人跨膜甾醇Δ8,Δ7-异构酶在毕赤酵母中的高水平异源表达。

High-level heterologous expression of the human transmembrane sterol Δ8,Δ7-isomerase in Pichia pastoris.

作者信息

Cai Hongmin, Yao Hebang, Li Tingting, Tang Yannan, Li Dianfan

机构信息

Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, National Center for Protein Science Shanghai, University of Chinese Academy of Sciences, Chinese Academy of Sciences, 333 Haike Road, Shanghai, 201210, China.

出版信息

Protein Expr Purif. 2019 Dec;164:105463. doi: 10.1016/j.pep.2019.105463. Epub 2019 Aug 2.

DOI:10.1016/j.pep.2019.105463

PMID:31381990

Abstract

Recombinant expression of human membrane proteins in large quantities remains a major challenge. Expression host is an important variable to screen for high-level production of membrane proteins. Using the green fluorescent protein (GFP) as a reporter, we screened the expression of a human multi-pass membrane protein called sterol Δ8-Δ7 isomerase in three different hosts: Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. The expression of the His-tagged isomerase was exceptionally high in P. pastoris, reaching ~200 mg L in standard flasks, and ~1,000 mg L in condensed culture that mimics fermentation. The heterogeneously expressed isomerase could be extracted fully with dodecyl maltoside, and the solubilized protein in the form of GFP fusion showed a sharp and symmetric peak on fluorescence-detection size exclusion chromatography. Our work provides a useful source for the purification of the recombinant isomerase.

摘要

大量重组表达人膜蛋白仍然是一项重大挑战。表达宿主是筛选膜蛋白高水平表达的一个重要变量。我们以绿色荧光蛋白（GFP）作为报告基因，在三种不同宿主中筛选了一种名为甾醇Δ8 - Δ7异构酶的人多跨膜蛋白的表达，这三种宿主分别是大肠杆菌、酿酒酵母和巴斯德毕赤酵母。His标签化的异构酶在巴斯德毕赤酵母中的表达量极高，在标准摇瓶中达到约200 mg/L，在模拟发酵的浓缩培养物中达到约1000 mg/L。异源表达的异构酶可用十二烷基麦芽糖苷完全提取，以GFP融合形式溶解的蛋白在荧光检测尺寸排阻色谱上显示出尖锐且对称的峰。我们的工作为重组异构酶的纯化提供了有用的来源。