Acton Thomas B, Xiao Rong, Anderson Stephen, Aramini James, Buchwald William A, Ciccosanti Colleen, Conover Ken, Everett John, Hamilton Keith, Huang Yuanpeng Janet, Janjua Haleema, Kornhaber Gregory, Lau Jessica, Lee Dong Yup, Liu Gaohua, Maglaqui Melissa, Ma Lichung, Mao Lei, Patel Dayaban, Rossi Paolo, Sahdev Seema, Shastry Ritu, Swapna G V T, Tang Yeufeng, Tong Saichiu, Wang Dongyan, Wang Huang, Zhao Li, Montelione Gaetano T
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA.
Methods Enzymol. 2011;493:21-60. doi: 10.1016/B978-0-12-381274-2.00002-9.
In this chapter, we concentrate on the production of high-quality protein samples for nuclear magnetic resonance (NMR) studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium and outline our high-throughput strategies for producing high-quality protein samples for NMR studies. Our strategy is based on the cloning, expression, and purification of 6×-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6×-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (>97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this chapter describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening, and structural genomics research.
在本章中，我们专注于为核磁共振（NMR）研究制备高质量的蛋白质样品。特别地，我们深入描述了NMR样品制备的最新进展以及它们与NMR硬件最新进展的协同应用。我们介绍了东北结构基因组学联盟的蛋白质制备平台，并概述了我们为NMR研究生产高质量蛋白质样品的高通量策略。我们的策略基于使用基于T7的大肠杆菌系统克隆、表达和纯化带有6×组氨酸标签的蛋白质，并在基本培养基中进行同位素富集。我们描述了96孔不依赖连接的克隆和分析表达系统、平行制备规模发酵以及高通量纯化方案。6×组氨酸亲和标签允许以平行高通量方式实施类似的两步纯化程序，该程序通常能产生足以用于NMR研究的纯度水平（>97%均一性）。利用这个平台，已将超过17500种不同目标蛋白质（或结构域）的蛋白质开放阅读框克隆为超过28000个构建体。其中近5000种蛋白质已被纯化至均一，数量达数十毫克（见总结统计数据，http://nesg.org/statistics.html），从而在蛋白质数据库中存入了超过950个新的蛋白质结构，包括400多个NMR结构。东北结构基因组学联盟的流程在生产原核和真核来源的蛋白质样品方面很有效。尽管本章描述了我们生产同位素富集蛋白质样品的整个流程，但重点是过去5年（美国国立综合医学科学研究所蛋白质结构计划的第二阶段）引入的主要更新。我们先进的自动化和/或平行克隆、表达、纯化及生物物理筛选技术适用于在大型独立实验室或由一小群合作研究人员用于结构生物学、功能蛋白质组学、配体筛选和结构基因组学研究。
