人胰岛素原密码子的优化及其在毕赤酵母中的表达

依据毕赤酵母偏好密码子,设计合成长效人胰岛素原( human proinsulin,HPI)基因序列,所合成的HPI基因全长为200 bp,并在其N端添加信号肽(EEAEAEAEPK)以提高目的蛋白表达.将改造后基因克隆到pPIC9K载体中,构建分泌表达型载体pPIC9K-HPI,转化到毕赤酵母(Pichia pastoris)GS115中.用遗传霉素G418梯度筛选获得高拷贝菌株,在甲醇诱导下表达分子质量为7.87 ku的HPI,利用金属螯合层析纯化蛋白质,胰蛋白酶酶切后利用人胰岛素试剂盒测得生物活性,HPI摇瓶最高产量可达35.4mg/L.该研究为获得大量长效胰岛素基因工程产品奠定研究基础.

Codon Optimization of Human Proinsulin and Expression in Pichia pastoris

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.