人基质金属蛋白酶2催化区的克隆与表达  

Cloning and Expression of the Catalytic Domain of Human MMP-2

作　　者：高媛[1] 王清明[2] 范国才[2] 陈吉中[2] 张德安[1] 陈惠鹏[2] 

机构地区：[1]吉林大学生命科学学院,长春13002 [2]军事医学科学院放射医学研究所,北京100850

出　　处：《中国生物化学与分子生物学报》2001年第5期631-635,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：北京市科委重点项目 (No .95 5 0 2 14 0 0 0 )

摘　　要：为从随机肽库中寻找具有基质金属蛋白酶 2 (MMP 2 )抑制活性的新型小肽抑制剂 ,应用PCR法从含有人MMP 2基因的质粒中扩增了人MMP 2的催化区 .序列分析结果表明无氨基酸突变 .然后构建人MMP 2催化区的表达载体pET MCD ,转化大肠杆菌BL2 1(DE3 ) ,经IPTG诱导表达人MMP 2催化区 .经包涵体分离、变性、金属螯合层析纯化和复性等过程 ,复性后的人MMP 2催化区具有较好的明胶水解活性 .Matrix metalloproteinase 2(MMP\|2) is an important matrix metalloproteinase in the degradation of basement membranes and denatured collagens(gelatin),and is believed to play a key role in cancer invasion and metastasis.In order to screen inhibitors of MMP\|2,cDNA of the catalytic domain of MMP\|2 (MCD) was amplified by polymerase chain reaction(PCR) and was cloned into expression vector pET\|28c.The constructed expression vector was then transformed in \%E.coli\% BL21(DE3).SDS PAGE analysis indicated that MCD expressed in \%E.coli\% existed as inclusion bodies.The inclusion bodies were separated and dissolved in 8 mol/L urea.Then MCD was refolded in the presence of Zn2+ and Ca 2+,and purified by chelating affinity chromatography.Gelatin zymography analysis showed that the refolded MCD had an excellent gelatinolytic activity.

关 键 词：MMP-2 包涵体 原核表达 基质金属蛋白酶2 催化区 克隆 

分 类 号：Q556.3[生物学—生物化学]