The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium.
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.
Xiao
Rong
R
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.
Anderson
Stephen
S
Aramini
James
J
Belote
Rachel
R
Buchwald
William A
WA
Ciccosanti
Colleen
C
Conover
Ken
K
Everett
John K
JK
Hamilton
Keith
K
Huang
Yuanpeng Janet
YJ
Janjua
Haleema
H
Jiang
Mei
M
Kornhaber
Gregory J
GJ
Lee
Dong Yup
DY
Locke
Jessica Y
JY
Ma
Li-Chung
LC
Maglaqui
Melissa
M
Mao
Lei
L
Mitra
Saheli
S
Patel
Dayaban
D
Rossi
Paolo
P
Sahdev
Seema
S
Sharma
Seema
S
Shastry
Ritu
R
Swapna
G V T
GV
Tong
Saichu N
SN
Wang
Dongyan
D
Wang
Huang
H
Zhao
Li
L
Montelione
Gaetano T
GT
Acton
Thomas B
TB
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
D011506:Proteins; D011994:Recombinant Proteins