Matrix metalloproteinases (MMPs) are a class of zinc and calcium-dependent endopeptidases responsible for degrading extracellular matrix (ECM) components. Their activity is critical for both normal biological function and pathological processes (Dejonckheere et al., Cytokine Growth Factor Rev 2011;22:73–81). In dental restorations, the release and subsequent acid activation of MMPs contributes to premature failure. In particular, MMP-8 accelerates degradation by cleaving the collagen matrix within the dentin substrate in incompletely infiltrated aged bonded dentin (Buzalaf et al., Adv Dent Res 2012;24:72–76), hastening the need for replacement of restorations. Therefore, development of a dental adhesive that better resists MMP-8 activity is of significant interest. We hypothesize that modification of the polymer surface with an inhibitor would disable MMP-8 activity. Here, we identify the metal abstraction peptide (MAP) as an inhibitor of MMP-8 and demonstrate that tethering MAP to methacrylate polymers effectively inhibits catalysis. Our findings indicate complete inhibition of MMP-8 is achievable using a grafting approach. This strategy has potential to improve longevity of dental adhesives and other polymers and enable rational design of a new generation of biocompatible materials.

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.

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