Preparation of protein samples for NMR structure, function, and small-molecule screening studies

Thomas B. Acton, Rong Xiao, Stephen Anderson, James Aramini, William A. Buchwald, Colleen Ciccosanti, Ken Conover, John Everett, Keith Hamilton, Yuanpeng Janet Huang, Haleema Janjua, Gregory Kornhaber, Jessica Lau, Dong Yup Lee, Gaohua Liu, Melissa Maglaqui, Lichung Ma, Lei Mao, Dayaban Patel, Paolo RossiSeema Sahdev, Ritu Shastry, G. V.T. Swapna, Yeufeng Tang, Saichiu Tong, Dongyan Wang, Huang Wang, Li Zhao, Gaetano T. Montelione

Research output: Chapter in Book/Report/Conference proceedingChapter

66 Scopus citations

Abstract

Abstract 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.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages21-60
Number of pages40
DOIs
StatePublished - 2011

Publication series

NameMethods in Enzymology
Volume493
ISSN (Print)0076-6879

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