Molecular cloning and sequence analysis of the gene encoding the collagenase from Cytophaga sp. L43-1 strain

Cytophaga sp. strain L43-1 secretes a collagenase [Y. Sasagawa et al., Biosci. Biotech. Biochem., 57, 1894-1898 (1993)]. A cog gene encoding the collagenase from this strain was cloned, and the nucleotides were sequenced. The structural gene of cog consisted of 3846 base pairs, which encoded a polypeptide consisting of 1282 amino acid residues with a predicted molecular mass of 130 kDa which was synthesized as a pre-matured enzyme. The deduced N-terminal 14 amino acids sequence, molecular mass of 120 kDa, and pI of 4.96 of the predicted matured enzyme were consistent with those previously found for the collagenase purified from the strain. The cog gene was expressed in Escherichia coli using the lac promoter and ribosomal binding sequence in plasmid vector pUC119 or pKK223-3, but not its own putative promoter and Shine-Dalgarno sequence. The consensus amino acid sequence (His-Glu-Xaa-Xaa-His) of an active site of the metal proteases including the collagenase from Vibrio arginolyticus and a series of human MMPs was found in the Cog protein of the strain.