The expression and refolding of isotopically labeled recombinant Matrilysin for NMR studies.
Matrilysin (MMP7) is the smallest member of matrix metalloproteinases (MMPs) family, which are collectively responsible for remodeling of connective tissue. MMP7 plays an essential role in cancer, innate immunity, and in inflammatory disorders, and has been justified as a novel drug target. Here, we report the gene synthesis, overexpression in Escherichia coli, purification and refolding of MMP7. The gene of Matrilysin was synthesized based on PCR method and overexpressed in E. coli in the form of inclusion bodies. The protein was subsequently purified and refolded to yield sufficient quantities for structural and functional studies. The purified protein was characterized by means of MALDI-TOF mass spectroscopy and dynamic light scattering (DLS) analysis. The MS data confirms the correctness of the primary sequence, while DLS experiment proves that the protein exists as a monomeric form. A significantly optimized protocol has been worked out to prepare (15)N and/or (13)C-labeled MMP7 in minimal medium with high yields for NMR studies. Under the various conditions optimized for the purification of MMP7, the yield of the purified protein is estimated to be 18-20 mg from 0.5 L of M9 minimal media. Finally, the (15)N-1H HSQC spectrum of uniformly (15)N-labeled MMP7 sample reveals that the protein is properly folded, and exists in a well-ordered structure.
Ou
Li
L
State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, PR China.
Ma
Jinbiao
J
Zheng
Xunhai
X
Chen
Xiang
X
Li
Guangyao
G
Wu
Houming
H
D002247:Carbon Isotopes / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling; D020783:Matrix Metalloproteinase 7 / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002247:Carbon Isotopes; D011994:Recombinant Proteins; C060932:MMP7 protein, human; D020783:Matrix Metalloproteinase 7