Purification And Refolding Of MMP-13 Catalytic Domain And Enzyme Kinetics

Matrix metalloproteinases(MMPs)are a family of extracellular zinc and calcium-dependent metalloendopeptidase collectively capable of precisely regulating degradation of the extracellular matrix(ECM),they can degradate almost all the proteins in ECM through cutting the internal peptides,and also can mediate the degradation of connective tissue and tissue remodeling.Therefore MMPs not only adjust the normal physiological processes,but also have important influence on the production and the development of arthritis,cancer,heart disease and some other diseases.So,the researches of MMPs and MMPIs have important significance on the detection and treatment of these diseases.This thesis investigated the induction expression,purification and refolding of MMP-13 catalytic domain,the inhibitor screen and inhibit dynamics.The obtained results might lay the foundation on the research of inhibition mechanism,inhibitors synthesis and the development of cancer drugs target on MMPs.The main rsults of this study include:1.The expression and purification of MMP-13 catalytic domain proteinRecombinant E.coli was used as expression system,MMP-13 catalytic domain was induced and expressed by IPTG in LB medium.It was found that the protein expression yield was up to 40%.We got the MMP-13 catalytic domain protein inclusion body through ultrasonication,centrifuging,inclusion body washing,and finally chose 6M urea as denaturant to dissolve inclusion body.Protein was purified by Ni2+-affinity chromatography under the optimized conditions.With NaCl exist we got the purity of 91.3%,the mass yield of 71.5%±2.1%,and we got the purity of 98.0%,the mass yield of 57.4%without NaCl2.Refolding of MMP-13 catalytic domain proteinIon exchange chromatography,size exclusion chromatography and dialysis were used to refold MMP-13.During ion exchange chromatography refolding,three gradients were first used for refolding at first,then size exclusion chromatography was used for desalting.We chose the best condition as follows:loading volume was 0.80 mL(protein concentration was 0.8527 mg/mL);flow rate was 0.80 mL/min;elution gradient time was 20 min;refolding balance buffer:5 mol/Lurea+1 mmol/L EDTA-Na2+20 mmol/L Tris-HCl(pH =8.0);refolding elution buffer:1 mol/L NaCl+1 mmol/L EDTA-Na2+20 mmol/L Tris-HCl(pH =7).The desalting condition:the flow rate was 1.00mL/min,the buffer was 20 mmol/L Tris-HCl(pH=7.5).The condition for derectly refolding of protein by SEC was the same as that in desalting.We got the refolded protein with the mass yield of 81.4%,activity recovery of 92.9%finally,the mass yield of desalting was 32.7%,so the tatal mass yield of ion exchange chromatography refolding was 26.6%.When SEC was used for refolding,the mass yield was 41.7%,activity recovery was 82.02%;the mass yield and activity recovery of dialysis was 58.6%and 65.3%respectively,and the purity of protein was 74.2%.These results indicated the activity recovery of ion exchange chromatography was higer,but the two steps refolding made the mass yield decrease,and diluted the protien.The mass yield of SEC was higer than that of ion exchange chromatography,but with lower activity recovery.3.The effect of inhibitor on purification and refoldingWe found that MMP-13 catalytic domain protein would degrade during the refolding process,reducing protein activity.EDTA,Al2(SO4)3 and K3[Fe(CN)6]were used as inhibitor added in refolding buffer.The results indicated that the inhibitor can prevent degradation,and also EDTA could prevent the degradation during storage.4.The research of enzyme inhibition dynamics of MMP-13 catalytic domain proteinFluorescence substrate method was used to analysis the enzyme kinetics.The kinetics parameters,such as Vmax and Km was obtained according to the Michaelis equation.The inhibition type of MMP-13 inhibitor was competitive inhibition according to Dixon mapping and CD results.