The Preparation Of MMP-13 Catalytic Domain Crystallization Samples And The Study Of The Inhibitory Mechanism Of New Inhibitors

Matrix metalloproteinase(MMPs)are zinc-dependent protein and peptide hydrolases,which constitute a separate family within the "metzincin" clan of metallopeptidases(MPs).MMPs are capable of degrading extracellar matrix(ECM)components to participate in physiological processes involving bone development,tissue remodeling and organs development.But the dysregulation of MMPs has a close connection with some pathologies process,such as osteoarthritis,diseases of the cardiovascular system,diseases of nervous system and neoplastic diseases.MMP-13,as a kind of MMPs,is largely expressed in inflammation and tumor pathologies.MMP-13 mainly effect pathologies in two ways,i:degrades the components of the basement membrane to promote tummor metastasis and invasion;ii:regulate some factors relevant to pathologies to indirectly participate in pathologies.Thus,the MMP-13 has been a biomarker for breast cancer and osteoarthritis.One of the therapeutic strategies for these diseases is the design of drugs and inhibitors to target their catalytic domain.In this thesis,two kinds of catalytic domain of MMP-13(cdMMP-13)were expressed in E.coli through recombinant technology.In addition,both of them were purified and refolded for screenning of crystallization conditions.At last,the inhibition mechanisms of new inhibitors of MMP-13 were investgated.The thesis includes the following contents:1.Preparation of single Histidtine tagged cdMMP-13(His-cdMMP-13)and screening of crystallization conditions.The His-cdMMP-13 was induced in E.coli BL21.The purification conditions of His-cdMMP-13 were optimized and the dynamic adsorption and static adsorption purification methods were compared.One time in the dynamic adsorption method,19-22mg protein samples can be loaded,and 13mg His-cdMMP-13 with purification of 98%can be recovered.This method is proper for preparing His-cdMMP-13 used for screening of inhibitors and optimization of crytallization conditions.One time in the static adsorption method,50-55mg protein samples can be loaded,and about 44mg His-cdMMP-13 with purification of 97%can be recoverd.This method is proper for sreening of crystallization conditions.In addition,His-cdMMP-13 can be refolded by desalting exclusion chromatography.The addtion of Al2(SO4)3 or K3[Fe(CN)6]of 10 u M can keep the refolded His-cdMMP-13 stable at 4oC.At last,crystallization conditions of the complex of refolded His-cdMMP-13 and its inhibitor were screened at 4oC,but none crystal formed after 384 conditions screened.2.Preparation of dual Histidtine tagged cdMMP-13(Dual-His-cdMMP-13)and screening of crystallization conditions.The plasmid containing DNA of cdMMP-13 was constructed through primers design,PCR,cutting and jointing DNA molecular.The expression product of BL21 was indentified and demonstrated that the Dual-His-cdMMP-13 was expressed as an inclusion body in BL21.The purification conditions of Dual-His-cdMMP-13 were optimized and the dynamic adsorption and static adsorption purification methods were compared.One time in the dynamic adsorption method,17-20mg protein samples can be loaded,and 12mg His-cdMMP-13 with purification of 98%can be recovered.This method is proper for preparing Dual-His-cdMMP-13 used for screening of inhibitors and optimization of crytallization conditions.One time in the static adsorption method,45-50mg protein samples can be loaded,and about 40mg His-cdMMP-13 with purification of 97%can be recoverd.This method is proper for sreening of crystallization conditions.In addition,Dual-His-cdMMP-13 can be refolded by desalting exclusion chromatography.Moreover,the Dual-His-cdMMP-13 is more stable than His-cdMMP-13 and can be stored at 16oC.At last,384 kinds of crystallization conditions of the Dual-His-cdMMP-13 and the complex of refolded His-cdMMP-13 and its inhibitor were screened at 16oC.In the 0.5 M NaCl,0.01 M MgCl2,0.01 M cetyl trimethyl ammonium bromide condition,there is a suspected crystal of the complex of refolded His-cdMMP-13 and its inhibitor,but the suspected crystal is too small and irregular to obtain the diffraction data.The condition of the suspected crystal should be optimized to obtain a better crystal.3.Inhibiton mechanisms of new inhibitorsDynamics experiments of inhibitors were conducted by using of fluorescence quenching substance.The results of dynamics experiments showed that the inhibition ability of K3[Fe(CN)6](IC50:0.5?M)is better than Al2(SO4)3(IC50:1.8?M),but the inhibition mechanisms of them,mixed inhibition,are same.In addtion,the inhibition ability of amygdalin for MMP-13 was investigated.The results showed that the inhibition ability of amygdalin(IC50:17.1 ?M)is weaker than Al2(SO4)3 and K3[Fe(CN)6].But considered the inhibition ability of K3[Fe(CN)6],it was assumed that the cyanogen(CN)organ might paly an assistant role in the inhibition of MMP-13.