Over-expression and refolding of isotopically labeled recombinant catalytic domain of human macrophage elastase (MMP-12) for NMR studies

摘要

Human macrophage elastase (MMP-12) plays an important role in inflammatory processes and is involved in a number of physiological or pathological situations, such as conversion of plasminogen into angiotatin, allergic airway inflammation, vascular remodeling or alteration, as well as emphysema, and has been justified as a novel drug target. Here, we report the over-expression in Escherichia coil, purification and refolding of MMP-12 catalytic domain for NMR studies. The primary sequence of expressed protein was identified by means of MALDI-TOF MS, and was confirmed by the MALDI-TOF MS data of trypsin-digested peptides. A significantly optimized protocol has been worked out to prepare N-15 and/or C-13-labeled MMP-12 catalytic domain, and the yield of the purified protein is estimated to 10-12 mg from 0.5 L of M9 minimal media. Finally, the N-15-H-1 HSQC spectrum of uniformly N-15-labeled MMP-12 catalytic domain indicates the presence of well-ordered and properly folded protein in a monomeric form. (C) 2007 Elsevier Inc. All rights reserved.