大肠杆菌PGDH末端缺失突变体的构建及抗反馈抑制效应分析  

Construction and Characterization of E.coli PGDH Mutants with Feedback-inhibition Resistance

作　　者：张绪梅[1] 郭长江[1] 刘云[2] 杨继军[1] 韦京豫[1] 徐琪寿[2] 

机构地区：[1]军事医学科学院卫生学环境医学研究所,天津300050 [2]北京放射医学研究所,北京100850

出　　处：《中国生物化学与分子生物学报》2006年第10期806-810,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金( No.30300010);天津市科技攻关项目(No.033182711)~~

摘　　要：为了获得具有抗反馈抑制性质的大肠杆菌磷酸甘油酸脱氢酶（PGDH,d-3-phosphoglycerate dehydrogenase,EC 1.1.1.95）,通过对其碱基序列和蛋白质结构分析,用PCR突变法构建突变酶M1（缺失第410位氨基酸）、M2（缺失407～410位氨基酸）、M3（缺失337～410位氨基酸）.M0（野生型）及各突变型基因与pET22b（＋）载体连接后,表达融合蛋白.在非变性条件下,由NTA-Ni镍离子螯合亲和层析柱纯化野生型和突变体的酶蛋白.酶活性测定结果表明,M1、M2蛋白酶均保持了原有的野生型磷酸甘油酸脱氢酶活性,且部分解除了终产物L-丝氨酸的反馈抑制作用;M3蛋白酶完全解除了终产物的反馈抑制作用,但酶本身的催化活性略有降低（为野生型的83%）.M0、M1、M2菌株PGDH与L-丝氨酸结合的Ki值分别约为7μmol/L、20μmol/L、50μmol/L,说明该酶C末端1～4个氨基酸残基对L-丝氨酸和调控区的结合有重要影响.In order to obtain E. coli PGDH mutants with feedback-inhibition resistance, PCR mutagenesis was used to construct three mutants on the basis of homology of protein structure and the related PGDH amino acid sequences： M1 （ serA mutant with aa410 deleted）, M2 （ serA mutant with aa407 - 410 deleted）, M3 （ serA mutant with aa337 -410 deleted）. The three mutant genes and wild type gene were inserted into prokaryotic fusion-expression vector pET22b （＋）. The expressed products were purified by NTA-Ni affinity chromatography resin in native condition. Catalytic activities of M3 retained only 83 % of the wild type, but the activities of M1 and M2 were retained. M3 showed the complete feedback-resistance, M1 and M2 also showed resistance to some extent. Ki value of M0, M1, M2 were about 7 μmol/L, 20 μmol/L, 50 μmol/L, respectively. This result means that PGDH C-terminal residues 1 - 4 have a profound effect on the interaction between L-serine and R regulatory region.

关 键 词：大肠杆菌 PGDH 蛋白纯化 缺失突变体 抗反馈抑制 

分 类 号：Q291[生物学—细胞生物学] Q786