Expression, purification and refolding of pro-MMP-2 from inclusion bodies of E. coli.

Autor: Zhang YN; Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, 210029, China., Liu JJ; Minigene Pharmacy Laboratory, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, China., Zhang W; Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, 210029, China., Qin HY; Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, 210029, China., Wang LT; Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, 210029, China., Chen YY; Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, 210029, China., Yuan L; Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, 210029, China., Yang F; Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, 210029, China. Electronic address: yangfen@njmu.edu.cn., Cao RY; Minigene Pharmacy Laboratory, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, China. Electronic address: caoronguenanjing@yahoo.com.cn., Wang XJ; Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, 210029, China. Electronic address: wangxuejun@njmu.edu.cn.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2023 Aug; Vol. 208-209, pp. 106278. Date of Electronic Publication: 2023 Apr 23.
DOI: 10.1016/j.pep.2023.106278
Abstrakt: MMP-2 has been reported as the most validated target for cancer progression and deserves further investigation. However, due to the lack of methods for obtaining large amounts of highly purified and bioactive MMP-2, identifying specific substrates and developing specific inhibitors of MMP-2 remains extremely difficult. In this study, the DNA fragment coding for pro-MMP-2 was inserted into plasmid pET28a in an oriented manner, and the resulting recombinant protein was effectively expressed and led to accumulation as inclusion bodies in E. coli. This protein was easy to purify to near homogeneity by the combination of common inclusion bodies purification procedure and cold ethanol fractionation. Then, our results of gelatin zymography and fluorometric assay revealed that pro-MMP-2 at least partially restored its natural structure and enzymatic activity after renaturation. We obtained approximately 11 mg refolded pro-MMP-2 protein from 1 L LB broth, which was higher than other strategies previously reported. In conclusion, a simple and cost-effective procedure for obtaining high amounts of functional MMP-2 was developed, which would contribute to the progress of studies on the gamut of biological action of this important proteinase. Furthermore, our protocol should be appropriate for the expression, purification, and refolding of other bacterial toxic proteins.
Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest.
(Copyright © 2023 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
Externí odkaz: