真核表达：结构蛋白质组学的进展
Eukaryotic expression: developments for structural proteomics.
作者信息
Aricescu A R, Assenberg R, Bill R M, Busso D, Chang V T, Davis S J, Dubrovsky A, Gustafsson L, Hedfalk K, Heinemann U, Jones I M, Ksiazek D, Lang C, Maskos K, Messerschmidt A, Macieira S, Peleg Y, Perrakis A, Poterszman A, Schneider G, Sixma T K, Sussman J L, Sutton G, Tarboureich N, Zeev-Ben-Mordehai T, Jones E Yvonne
机构信息
Division of Structural Biology and Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, Oxford, England.
出版信息
Acta Crystallogr D Biol Crystallogr. 2006 Oct;62(Pt 10):1114-24. doi: 10.1107/S0907444906029805. Epub 2006 Sep 19.
The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.
生产足够数量的蛋白质是确定结构的必要前奏，但对于许多病毒蛋白和人类蛋白而言，使用原核表达系统无法实现这一点。欧洲结构蛋白质组学（SPINE）联盟的多个团队已经开发并实施了用于真核系统中克隆、表达筛选和蛋白质生产的高通量（HTP）方法。研究聚焦于三个系统：酵母（巴斯德毕赤酵母和酿酒酵母）、杆状病毒感染的昆虫细胞以及哺乳动物细胞中的瞬时表达。描述了适用于高通量克隆的载体，并报告了它们在表达筛选和蛋白质生产流程中的使用结果。评估了共表达策略、硒代蛋氨酸标记（在所有三个真核系统中）以及糖基化控制（针对哺乳动物细胞中的分泌蛋白）。
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