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Updated: Aug 1, 2025
Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography
Published on: March 30, 2022
Expression, purification and refolding of pro-MMP-2 from inclusion bodies of E. coli
Yu Nan Zhang1, Jia Jian Liu2, Wei Zhang1
1Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, 210029, China.
Abstract:
MMP-2 has been reported as the most validated target for cancer progression and deserves further investigation. However, due to the lack of methods for obtaining large amounts of highly purified and bioactive MMP-2, identifying specific substrates and developing specific inhibitors of MMP-2 remains extremely difficult. In this study, the DNA fragment coding for pro-MMP-2 was inserted into plasmid pET28a in an oriented manner, and the resulting recombinant protein was effectively expressed and led to accumulation as inclusion bodies in E. coli. This protein was easy to purify to near homogeneity by the combination of common inclusion bodies purification procedure and cold ethanol fractionation. Then, our results of gelatin zymography and fluorometric assay revealed that pro-MMP-2 at least partially restored its natural structure and enzymatic activity after renaturation. We obtained approximately 11 mg refolded pro-MMP-2 protein from 1 L LB broth, which was higher than other strategies previously reported. In conclusion, a simple and cost-effective procedure for obtaining high amounts of functional MMP-2 was developed, which would contribute to the progress of studies on the gamut of biological action of this important proteinase. Furthermore, our protocol should be appropriate for the expression, purification, and refolding of other bacterial toxic proteins.
Insights
Researchers developed a cost-effective method to produce large quantities of active Matrix Metalloproteinase-2 (MMP-2) for cancer research. This breakthrough facilitates the study of MMP-2
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Matrix Metalloproteinase-2 (MMP-2) is a key target in cancer progression research.
- Current challenges in cancer research stem from difficulties in obtaining sufficient purified, bioactive MMP-2.
- This hinders the identification of MMP-2 substrates and the development of specific inhibitors.
Purpose of the Study:
- To develop a simple, cost-effective method for producing high yields of functional MMP-2.
- To enable further investigation into MMP-2's biological functions and therapeutic targeting.
Main Methods:
- Engineered E. coli for oriented expression of the pro-MMP-2 DNA fragment using plasmid pET28a.
- Purified recombinant pro-MMP-2 from inclusion bodies using standard procedures and cold ethanol fractionation.
- Renatured the purified protein and confirmed its biological activity via gelatin zymography and fluorometric assays.
Main Results:
- Successfully expressed and purified pro-MMP-2 to near homogeneity.
- Renaturation restored significant enzymatic activity and natural structure to the MMP-2 protein.
- Achieved high yields of approximately 11 mg of refolded pro-MMP-2 per liter of culture medium.
Conclusions:
- A straightforward and economical protocol for producing substantial amounts of active MMP-2 has been established.
- This method overcomes previous limitations, supporting advanced research on MMP-2's biological roles.
- The protocol is potentially adaptable for expressing, purifying, and refolding other bacterial toxic proteins.
