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 Rossi; Seema Sahdev; Ritu Shastry; G.V.T Swapna; Yeufeng Tang; Saichiu Tong; Dongyan Wang; Huang Wang; Li Zhao; Gaetano T Montelione

doi:10.1016/B978-0-12-381274-2.00002-9

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Preparation of Protein Samples for NMR Structure, Function, and Small-Molecule Screening Studies

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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, …

Methods in Enzymology, pp.21-60

Elsevier Science & Technology

2011

DOI: https://doi.org/10.1016/B978-0-12-381274-2.00002-9

PMCID: PMC4110644

PMID: 21371586

Abstract

Parallel protein purification

High-throughput protein production

Condensed single protein production

T7 Escherichia coli expression system

Laboratory information management system

Structural genomics

Ligation-independent cloning

Total gene synthesis

Protein structure initiative

NMR microprobe screening

Wheat germ cell free

NMR

Multiple displacement amplification

Construct optimization

HDX-MS

Disorder prediction

6×-His tag

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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Title: Preparation of Protein Samples for NMR Structure, Function, and Small-Molecule Screening Studies
Creators: Thomas B Acton - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Rong Xiao - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Stephen Anderson - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
James Aramini - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
William A Buchwald - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Colleen Ciccosanti - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Ken Conover - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
John Everett - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Keith Hamilton - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Yuanpeng Janet Huang - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Haleema Janjua - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Gregory Kornhaber - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Jessica Lau - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Dong Yup Lee - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Gaohua Liu - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Melissa Maglaqui - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Lichung Ma - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Lei Mao - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Dayaban Patel - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Paolo Rossi - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Seema Sahdev - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Ritu Shastry - Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey, USA
G.V.T Swapna - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Yeufeng Tang - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Saichiu Tong - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Dongyan Wang - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Huang Wang - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Li Zhao - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Gaetano T Montelione - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers University, Piscataway, New Jersey, USA
Publication Details: Methods in Enzymology, pp.21-60
Date published: 2011
Publisher: Elsevier Science & Technology
Academic Unit: Biochemistry and Molecular Biology (RWJMS); Center for Advanced Biotechnology and Medicine; Molecular Biology and Biochemistry (SAS); Medicine - Hematology (RWJMS)
Language: English
Resource Type: Book chapter
Identifiers: 991031653966704646