Expression of SARS-coronavirus spike glycoprotein in Pichia pastoris.
Expression of SARS-coronavirus spike glycoprotein in Pichia pastoris.
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DOI：
10.1007/s11262-008-0292-3
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发表时间：
2009-03
期刊：
影响因子：
1.6
通讯作者：
Tsui SK
中科院分区：
文献类型：
--
作者：
Chuck CP;Wong CH;Chow LM;Fung KP;Waye MM;Tsui SK
为了建立一种快速、经济、高效表达SARS冠状病毒S蛋白的方法，本研究以SARS冠状病毒S蛋白为研究对象。在大肠杆菌BL 21和酵母KM 71 H中表达了6个S糖蛋白片段。经亲和层析纯化后，蛋白质的身份进行了确认，蛋白质印迹分析，N-末端测序和质谱。在E.大肠杆菌的可溶性低，并与GroEL结合。它们仍然形成可溶性聚集体，即使当GroEL被尿素去除。在毕赤酵母中表达的蛋白质是相对可溶的。RBD的最大收率达到46 mg/l，纯度大于95%。Pull-down分析显示ACE 2特异性地从细胞裂解物中捕获，表明RBD具有生物活性。然后对糖基化和去糖基化的RBD进行SEC，结果显示去糖基化的RBD再次形成可溶性聚集体。综上所述，S蛋白的纯的和生物活性的RBD可以在巴斯德毕赤酵母中表达，并且巴斯德毕赤酵母表达平台将是表达病毒蛋白，特别是介导组织嗜性和病毒进入的高度糖基化的表面蛋白的良好替代方案。
To establish a rapid and economical method for the expression of viral proteins in high yield and purity by Pichia pastoris, the S protein of the SARS-CoV was selected in this study. Six S glycoprotein fragments were expressed in Escherichia coli BL21 and yeast KM71H strains. After purification by affinity chromatography, the protein identities were confirmed by western blot analysis, N-terminal sequencing and mass spectrometry. The proteins expressed in E. coli were low in solubility and bound by GroEL. They still formed soluble aggregates even when the GroEL was removed by urea. The proteins expressed in P. pastoris were relatively soluble. The maximal yield of the RBD reached 46 mg/l with purity greater than 95%. Pull-down assay revealed that ACE2 was specifically captured from cell lysate, indicating that the RBD was biologically active. The glycosylated and deglycosylated RBD was then subjected to SEC and results showed that deglycosylated RBD formed soluble aggregates again. Taken together, pure and biological active RBD of the S protein could be expressed in P. pastoris, and the P. pastoris expression platform will be a good alternative for the expression of viral proteins, in particular, the highly glycosylated surface proteins that mediate the tissue tropism and viral entry.
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影响因子：
64.8
作者：
Li W;Moore MJ;Vasilieva N;Sui J;Wong SK;Berne MA;Somasundaran M;Sullivan JL;Luzuriaga K;Greenough TC;Choe H;Farzan M
通讯作者：
Farzan M
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1.6
作者：
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1.6
作者：
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通讯作者：
Forstner, M
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3.1
作者：
Han, XQ;Bartlam, M;Rao, ZH
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