Over-expression and refolding of isotopically labeled recombinant catalytic domain of human macrophage elastase (MMP-12) for NMR studies.
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.
Zheng
Xunhai
X
State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Science, Shanghai 200032, PR China.
Ou
Li
L
Tong
Xiaotian
X
Zhu
Jing
J
Wu
Houming
H
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D053508:Matrix Metalloproteinase 12 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D012996:Solutions; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011994:Recombinant Proteins; D012996:Solutions; C507122:MMP12 protein, human; D053508:Matrix Metalloproteinase 12