Expression and characterization of common carp (Cyprinus carpio) matrix metalloproteinase-2 and its activity against type I collagen

• MMP-2 full-length gene was successfully cloned from common carp (Cyprinus carpio).
• The recombinant catalytic domain of MMP-2 was expressed in Escherichia coli and purified.
• The enzyme characteristics of recombinant MMP-2 were similar to native MMP-2 and degraded collagen effectively.

Matrix metalloproteinases (MMPs) play essential roles in the metabolism of animal collagen while few reports are available for MMPs in aquatic animals. In this study, we report the complete sequence of matrix metalloproteinase-2 (MMP-2) gene from common carp (Cyprinus carpio) skeletal muscle. The full-length cDNA of MMP-2 was 2792 bp which contains an open reading frame of 1974 bp, corresponding to a protein of 657 amino acid residues. Based on the structural feature of MMP-2, the gene of the catalytic domain containing 351 amino acid residues was cloned and expressed in Escherichia coli. SDS-PAGE showed that the truncated recombinant MMP-2 (trMMP-2) with molecular mass of approximately 38 kDa was in the form of inclusion body. The trMMP-2 was further purified by immobilized metal ion affinity chromatography. After renaturation, similar to native MMP-2, the trMMP-2 exhibited high hydrolyzing activity toward gelatin as appeared on gelatin zymography and optimal activity was at pH 8.0 and 40 °C. The activity of the trMMP-2 was completely suppressed by metalloproteinase inhibitors, including EDTA, EGTA and 1,10-phenanthroline while other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca2+ was necessary for the gelatinolytic activity, suggesting it is a calcium-dependent metalloproteinase. Moreover, the trMMP-2 effectively hydrolyzed native type I collagen at 37 °C and even at 4 °C, implying its potential application value as a collagenase for preparation of biologically active oligopeptides.