Modification of SARS-CoV S1 gene render expression in Pichia pastoris
Modification of SARS-CoV S1 gene render expression in Pichia pastoris
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DOI：
10.1007/s11262-006-0072-x
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发表时间：
2006-12-01
期刊：
影响因子：
1.6
通讯作者：
Guo, Aizhen
中科院分区：
文献类型：
--
作者：
Lu, Haisong;Yang, Guoliang;Guo, Aizhen
采用Over-Lap PCR方法，用多对引物扩增出含有受体结合区的S1基因片段。基于相同的氨基酸序列，在A + T丰富区修饰天然S1基因; n.t.777-1683、n.t.1041-1050、n.t.1236-1248、n.t.1317-1335、n.t.1590-1605。将改造后的基因克隆到酵母表达载体pPIC 9 K中。将重组质粒pPIC 9 K-S1转化到毕赤酵母GS115中，用甲醇诱导表达。SDS-PAGE证实重组SI在诱导的GS 115上清中分泌。蛋白质产量达到69 mg/l。ELISA和Westernblot检测表明，S1蛋白与SARS患者恢复期血清有特异性反应。配体印迹实验表明，重组S1蛋白能与SARS-CoV的受体ACE 2发生反应。表达的S1分子量约为70 kDa，高于预期的30 kDa。PNGase F去糖基化产生30 kDa的蛋白条带。综上所述，S1基因的修饰使得S1在巴斯德毕赤酵母GS 115中得到高水平表达，并且该蛋白以高糖基化的生物活性形式分泌。
S1 gene fragment containing receptor-binding region was amplified by several sets of primers using Over-Lap PCR. The native S1 gene was modified at A + T abundant regions; n.t.777-1683, n.t.1041-1050, n.t.1236-1248, n.t.1317-1335, n.t.1590-1605; based on the same amino acid sequences. The modified gene was cloned into a yeast expression vector pPIC9K. The resultant plasmid pPIC9K- S1 was transformed into Pichia pastoris GS 115 and the protein expression was induced with methanol. SDS-PAGE confirmed that the recombinant SI was secreted in the supernatant of induced GS 115. The protein yield reached 69 mg/l. ELISA and Western blot demonstrated that the S1 could react with the convalescent sera of people infected by SARS-CoV. Furthermore, ligand blot assay showed that the recombinant S1 could react with ACE2, the receptor of SARS-CoV. The molecular mass of expressed S1 was about 70 kDa, which was higher than that of the 30 kDa expected. PNGase F deglycosylation resulted in a protein band of 30 kDa. In conclusion, the S1 gene modification rendered the high-level expression of S1 in P. pastoris GS 115 and the protein was secreted as a biologically active form which was hyperglycosylated.