title: Expression and characterization of common carp (Cyprinus carpio) matrix metalloproteinase-2 and its activity against type I collagen.
abstract: 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 2792bp which contains an open reading frame of 1974bp, 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 38kDa 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 Ca(2+) 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.
authors.affiliation: College of Biological Engineering, Engineering Research Center for Aquatic Products Processing of Fujian Province, Jimei University, Xiamen 361021, China.
authors.forename: Ci
authors.initials: C
authors.lastname: Wang
authors.affiliation: College of Biological Engineering, Engineering Research Center for Aquatic Products Processing of Fujian Province, Jimei University, Xiamen 361021, China.
authors.forename: Chun-Lan
authors.initials: CL
authors.lastname: Zhan
authors.affiliation: College of Biological Engineering, Engineering Research Center for Aquatic Products Processing of Fujian Province, Jimei University, Xiamen 361021, China.
authors.forename: Qiu-Feng
authors.initials: QF
authors.lastname: Cai
authors.affiliation: College of Biological Engineering, Engineering Research Center for Aquatic Products Processing of Fujian Province, Jimei University, Xiamen 361021, China.
authors.forename: Cui-Hong
authors.initials: CH
authors.lastname: Du
authors.affiliation: College of Biological Engineering, Engineering Research Center for Aquatic Products Processing of Fujian Province, Jimei University, Xiamen 361021, China.
authors.forename: Guang-Ming
authors.initials: GM
authors.lastname: Liu
authors.affiliation: College of Biological Engineering, Engineering Research Center for Aquatic Products Processing of Fujian Province, Jimei University, Xiamen 361021, China.
authors.forename: Wen-Jin
authors.initials: WJ
authors.lastname: Su
authors.affiliation: College of Biological Engineering, Engineering Research Center for Aquatic Products Processing of Fujian Province, Jimei University, Xiamen 361021, China. Electronic address: mjcao@jmu.edu.cn.
authors.forename: Min-Jie
authors.initials: MJ
authors.lastname: Cao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002347:Carps / Q000235:genetics; D020134:Catalytic Domain; D003001:Cloning, Molecular; D024042:Collagen Type I / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology*; D016415:Sequence Alignment
chemical_list: D024042:Collagen Type I; D004791:Enzyme Inhibitors; D020778:Matrix Metalloproteinase 2; D002118:Calcium
keywords: Catalytic domain; Cloning; Collagen; Common carp; Gelatin zymography; Matrix metalloproteinase-2