selected from the group consisting of E. coli, Streptomyces, Pseudomonas, Serratia marcescens, Salmonella typhimurium , a yeast cell, plant cells, thymocytes, Chinese hamster ovary cell (CHO), COS cell, and Lactococcus lacti.
8. A method of producing collagenase I comprising culturing the cell of claim 5 under conditions suitable for expression of the nucleic acid and recovering the collagenase I.
9. The recombinant nucleic acid molecule of claim 1 , wherein the recombinant nucleic acid molecule is fused to a marker sequence.
10. The recombinant nucleic acid molecule of claim 9 , wherein the marker sequence encodes a polypeptide selected from the group consisting of: a glutathione-S-transferase (GST) fusion protein, a hemaglutin A (HA) polypeptide marker from influenza, and hexa-histidine peptide.
11. The vector of claim 3 , wherein the vector is introduced into a host cell using conventional transformation or transfection techniques.
12. The recombinant nucleic acid molecule of claim 1 having a biological activity of a naturally-occurring collagenase.