蛋白质N端豆蔻酰化修饰的基因工程表达偶联加工系统  

Coexpression System for Protein N myristoylation in Escherichia coli

作　　者：段治军[1] 陈长征[1] 杨新颖[1] 李伯良[1] 王德宝[1] 

机构地区：[1]中国科学院上海生物化学研究所

出　　处：《生物工程学报》1996年第S1期15-21,共7页Chinese Journal of Biotechnology

摘　　要：对酵母ＮＭＴ基因在大肠杆菌中表达进行较详细的研究，进而构建了复制子为ｐ１５Ａ并含卡那霉素抗性基因的相容性表达质粒ｐＫＺＭＴ，将其与表达质粒ｐＣＺｍＣα１共转化进大肠杆菌ＢＬ２１（ＤＥ３）Ｆ′，进行双质粒表达偶联加工修饰研究，其中ｐＣＺｍＣα１表达底物蛋白小鼠ｃＡＭＰ依赖的蛋白激酶催化亚基α（ＰＫＡｍＣα）。ＳＤＳＰＡＧＥ及Ｗｅｓｔｅｒｎｂｌｏｔ分析表明，双质粒表达系统中，ＰＫＡｍＣα都得到了稳定的高表达，尤其在２３℃低温诱导表达时，表达产物的可溶性部分明显增多；而酵母ＮＭＴ被控制在有利于活性功能的可溶性低水平表达。［３Ｈ］ｍｙｒｉｓｔｉｃａｃｉｄ标记测定及放射自显影的结果显示，在大肠杆菌中表达的重组ＰＫＡｍＣα被豆蔻酰化修饰。High level expression of YSCNMT was achieved in E.coli . Then, two compatible expression plasmids pKNMT and pCZmCα1 were constructed. The former includes the coding region of YSCNMT gene, the p15A origin of replication and a kanamycin resistance gene, while the latter contains the coding region of PKA mC α gene. Both of the plasmids were used to cotransform E.coli strain BL21(DE3)F′ and a double transformat was then obtained. Analyzed by SDS PAGE and Western Blot, it was observed that PKA mC α was highly coexpressed with the dual plasmid system and its soluble form was sapparently increased when the coexpression was induced at 23℃; while the inducible expression of YSCNMT was controlled to a low level in oreder to form soluble functional product. The results of autoradiography after labeling with myristic acid in vivo suggested that the recombinant PKA mC α was myristoylated within the dual plasmid system.

关 键 词：蛋白质N端豆蔻酰化 PKAmCα 偶联表达 双质粒系统 

分 类 号：Q51[生物学—生物化学]