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 PatelPaolo 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

Research output: Contribution to journalArticlepeer-review

96 Scopus citations

Abstract

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 10. years, more than 16,000 of these expressed protein, and more than 4400 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 5. 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.

Original languageEnglish (US)
Pages (from-to)21-33
Number of pages13
JournalJournal of Structural Biology
Volume172
Issue number1
DOIs
StatePublished - Oct 2010

ASJC Scopus subject areas

  • Structural Biology

Keywords

  • 6X-His tag
  • Construct optimization
  • Disorder prediction
  • HDX-MS
  • High-throughput protein production
  • Laboratory Information Management System
  • Ligation-independent cloning
  • Multiple Displacement Amplification
  • NMR
  • NMR microprobe screening
  • Parallel protein purification
  • Protein Structure Initiative
  • Structural genomics
  • T7 Escherichia coli expression system
  • Wheat germ cell-free
  • X-ray crystallography

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