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.
出处
《生物工程学报》
CAS
CSCD
北大核心
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~~
关键词
甜蛋白
毕氏酵母
表达
活性检测
des-pGlu1-Brazzein
Pichia pastoris
expression
biological activity
