植物甜蛋白des-pGlu1-Brazzein的密码子优化及其在毕.PDFVIP

生 物 工 程 学 报 Chin J Biotech 2011, August 25; 27(8): 1158−1163 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ ©2011 CJB, All rights reserved. 农业生物技术 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 李春丽，韩露，郑振宇，赵卫东 河南农业大学牧医工程学院，郑州 450002 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。 关键词: 甜蛋白，毕氏酵母，表达，活性检测 Optimization of plant des-pGlu1-Brazzein gene according to yeasty biased codons and its expression in Pichia pastoris Chunli Li, Lu Han, Zhenyu Zheng, and Weidong Zhao College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China 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. Keywords: des-pGlu1-Brazzein, Pichia pastoris, expression, biological activity 甜蛋白是从热带植物中提取的一类蛋白，其甜 用。目前发现的甜蛋白有7 种，其中Brazzein ( 巴西 度高，热量低；甜味纯正，口感好；食用该类蛋白 甜) 是Ming 和Hellekant 从西非植物Pentadiplandra 不会使体内血糖升高，不会引起口腔蛀牙，无毒副作 brazzeana Baillion 的果实中分离得到的一种甜度极 Received: Dec