title: The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium.
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.
authors.affiliation: 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, NJ 08854, USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Xiao
authors.forename: Stephen
authors.initials: S
authors.lastname: Anderson
authors.forename: James
authors.initials: J
authors.lastname: Aramini
authors.forename: Rachel
authors.initials: R
authors.lastname: Belote
authors.forename: William A
authors.initials: WA
authors.lastname: Buchwald
authors.forename: Colleen
authors.initials: C
authors.lastname: Ciccosanti
authors.forename: Ken
authors.initials: K
authors.lastname: Conover
authors.forename: John K
authors.initials: JK
authors.lastname: Everett
authors.forename: Keith
authors.initials: K
authors.lastname: Hamilton
authors.forename: Yuanpeng Janet
authors.initials: YJ
authors.lastname: Huang
authors.forename: Haleema
authors.initials: H
authors.lastname: Janjua
authors.forename: Mei
authors.initials: M
authors.lastname: Jiang
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kornhaber
authors.forename: Dong Yup
authors.initials: DY
authors.lastname: Lee
authors.forename: Jessica Y
authors.initials: JY
authors.lastname: Locke
authors.forename: Li-Chung
authors.initials: LC
authors.lastname: Ma
authors.forename: Melissa
authors.initials: M
authors.lastname: Maglaqui
authors.forename: Lei
authors.initials: L
authors.lastname: Mao
authors.forename: Saheli
authors.initials: S
authors.lastname: Mitra
authors.forename: Dayaban
authors.initials: D
authors.lastname: Patel
authors.forename: Paolo
authors.initials: P
authors.lastname: Rossi
authors.forename: Seema
authors.initials: S
authors.lastname: Sahdev
authors.forename: Seema
authors.initials: S
authors.lastname: Sharma
authors.forename: Ritu
authors.initials: R
authors.lastname: Shastry
authors.forename: G V T
authors.initials: GV
authors.lastname: Swapna
authors.forename: Saichu N
authors.initials: SN
authors.lastname: Tong
authors.forename: Dongyan
authors.initials: D
authors.lastname: Wang
authors.forename: Huang
authors.initials: H
authors.lastname: Wang
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Acton
mesh_terms: D003001:Cloning, Molecular; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D023281:Genomics / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011506:Proteins; D011994:Recombinant Proteins