植物甜蛋白des-pGlu1-Brazzein的密码子优化及其在毕氏酵母中的表达  

Optimization of plant des-pGlu1-Brazzein gene according to yeasty biased codons and its expression in Pichia pastoris

作　　者：李春丽[1] 韩露[1] 郑振宇[1] 赵卫东[1] 

机构地区：[1]河南农业大学牧医工程学院,郑州450002

出　　处：《生物工程学报》2011年第8期1158-1163,共6页Chinese Journal of Biotechnology

基　　金：河南省教育厅基础研究项目(No.2004922043);河南农业大学博士基金项目资助~~

摘　　要：根据甜蛋白des-pGlu1-Brazzein的成熟区氨基酸序列,按照酵母密码子的偏好优化其编码序列,合成了4对末端具粘合位点的寡聚核苷酸序列,经连接、PCR扩增后得到了179 bp的des-pGlu1-Brazzein编码序列,插入到pPIC9K载体中,构建了重组表达载体pPIC9K-Bra。经酶切、电击转化到毕赤酵母菌GS115中。酵母工程菌株经筛选鉴定后诱导表达,目的蛋白的表达量约占上清总蛋白的51.6%,分离纯化后的des-pGlu1-Brazzein具有一定的甜度。According to the amino acid sequence of des-pGlu1-Brazzein,4 pairs of oligonucleotide with cosmic site were synthesized by using yeasty biased codons.After linkage and PCR,the 179 bp code area of des-pGlu1-Brazzein was obtained and inserted into pPIC9K,which resulted in the recombinant expression vector pPIC9K-Bra.By digestion with Sal I,the lined pPIC9K-Bra was transformed into Pichia pastoris GS115 by electric shock.The results of expression indicted that the secreted target protein accounted for 51.6% of total protein in the supernatant and showed biological activity after purification.

关 键 词：甜蛋白 毕氏酵母 表达 活性检测 

分 类 号：Q946[生物学—植物学]