Soluble Expression of Recombinant Bladder Cancer Biomarker Matrix Metalloproteinase-1 and Analysis of Urinary Enzyme by Gelatin Zymography

• Published in: Fēnxī huàxué Vol. 43; no. 4; pp. 582 - 587
• Main Authors: Jin, Xue-Fei, Kong, Xiang-Bo, Zhang, Dan, Liu, Ting-Ting, Wang, Kai-Chen
• Format: Journal Article
• Language: Chinese; English
• Published: 01.01.2015
• Subjects: Biomarkers; Cleavage; Escherichia coli; Genetic engineering; Human performance; Proteins; Recombinant; Screens; Solubility
• DOI: 10.11895/j.issn.0253-3820.140706
• ISSN: 0253-3820

To obtain more efficient matrix metalloproteinase-1 (MMP1) targets for small-molecule fluorescence probe screen, the cDNA of MMP1 was isolated from human embryonic kidney 293 (HEK293) cells, and soluble expression of MMP1 has been performed and compared with recombinant MMP1 refolded from inclusion body in E. coli by genetic engineering. The activities of purified soluble MMP1 were compared with in-vivo MMP1 (urinary MMP1) by zymography. The soluble MMP1 was expressed as fusion protein of MMP1 and thioredoxin (TrxA-MMP1). By introducing a cleavage site in TrxA-MMP1 fusion protein, the soluble MMPL was obtained through hydrolysis by enterokinase. The results exhibited TrxA could obviously increase the solubility of MMP1, and soluble MMP1 showed 1.54 fold of gelatin-degradation activity as the refolded MMP1, which was more close to the activity of urinary MMP1 (uMMP1). In conclusion, we successfully detected the activity of in vitro recombinant MMP1 and in vivo MMP1 by zymography, which was a more reliable target for small-molecule probe screen.