The High-Throughput Protein Sample Production Platform of the Northeast Structural Genomics Consortium

Rong Xiao; Stephen Anderson; James Aramini; Rachel Belote; William A Buchwald; Colleen Ciccosanti; Ken Conover; John K Everett; Keith Hamilton; Yuanpeng Janet Huang; Haleema Janjua; Mei Jiang; Gregory J Kornhaber; Dong Yup Lee; Jessica Y Locke; Li-Chung Ma; Melissa Maglaqui; Lei Mao; Saheli Mitra; Dayaban Patel; Paolo Rossi; Seema Sahdev; Seema Sharma; Ritu Shastry; G.V.T Swapna; Saichu N Tong; Dongyan Wang; Huang Wang; Li Zhao; Gaetano T Montelione; Thomas B Acton

doi:10.1016/j.jsb.2010.07.011

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The High-Throughput Protein Sample Production Platform of the Northeast Structural Genomics Consortium

Journal article

Open access

Peer reviewed

The High-Throughput Protein Sample Production Platform of the Northeast Structural Genomics Consortium

Rong Xiao, Stephen Anderson, James Aramini, Rachel Belote, William A Buchwald, Colleen Ciccosanti, Ken Conover, John K Everett, Keith Hamilton, Yuanpeng Janet Huang, …

Journal of structural biology, Vol.172(1), pp.21-33

10/2010

DOI: https://doi.org/10.1016/j.jsb.2010.07.011

PMCID: PMC4110633

PMID: 20688167

Abstract

Parallel protein purification

Laboratory Information Management System

Protein Structure Initiative

6X-His tag

Wheat Germ Cell Free

NMR microprobe screening

X-ray crystallography

NMR

Structural Genomics

High throughput protein production

Construct optimization

HDX-MS

Disorder prediction

Ligation independent cloning

Multiple Displacement Amplification

T7 Escherichia Coli expression system

We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (> 97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as > 26,000 constructs. Over the past nine years, more than 16,000 of these expressed protein, and more than 4,400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html ). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli . This paper summarizes some of the updates made to the protein production pipeline in the last five years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities.

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Title: The High-Throughput Protein Sample Production Platform of the Northeast Structural Genomics Consortium
Creators: Rong Xiao - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Stephen Anderson - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
James Aramini - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Rachel Belote - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
William A Buchwald - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Colleen Ciccosanti - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Ken Conover - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
John K Everett - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Keith Hamilton - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Yuanpeng Janet Huang - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Haleema Janjua - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Mei Jiang - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Gregory J Kornhaber - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Dong Yup Lee - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Jessica Y Locke - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Li-Chung Ma - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Melissa Maglaqui - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Lei Mao - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Saheli Mitra - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Dayaban Patel - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Paolo Rossi - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Seema Sahdev - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Seema Sharma - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Ritu Shastry - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
G.V.T Swapna - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Saichu N Tong - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Dongyan Wang - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Huang Wang - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Li Zhao - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Gaetano T Montelione - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Thomas B Acton - Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854
Publication Details: Journal of structural biology, Vol.172(1), pp.21-33
Date published: 10/2010
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: Journal article
Identifiers: 991031653888804646