Euprosthenops australis牵引丝蛋白的原核表达  

Prokaryotic Expression of Euprosthenops australis Dragline Silk Protein

作　　者：张秀丽[1] 陈格飞[1] 林瑛[1] 李佳[1] 孟清[1] 

机构地区：[1]东华大学生物科学与技术研究所,中国上海201620

出　　处：《生命科学研究》2013年第1期14-19,62,共7页Life Science Research

基　　金：国家高技术研究发展计划(863计划)资助项目(2006AA03Z451);国家自然科学基金资助项目(31070698);教育部博士点基金资助项目(20120075110007)

摘　　要：参照GenBank收录的牵引丝蛋白编码序列,经过密码子优化,人工合成3段cDNA序列:NT、CT和4Rep,由Ⅰ型限制性内切酶BsaⅠ和BspMⅠ介导无缝拼接,分别构建4RepCT、NT4RepCT、NT8RepCT和NT12RepCT重组模块.通过改造表达载体和优化培养温度,成功提高了4RepCT的表达量(30 mg/L).另外还首次成功表达NT4RepCT和NT8RepCT重组蛋白模块,表达量分别为21 mg/L和8.5 mg/L.4RepCT表达量的提高及NT4RepCT和NT8RepCT的成功表达证实了融合标签TRX有利于提高蛛丝蛋白的表达水平,为E.australis全长牵引丝蛋白的表达奠定了基础.According to major ampullate spidroin gene sequences published by GenBank, three cDNA se- quences, NT, CT and 4Rep, were chemically synthesized after codon optimization. Mediated by Bsa I and BspM I restriction enzyme, 4RepCT, NT4RepCT, NTSRepCT and NT12RepCT modulus were constructed without inducing extra amino acids. With modifying expression vector and optimizing incubation temperature, the expression level of 4RepCT was successfully improved to 30 mg/L. In addition, recombinant NT4RepCT and NT8RepCT proteins were also firstly produced with expression levels of 21 mg/L and 8.5 mg/L respectively. The improved expression level of 4RepCT together with NT4RepCT and NT8RepCT successful expression indicates thioredoxin is a useful fusion tag for spider silk spidroin expression, and would be helpful for fully exploring E.australis dragline silk proteins.

关 键 词：4Rep 基因克隆 融合蛋白 原核表达 

分 类 号：Q785[生物学—分子生物学]