title: Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium.
abstract: 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.
authors.affiliation: Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854, USA.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Acton
authors.forename: Kristin C
authors.initials: KC
authors.lastname: Gunsalus
authors.forename: Rong
authors.initials: R
authors.lastname: Xiao
authors.forename: Li Chung
authors.initials: LC
authors.lastname: Ma
authors.forename: James
authors.initials: J
authors.lastname: Aramini
authors.forename: Michael C
authors.initials: MC
authors.lastname: Baran
authors.forename: Yi-Wen
authors.initials: YW
authors.lastname: Chiang
authors.forename: Teresa
authors.initials: T
authors.lastname: Climent
authors.forename: Bonnie
authors.initials: B
authors.lastname: Cooper
authors.forename: Natalia G
authors.initials: NG
authors.lastname: Denissova
authors.forename: Shawn M
authors.initials: SM
authors.lastname: Douglas
authors.forename: John K
authors.initials: JK
authors.lastname: Everett
authors.forename: Chi Kent
authors.initials: CK
authors.lastname: Ho
authors.forename: Daphne
authors.initials: D
authors.lastname: Macapagal
authors.forename: Paranji K
authors.initials: PK
authors.lastname: Rajan
authors.forename: Ritu
authors.initials: R
authors.lastname: Shastry
authors.forename: Liang-Yu
authors.initials: LY
authors.lastname: Shih
authors.forename: G V T
authors.initials: GV
authors.lastname: Swapna
authors.forename: Michael
authors.initials: M
authors.lastname: Wilson
authors.forename: Margaret
authors.initials: M
authors.lastname: Wu
authors.forename: Mark
authors.initials: M
authors.lastname: Gerstein
authors.forename: Masayori
authors.initials: M
authors.lastname: Inouye
authors.forename: John F
authors.initials: JF
authors.lastname: Hunt
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
mesh_terms: 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
chemical_list: D011506:Proteins