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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0686-9) contains supplementary material, which is available to authorized users.

Functional Production of Catalytic Domains of Human MMPs in Escherichia coli Periplasm

“…Using protease deficient host BL21 reduced the amount of unwanted truncations. In addition, although cdMMPs do not have disulfide bonds, we found that co-expression of molecular chaperon DsbC (a disulfide isomerase) significantly improved the yields of cdMMPs (Fig 2B), a similar observation for TIMPs [15]. …”

Reducing proteolytic liability of a MMP‐14 inhibitory antibody by site‐saturation mutagenesis

“…N‐terminal domain of TIMP‐2 (nTIMP‐2) was prepared from periplasmic expression . Purified cdMMP‐14 was biotinylated by using EZ‐Link Sulfo‐NHS‐LC kit (Thermo Fisher).…”

The Preparation and Solution NMR Spectroscopy of Human Glycoproteins Is Accessible and Rewarding

“…Lastly, the prokaryotic cytosol is reductive and incompatible with disulfide bond formation that occurs in the oxidative endoplasmic reticulum of eukaryotes. Currently, no holistic prokaryotic expression strategy to produce uniformly labeled mammalian glycoproteins exists, though multiple groups are engineering E. coli to surmount this limitation (Lee, Nam, Nuhn, Wang, Schneider, & Ge, 2017; Schein et al, 1992; Valderrama-Rincon et al, 2012; Wang & Amin, 2014). Eukaryotic microbes, namely the yeasts, have an analogous oxidative secretory system with glycosylation machinery.…”