CHARACTERIZATION OF THE PHE-81 AND VAL-82 HUMAN FIBROBLAST COLLAGENASE CATALYTIC DOMAIN PURIFIED FROM ESCHERICHIA-COLI

Soluble recombinant human fibroblast collagenase catalytic domain was highly expressed and purified from Escherichia coil, The expression co nstruct utilized the T7 gene 10 promoter for transcription of a two-ci stron messenger RNA which encoded the ubiquitin-collagenase catalytic domain fusion protein as the second cistron. The ubiquitin domain was attached to the collagenase catalytic domain with the linker sequences Gly-Gly-Thr-Gly-Asp-Val-Ala-Gln (wild type) or Gly-Gly-Thr-Gly-Asp-Va l-Gly-His (mutant) which served as cleavage sites for in vitro activat ion. The last four residues of the linker were included based on the c rystal structure of human prostromelysin-l catalytic domain. Soluble f usion proteins purified from E. coli retained the proteolytic activity of the collagenase catalytic domain, The collagenase catalytic domain was released by either autoproteolytic or stromelysin-1-catalyzed cle avage, purified to homogeneity, and separately possess Phe-81, Val-82, or Leu-83 as the amino-terminal residue. Very similar K-cat/K-m value s were determined for the Phe-81 and Val-82 forms using continuous flu orogenic and chromogenic peptide cleavage assays.