Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium.
In this chapter 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 using Escherichia coli host vectors. The platform is centered on 6X-His affinity-tagged protein constructs, allowing for a similar purification procedure for most targets, and the implementation of high-throughput parallel methods. In most cases, these affinity-purified proteins are sufficiently homogeneous that a single subsequent gel filtration chromatography step is adequate to produce protein preparations that are greater than 98% pure. Using this platform, over 1000 different proteins have been cloned, expressed, and purified in tens of milligram quantities over the last 36-month period (see Summary Statistics for All Targets, ). Our experience using a hierarchical multiplex expression and purification strategy, also described in this chapter, has allowed us to achieve success in producing not only protein samples but also many three-dimensional structures. As of December 2004, the NESG Consortium has deposited over 145 new protein structures to the Protein Data Bank (PDB); about two-thirds of these protein samples were produced by the NESG Protein Production Facility described here. The methods described here have proven effective in producing quality samples of both eukaryotic and prokaryotic proteins. These improved robotic and?or parallel cloning, expression, protein production, and biophysical screening technologies will be of broad value to the structural biology, functional proteomics, and structural genomics communities.
Acton
Thomas B
TB
Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854, USA.
Gunsalus
Kristin C
KC
Xiao
Rong
R
Ma
Li Chung
LC
Aramini
James
J
Baran
Michael C
MC
Chiang
Yi-Wen
YW
Climent
Teresa
T
Cooper
Bonnie
B
Denissova
Natalia G
NG
Douglas
Shawn M
SM
Everett
John K
JK
Ho
Chi Kent
CK
Macapagal
Daphne
D
Rajan
Paranji K
PK
Shastry
Ritu
R
Shih
Liang-Yu
LY
Swapna
G V T
GV
Wilson
Michael
M
Wu
Margaret
M
Gerstein
Mark
M
Inouye
Masayori
M
Hunt
John F
JF
Montelione
Gaetano T
GT
D002850:Chromatography, Gel; D003001:Cloning, Molecular / Q000379:methods*; D019295:Computational Biology / Q000379:methods; D009682:Magnetic Resonance Spectroscopy; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification; D012371:Robotics / Q000379:methods*; D012984:Software
D011506:Proteins