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

By R. Xiao, S. Anderson, J. Aramini, R. Belote, W. Buchwald, C. Ciccosanti, K. Conover, J. Everett, K. Hamilton, Yuanpeng Janet Huang and 21 more. 2010

124 citations recorded by the source corpus.

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.

Subfields: Materials Chemistry, Molecular Biology, Spectroscopy

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In Materials Chemistry

  1. Torsten Schwede
  2. David Baker
  3. Sergey Ovchinnikov
  4. Yang Zhang
  5. Frank DiMaio
  6. Lorenza Bordoli
  7. Gaetano T. Montelione
  8. Ivan V. Anishchenko

In Molecular Biology

  1. Torsten Schwede
  2. Yang Zhang
  3. David Baker
  4. Sergey Ovchinnikov
  5. Frank DiMaio
  6. Lorenza Bordoli
  7. Ivan V. Anishchenko
  8. Andriy Kryshtafovych

In Spectroscopy

  1. David Baker
  2. Sergey Ovchinnikov
  3. Frank DiMaio
  4. Gaetano T. Montelione
  5. Christoffer H Norn
  6. Lorenza Bordoli
  7. Torsten Schwede
  8. Ivan V. Anishchenko

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