Over-expression and refolding of isotopically labeled recombinant catalytic domain of human macrophage elastase (MMP-12) for NMR studies |
| |
Authors: | Zheng Xunhai Ou Li Tong Xiaotian Zhu Jing Wu Houming |
| |
Affiliation: | State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Science, Shanghai 200032, PR China. |
| |
Abstract: | 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 angiostatin, 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 15N and/or 13C-labeled MMP-12 catalytic domain, and the yield of the purified protein is estimated to 10-12 mg from 0.5L of M9 minimal media. Finally, the 15N-1H HSQC spectrum of uniformly 15N-labeled MMP-12 catalytic domain indicates the presence of well-ordered and properly folded protein in a monomeric form. |
| |
Keywords: | Human macrophage elastase Isotope labeling MMP-12 NMR Over-expression Refolding |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|