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 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.
Gehring
M R
MR
Agouron Pharmaceuticals, Inc., San Diego, California 92121, USA.
Condon
B
B
Margosiak
S A
SA
Kan
C C
CC
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
D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D010649:Phenylalanine; D017364:Collagenases; D020781:Matrix Metalloproteinase 1; D014633:Valine