厦门大学学报（自然科学版）2012，Vol.51Issue(1)：101-106,6.
人胰岛素原密码子的优化及其在毕赤酵母中的表达
Codon Optimization of Human Proinsulin and Expression in Pichia pastoris
摘要
Abstract
According to the preferential codon of Pichia pastoris ,the cDNA of human proinsulin (HPI) with the full length of 200 bp was designed and synthesized. It contains the singnal peptide (EEAEAEAEPK) in N terminal in order to increase the protein expression level. The HPI was cloned into shuttle vector pPIC9K to construct recombinant expression plasmid pPIC9K-HPI. It was then transformed into the Pichia pastoris GS115 by electrotransformation. A transformant with a high copy number of HPI gene was obtained by G418 concentration gradient screening, and then started to express HPI protein after induction with methanol in shake flasks for 120 h. The HPI was digested by trypsin at 4 ℃ for 12 h after being purified by metal chelate chromatography. The HPI reached 35. 4 mg/L,and showed the biological activity detected by ELISA kit.关键词
胰岛素原/基因改造/高拷贝/毕赤酵母Key words
proinsulin/gene modified/high-copy/Pichia pastoris分类
生物科学引用本文复制引用
史莹飞,敬科举,凌雪萍,卢英华..人胰岛素原密码子的优化及其在毕赤酵母中的表达[J].厦门大学学报（自然科学版）,2012,51(1):101-106,6.基金项目
国家自然科学基金项目(3107488) （3107488）
福建省自然科学基金项目(2011J0l058) （2011J0l058）
