title: Characterization of the Phe-81 and Val-82 human fibroblast collagenase catalytic domain purified from Escherichia coli.
abstract: Soluble recombinant human fibroblast collagenase catalytic domain was highly expressed and purified from Escherichia coli. The expression construct utilized the T7 gene 10 promoter for transcription of a two-cistron 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-Val-Gly-His (mutant) which served as cleavage sites for in vitro activation. The last four residues of the linker were included based on the crystal structure of human prostromelysin-1 catalytic domain. Soluble fusion 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 cleavage, purified to homogeneity, and separately possess Phe-81, Val-82, or Leu-83 as the amino-terminal residue. Very similar kcat/Km values were determined for the Phe-81 and Val-82 forms using continuous fluorogenic and chromogenic peptide cleavage assays.
authors.affiliation: Agouron Pharmaceuticals, Inc., San Diego, California 92121, USA.
authors.forename: M R
authors.initials: MR
authors.lastname: Gehring
authors.forename: B
authors.initials: B
authors.lastname: Condon
authors.forename: S A
authors.initials: SA
authors.lastname: Margosiak
authors.forename: C C
authors.initials: CC
authors.lastname: Kan
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017364:Collagenases / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D020781:Matrix Metalloproteinase 1; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010649:Phenylalanine / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D013379:Substrate Specificity; D014452:Ubiquitins / Q000737:chemistry; D014633:Valine / Q000737:chemistry
chemical_list: D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D010649:Phenylalanine; D017364:Collagenases; D020781:Matrix Metalloproteinase 1; D014633:Valine