title: Cloning, expression, purification, and characterization of the catalytic domain of sika deer MMP-13.
abstract: Matrix metalloproteinase 13 is one of three mammalian collagenases that are capable of initiating the degradation of interstitial collagens during wound healing. Herein, we report for the first time the molecular cloning of the catalytic domain (CD) of sika deer MMP-13, followed by protein expression in Escherichia coli and purification by affinity chromatography. The final yield was approximately 90.4 mg per liter of growth culture with a purity of 91.6%. The mass recovery during the purification and renaturation were 70.2% and 81.5%, respectively. Using gelatin zymography and a degradation assay, we found that the refolded sika deer MMP-13 (CD) could digest gelatin. The optimal pH and temperature for the enzyme bioactivity was 8.0 and 37 °C, respectively. The Km value for the enzyme-catalyzed digestion of gelatin was 136+/-8 μg/mL, and the Vmax was 4.12 × 10(3) U/μg. sdMMP13 (CD) was able to completely degrade collagen II and gelatin, and partially degrade fibronectin. The sdMMP-13 (CD) activity was significantly inhibited by several chemicals including 1, 10-phenanthroline, EDTA, Fe(2+), Cu(2+), and Mn(2+).
authors.affiliation: Traditional Chinese Medicine and Biotechnology Research and Development Center, Changchun University of Traditional Chinese Medicine, Changchun, Jilin, People's Republic of China.
authors.forename: Xueliang
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Traditional Chinese Medicine and Biotechnology Research and Development Center, Changchun University of Traditional Chinese Medicine, Changchun, Jilin, People's Republic of China.
authors.forename: Jiawen
authors.initials: J
authors.lastname: Wang
authors.affiliation: Traditional Chinese Medicine and Biotechnology Research and Development Center, Changchun University of Traditional Chinese Medicine, Changchun, Jilin, People's Republic of China.
authors.forename: Meichen
authors.initials: M
authors.lastname: Liu
authors.affiliation: Traditional Chinese Medicine and Biotechnology Research and Development Center, Changchun University of Traditional Chinese Medicine, Changchun, Jilin, People's Republic of China.
authors.forename: Siming
authors.initials: S
authors.lastname: Wang
authors.affiliation: Traditional Chinese Medicine and Biotechnology Research and Development Center, Changchun University of Traditional Chinese Medicine, Changchun, Jilin, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Traditional Chinese Medicine and Biotechnology Research and Development Center, Changchun University of Traditional Chinese Medicine, Changchun, Jilin, People's Republic of China. Electronic address: cnzhaoyu1972@126.com.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D003670:Deer / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D053509:Matrix Metalloproteinase 13 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D053509:Matrix Metalloproteinase 13
keywords: Catalytic domain; Characterization; MMP-13; Purification; Sika deer