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.

The mechanism of matrix metalloproteinase-8 (MMP-8) inhibition was investigated using ellipsometric measurements of the interaction of MMP-8 with a surface bound peptide inhibitor, tether-metal abstraction peptide (MAP), bound to self-assembled monolayer films. MMP-8 is a collagenase whose activity and dysregulation have been implicated in a number of disease states, including cancer metastasis, diabetic neuropathy, and degradation of biomedical reconstructions, including dental restorations. Regulation of activity of MMP-8 and other matrix metalloproteinases is thus a significant, but challenging, therapeutic target. Strong inhibition of MMP-8 activity has recently been achieved via the small metal binding peptide tether-MAP. Here, the authors elucidate the mechanism of this inhibition and demonstrate that it occurs through the direct interaction of the MAP Tag and the Zn 2þ binding site in the MMP-8 active site. This enhanced understanding of the mechanism of inhibition will allow the design of more potent inhibitors as well as assays important for monitoring critical MMP levels in disease states.

Biologics can be an improvement to small molecule drugs, providing high specificity for an identified target, lowering toxicity and limiting side effects. To achieve effective delivery, the biologic must have sufficient time to reach the target tissue. A prolonged half-life in the circulating environment is desired, but often serum stability is limited by proteases. Proteolysis in the serum causes degradation and inactivation as the biologic is fragmented and more rapidly cleared from the body. To improve the circulating half-life, large, hydrophilic polymers may be conjugated or stable fusion tags may be engineered to increase the effective size of the peptide and to hinder degradation by proteases. Improved resistance to proteases is essential for effective delivery. Here, a proof of concept study is presented using a metal-binding tripeptide tag known as the claMP Tag to create an inline conjugate and the ability of the tag to inhibit proteolysis was examined.