Thioredoxin fusion construct enables high-yield production of soluble, active matrix metalloproteinase-8 (MMP-8) in Escherichia coli

Abstract

Matrix metalloproteinases (MMPs) are crucial proteases in maintaining the health and integrity of many tissues, however their dysregulation often facilitates disease progression. In disease states these remodeling and repair functions support, for example, metastasis of cancer by both loosening the matrix around tumors to enable cellular invasion and by affecting proliferation and apoptosis, and they promote degradation of biological restorations by weakening the substrate to which the restoration is attached. As such, MMPs are important therapeutic targets. MMP-8 participates in cancer, arthritis, asthma and failure of dental fillings. MMP-8 differs from other MMPs in that it has an insertion that enlarges its active site. To elucidate the unique features of MMP-8 and develop selective inhibitors to this therapeutic target, a stable and active form of the enzyme is needed. MMP-8 has been difficult to express at high yield in a soluble, active form. Typically recombinant MMPs accumulate in inclusion bodies and complex methods are applied to refold and purify protein in acceptable yield. Presented here is a streamlined approach to produce in E. coli a soluble, active, stable MMP-8 fusion protein in high yield. This fusion shows much greater retention of activity when stored refrigerated without glycerol. A variant of this construct that contains the metal binding claMP Tag was also examined to demonstrate the ability to use this tag with a metalloprotein. SDS-PAGE, densitometry, mass spectrometry, circular dichroism spectroscopy and an activity assay were used to analyze the chemical integrity and function of the enzyme.

Mechanistic investigations of matrix metalloproteinase-8 inhibition by metal abstraction peptide

“…17 In the present study, two variants of MMP-8 were used. Both contain an N-terminal thioredoxin-S Tag fusion partner and a polyhistidine tag for purification (MMP-8 fusion), and in addition, one contained the metal-binding claMP Tag and a spacer sequence inserted between the fusion partner and MMP-8 (claMP-link-MMP-8 fusion).…”

“…Our group has recently developed methods of preparing a relatively stable, active, inhibitable MMP-8 fusion construct. 17 Two different protein variants were used in the investigations described here: claMP-link-MMP-8 fusion and MMP-8 fusion. 17 Each of these proteins has been characterized extensively and shown to be catalytically active and inhibited by the standard inhibitor NNGH, both in the fusion construct and following cleavage to release the tags.…”

“…17 Two different protein variants were used in the investigations described here: claMP-link-MMP-8 fusion and MMP-8 fusion. 17 Each of these proteins has been characterized extensively and shown to be catalytically active and inhibited by the standard inhibitor NNGH, both in the fusion construct and following cleavage to release the tags. Once the fusion partner in each of these constructs is cleaved to release the MMP-8 domain, significant fragmentation of the enzyme is observed over time along with a decrease in activity.…”

“…Experiments were run within 4 days after cleavage, and protein was typically stored refrigerated until samples were used to minimize the amount of degradation. Based on the characterization of activity and stability presented in McNiff et al, 17 sufficient quantities of active MMP-8 are present in these samples to investigate the mechanism of interaction between MMP-8 and tether-MAP. …”

Selection of High Producers From Combinatorial Libraries for the Production of Recombinant Proteins in Escherichia coli and Vibrio natriegens

“…These elements were further combined with four different N-terminal affinity tags for purification: glutathione-S-transferase (GST), maltose-binding protein (MBP), the small ubiquitin-like modifier (SUMO), and thioredoxin. All four tags have been shown to improve the solubility of recombinant proteins (Young et al, 2012), and affinity chromatography methods are available based, respectively, on immobilized glutathione (Schäfer et al, 2015), amylose (Reuten et al, 2016; Han et al, 2018), SUMO-specific antibodies (Butt et al, 2005), and the formation of reversible disulfide bonds (Mambetisaeva et al, 1997; McNiff et al, 2016). All four fusion tags were also combined with an additional N-terminal His 6 tag for purification by immobilized metal affinity chromatography (IMAC) (Loughran et al, 2017), resulting in eight different purification tag versions in the library.…”