TY - CHAP
T1 - Preparation of protein samples for NMR structure, function, and small-molecule screening studies
AU - Acton, Thomas B.
AU - Xiao, Rong
AU - Anderson, Stephen
AU - Aramini, James
AU - Buchwald, William A.
AU - Ciccosanti, Colleen
AU - Conover, Ken
AU - Everett, John
AU - Hamilton, Keith
AU - Huang, Yuanpeng Janet
AU - Janjua, Haleema
AU - Kornhaber, Gregory
AU - Lau, Jessica
AU - Lee, Dong Yup
AU - Liu, Gaohua
AU - Maglaqui, Melissa
AU - Ma, Lichung
AU - Mao, Lei
AU - Patel, Dayaban
AU - Rossi, Paolo
AU - Sahdev, Seema
AU - Shastry, Ritu
AU - Swapna, G. V.T.
AU - Tang, Yeufeng
AU - Tong, Saichiu
AU - Wang, Dongyan
AU - Wang, Huang
AU - Zhao, Li
AU - Montelione, Gaetano T.
PY - 2011
Y1 - 2011
N2 - 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.
AB - 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.
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U2 - 10.1016/B978-0-12-381274-2.00002-9
DO - 10.1016/B978-0-12-381274-2.00002-9
M3 - Chapter
C2 - 21371586
AN - SCOPUS:79952411326
T3 - Methods in Enzymology
SP - 21
EP - 60
BT - Methods in Enzymology
PB - Academic Press Inc.
ER -