Isolation,Purification And Renaturation Of MT3-MMP

Extracellular matrix is important as a scaffold for tissue structure and also as an environment regulating cell function in tissues. Matrix Metalloproteinases (MMPs) are a family of zinc metallo-endopeptidases secreted by cells, and are responsible for much of the turnover of matrix components. The MMPs are involved in a wide range of proteolytic events, in normal and pathological circumstances. Normal physiological roles for the MMPs include neurite growth, cell migration, bone elongation, wound healing, angiogenesis, ovulation, sperm maturation, uterine involution, menstruation, enamel formation, antigen processing and presentation, mammary gland development, hair follicle development, and embryo implantation. Pathological processes involving MMPs include tumor growth and migration, fibrosis, arthritis, glaucoma, lupus scleroderma, cirrhosis, multiple sclerosis, aortic aneurysms, infertility, and many more diseases. It is fair to say that a proteolytic event is key to a wide range of biological processes, including tissue remodeling and also modification or release of biological factors. The MMPs are over-expressed during invasion and metastasis and degrade extracellular matrix, lead to invasion and metastasis of tumor. So, one of the important future prospects is to address the inhibitors that act only on specific MMPs for the treatment of cancer.We have carried out these works: 1. We have optimized the condition for the high-level expression of MT3-MMP. The results indicate that cultivate on the table, the MT3-MMP expression amount is high when cells were induced by adding 0.5 mM IPTG for four hours when the cell density reached an A600 of 0.3-0.4. The inclusion bodies were purified through washing. Then the inclusion bodies were passed through IMAC and HPLC to further purify them from their associated impurities. 2. Dialysis for refolding. Dialysis using ultra-filtration membranes have been used to reduce high denaturant concentrations. 3. Protein refolding by gel filtration chromatography. GFC restricts the available pore volume for various protein forms in the gel matrix, thus, facilitating the separation of correctly folded and aggregated species. We have chosen Sephadex G-75 as gel matrix, conducted the research about refolding of MT3-MMP and realized its refolding. 4. Refolding by IMAC, this method can realize the purification and refolding at the same time, but the results of refolding are not ideal.