硫氧还原蛋白突变基因用于鲑鱼降钙素的高效可溶性融合表达  

Mutated thioredoxin gene used in high and soluble fusion expression of salmon calcitonin

作　　者：刘深基[1] 陈松森[1] 狄旭[1] 熊安琪[1] 张友鸿[1] 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所,北京100005

出　　处：《基础医学与临床》2001年第1期24-27,共4页Basic and Clinical Medicine

摘　　要：为了化学裂解后重组多肽的分离纯化 ,用双引物定点突变的方法将表达质粒pTrx sCT Gly中硫氧还原蛋白 (Trx)与鲑鱼降钙素 (sCT)的融合基因的第 38位Met突变为Ala,并将突变后的基因克隆到pET30a中 ,然后转入大肠杆菌BL2 1,以IPTG诱导 ,使降钙素与硫氧还原蛋白融合表达 ,融合蛋白占菌体总蛋白的 5 6 %左右。并发现本原核表达体系存在“表达渗漏”现象 ,此现象为在菌体的对数生长期中 ,外源蛋白极少表达 ,随着培养时间的延长 ,外源蛋白逐步积累 ,达到 5 1%的高水平。该发现在基因工程生产重组蛋白工艺中具有应用价值。In order to purify the CNBr cleaved recombinant peptide easily, the methionine at position 38 of the thioredoxin and sCT Gly fusion protein was mutated into alanine by two primer site directed mutation method. Then the mutated gene was cloned into the expressing vector pET30a. The recombinant plasmid was transferred into E coli strand BL21(DE3). The expressing level of the recombinant fusion protein can reach 56% of the total bacteria protein with IPTG induction. In the mean time, we found the “expression leakage” phenomenon. The bacteria seldom express the recombinant protein in its logarithmic growth period. Only when the culture period is long enough, the recombinant protein can accumulate to 51% of the total bacteria protein. This kind of “expression leakage” phenomenon is very useful in large scale preparation of the recombinant protein.

关 键 词：鲑鱼降钙素 定点突变 高效表达 硫氧还原蛋白 

分 类 号：R346[医药卫生—基础医学]