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

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。

Abstract:

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.