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Updated: Aug 11, 2026
Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins
Published on: December 12, 2013
The expression and refolding of isotopically labeled recombinant Matrilysin for NMR studies
Li Ou1, Jinbiao Ma, Xunhai Zheng
1State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, PR China.
Abstract:
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.
Insights
Researchers synthesized and purified matrilysin (MMP7), a key protein in cancer and inflammation. This optimized protocol yields high quantities of properly folded MMP7 for further structural and functional studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Matrilysin (MMP7) is the smallest matrix metalloproteinase (MMP), crucial for connective tissue remodeling.
- MMP7 is implicated in cancer, innate immunity, and inflammatory disorders, making it a potential drug target.
Purpose of the Study:
- To report the gene synthesis, overexpression, purification, and refolding of MMP7.
- To establish an optimized protocol for producing high yields of labeled MMP7 for NMR studies.
Main Methods:
- Gene synthesis via PCR and overexpression in Escherichia coli.
- Protein purification and refolding from inclusion bodies.
- Characterization using MALDI-TOF mass spectrometry and dynamic light scattering (DLS).
- Preparation of (15)N and/or (13)C-labeled MMP7 in M9 minimal media.
Main Results:
- Successfully synthesized and expressed MMP7 in E. coli, yielding inclusion bodies.
- Purified and refolded MMP7 confirmed by mass spectrometry for primary sequence accuracy.
- DLS analysis indicated MMP7 exists as a monomer.
- Optimized protocol achieved 18-20 mg of purified MMP7 per 0.5 L of minimal media.
- NMR analysis of (15)N-labeled MMP7 confirmed proper folding and a well-ordered structure.
Conclusions:
- An efficient protocol for MMP7 production and purification was developed.
- The study provides sufficient quantities of well-folded MMP7 for structural and functional investigations.
- The findings support MMP7 as a viable target for drug development in cancer and inflammatory diseases.
