甲醇利用菌SDM11中glyA基因的克隆及其在大肠杆菌中的表达  

Molecular Cloning and Expression of a glyA Gene from Methylobacterium.sp SDM11

作　　者：孔庆胜[1] 张向阳[1] 韩晓琳[1] 林强[2] 董全林[2] 

机构地区：[1]济宁医学院,日照276826 [2]华中科技大学,武汉430071

出　　处：《生物技术通报》2010年第5期141-144,157,共5页Biotechnology Bulletin

基　　金：山东省教育厅资助项目(J07YD16)

摘　　要：对甲醇降解菌Methylobacterium.sp SDM11中的glyA基因进行克隆及特性研究,以获得更多的丝氨酸羟甲基转移酶(serine hydroxymethyltransferase,SHMT)资源。根据GenBank中已报道的Methylobacterium extorquensAM1中的glyA基因序列(登录号:L33463)设计引物,以SDM11的基因组DNA为模板,PCR扩增glyA基因。利用pETblue-2载体将该基因在大肠杆菌BL21(DE3)中得到表达。PCR扩增到一个1.40 kb大小的DNA片段,经过blast软件比对分析,发现该片段与已报道的Methylobacterium extorquensAM1的glyA基因的序列相似性为95%,氨基酸序列的相似性为98%。该基因编码468个氨基酸,预测的分子量大小为52.2 kD,等电点为7.02,发现纯化后的目标蛋白具有SHMT酶活性,并初步测定了酶活力。To clone glyA gene from Methylobacterium. sp SDM11 and study its properties to enrich the gene resources for serine hydroxymethyltransferase （SHMT） , primers were designed based on the glyA sequence of the reported Methylobacterium extorquens AM1 （accession number is L33463）in GenBank,and an about 1.4 kb DNA fragment was amplified with the genomic DNA of SDMll as a template. The sequence analyzied result showed that this fragment had an ORF encoded 468 amino acids,and had 95% and 98% iden- tities with the glyA gene of M. extorquens AM1 respectively. The predicted molecular is 52.2 kD and its isoelectric point is 7.02. The ORF was amplified by PCR,and cloned into expression vector pETBlue-2,then transferred into E. coli Tunner（ DE3 ）. After introduced by IPTG,the target protein was expressed,which showed SHMT activity after purification.

关 键 词：SDM11 glyA基因 克隆 表达 SHMT酶 

分 类 号：Q78[生物学—分子生物学]