title: Direct production of functional matrix metalloproteinase--14 without refolding or activation and its application for in vitro inhibition assays.
abstract: Human matrix metalloproteinase (MMP)-14, a membrane-bound zinc endopeptidase, is one of the most important cancer targets because it plays central roles in tumor growth and invasion. Large amounts of active MMP-14 are required for cancer research and the development of chemical or biological MMP-14 inhibitors. Current methods of MMP-14 production through refolding and activation are labor-intensive, time-consuming, and often associated with low recovery rates, lot-to-lot variation and heterogeneous products. Here, we report direct production of the catalytic domain of MMP-14 in the periplasmic space of Escherichia coli. 0.5 mg/L of functional MMP-14 was produced without tedious refolding or problematic activation process. MMP-14 prepared by simple periplasmic treatment can be readily utilized to evaluate the potencies of chemical and antibody-based inhibitors. Furthermore, co-expression of both MMP-14 and antibody Fab fragments in the periplasm facilitated inhibitory antibody screening by avoiding purification of MMP-14 or Fabs. We expect this MMP-14 expression strategy can expedite the development of therapeutic drugs targeting MMPs with biological significance.
authors.affiliation: Department of Chemical and Environmental Engineering, University of California, 900 University Ave Riverside, Riverside, 92521, California.
authors.forename: Dong Hyun
authors.initials: DH
authors.lastname: Nam
authors.affiliation: Department of Chemical and Environmental Engineering, University of California, 900 University Ave Riverside, Riverside, 92521, California. xge@engr.ucr.edu.
authors.forename: Xin
authors.initials: X
authors.lastname: Ge
mesh_terms: D000970:Antineoplastic Agents / Q000302:isolation & purification; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D053511:Matrix Metalloproteinase 14 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D053511:Matrix Metalloproteinase 14
keywords: MMP; inhibitory antibody; periplasmic expression