Direct expression of active human tissue inhibitors of metalloproteinases by periplasmic secretion in Escherichia coli

Microbial Cell Factories

Research

Juan Wang¹  Jacob A. M. Nuhn¹  Ian C. Schneider²  Ki Baek Lee³  Xin Ge³  Dong Hyun Nam³ 

[1] Department of Chemical and Biological Engineering, Iowa State University, 3053 Sweeney, 50011, Ames, IA, USA;Department of Chemical and Biological Engineering, Iowa State University, 3053 Sweeney, 50011, Ames, IA, USA;Genetics Development, and Cell Biology Department, Iowa State University, 1210 Molecular Biology, 50011, Ames, IA, USA;Department of Chemical and Environmental Engineering, University of California, Riverside, 900 University Ave, 92521, Riverside, CA, USA;

关键词: Tissue inhibitor of metalloproteinase;    Matrix metalloproteinase;    Periplasmic expression;    Disulfide isomerase;    Contact guidance;   

DOI  :  10.1186/s12934-017-0686-9

 received in 2016-12-16, accepted in 2017-04-21,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundAs regulators of multifunctional metalloproteinases including MMP, ADAM and ADAMTS families, tissue inhibitors of metalloproteinases (TIMPs) play a pivotal role in extracellular matrix remodeling, which is involved in a wide variety of physiological processes. Since abnormal metalloproteinase activities are related to numerous diseases such as arthritis, cancer, atherosclerosis, and neurological disorders, TIMPs and their engineered mutants hold therapeutic potential and thus have been extensively studied. Traditional productions of functional TIMPs and their N-terminal inhibitory domains (N-TIMPs) rely on costly and time-consuming insect and mammalian cell systems, or tedious and inefficient refolding from denatured inclusion bodies. The later process is also associated with heterogeneous products and batch-to-batch variation.ResultsIn this study, we developed a simple approach to directly produce high yields of active TIMPs in the periplasmic space of Escherichia coli without refolding. Facilitated by disulfide isomerase (DsbC) co-expression in protease-deficient strain BL21 (DE3), N-TIMP-1/-2 and TIMP-2 which contain multiple disulfide bonds were produced without unwanted truncations. 0.2–1.4 mg purified monomeric TIMPs were typically yielded per liter of culture media. Periplasmically produced TIMPs exhibited expected inhibition potencies towards MMP-1/2/7/14, and were functional in competitive ELISA to elucidate the binding epitopes of MMP specific antibodies. In addition, prepared N-TIMPs were fully active in a cellular context, i.e. regulating cancer cell morphology and migration in 2D and 3D bioassays.ConclusionPeriplasmic expression in E. coli is an excellent strategy to recombinantly produce active TIMPs and N-TIMPs.

【 授权许可】

CC BY   
© The Author(s) 2017