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Article
Direct expression of active human tissue inhibitors of metalloproteinases by periplasmic secretion in Escherichia coli
Microbial Cell Factories
  • Ki Baek Lee, University of California, Riverside
  • Dong Hyun Nam, University of California, Riverside
  • Jacob A. M. Nuhn, Iowa State University
  • Juan Wang, Iowa State University
  • Ian C. Schneider, Iowa State University
  • Xin Ge, University of California, Riverside
Document Type
Article
Publication Version
Published Version
Publication Date
4-28-2017
DOI
10.1186/s12934-017-0686-9
Abstract

Background

As 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. Results

In 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. Conclusion

Periplasmic expression in E. coli is an excellent strategy to recombinantly produce active TIMPs and N-TIMPs.

Comments

This article is published as Lee, Ki Baek, Dong Hyun Nam, Jacob AM Nuhn, Juan Wang, Ian C. Schneider, and Xin Ge. "Direct expression of active human tissue inhibitors of metalloproteinases by periplasmic secretion in Escherichia coli." Microbial Cell Factories 16:73 (2017).

Creative Commons License
Creative Commons Attribution 4.0
Copyright Owner
The Authors
Language
en
File Format
application/pdf
Citation Information
Ki Baek Lee, Dong Hyun Nam, Jacob A. M. Nuhn, Juan Wang, et al.. "Direct expression of active human tissue inhibitors of metalloproteinases by periplasmic secretion in Escherichia coli" Microbial Cell Factories Vol. 16 Iss. 73 (2017)
Available at: http://works.bepress.com/ian_schneider/15/