Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium

Thomas B. Acton, Kristin C. Gunsalus, Rong Xiao, Li Chung Ma, James Aramini, Michael C. Baran, Yi Wen Chiang, Teresa Climent, Bonnie Cooper, Natalia G. Denissova, Shawn M. Douglas, John K. Everett, Chi Kent Ho, Daphne Macapagal, Paranji K. Rajan, Ritu Shastry, Liang Yu Shih, G. V T Swapna, Michael Wilson, Margaret Wu & 4 others Mark Gerstein, Masayori Inouye, John F. Hunt, Gaetano T. Montelione

Research output: Contribution to journalArticle

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, http://www.nmr.cabm. rutgers.edu/bioinformatics/ZebaView/). 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.

Original languageEnglish (US)
Pages (from-to)210-243
Number of pages34
JournalMethods in Enzymology
Volume394
DOIs
StatePublished - 2005

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Production platforms
Cloning
Robotics
Genomics
Organism Cloning
Proteins
Purification
Bioinformatics
Chromatography
Computational Biology
Proteomics
Escherichia coli
Gel Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium. / Acton, Thomas B.; Gunsalus, Kristin C.; Xiao, Rong; Ma, Li Chung; Aramini, James; Baran, Michael C.; Chiang, Yi Wen; Climent, Teresa; Cooper, Bonnie; Denissova, Natalia G.; Douglas, Shawn M.; Everett, John K.; Ho, Chi Kent; Macapagal, Daphne; Rajan, Paranji K.; Shastry, Ritu; Shih, Liang Yu; Swapna, G. V T; Wilson, Michael; Wu, Margaret; Gerstein, Mark; Inouye, Masayori; Hunt, John F.; Montelione, Gaetano T.

In: Methods in Enzymology, Vol. 394, 2005, p. 210-243.

Research output: Contribution to journalArticle

Acton, TB, Gunsalus, KC, Xiao, R, Ma, LC, Aramini, J, Baran, MC, Chiang, YW, Climent, T, Cooper, B, Denissova, NG, Douglas, SM, Everett, JK, Ho, CK, Macapagal, D, Rajan, PK, Shastry, R, Shih, LY, Swapna, GVT, Wilson, M, Wu, M, Gerstein, M, Inouye, M, Hunt, JF & Montelione, GT 2005, 'Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium', Methods in Enzymology, vol. 394, pp. 210-243. https://doi.org/10.1016/S0076-6879(05)94008-1
Acton, Thomas B. ; Gunsalus, Kristin C. ; Xiao, Rong ; Ma, Li Chung ; Aramini, James ; Baran, Michael C. ; Chiang, Yi Wen ; Climent, Teresa ; Cooper, Bonnie ; Denissova, Natalia G. ; Douglas, Shawn M. ; Everett, John K. ; Ho, Chi Kent ; Macapagal, Daphne ; Rajan, Paranji K. ; Shastry, Ritu ; Shih, Liang Yu ; Swapna, G. V T ; Wilson, Michael ; Wu, Margaret ; Gerstein, Mark ; Inouye, Masayori ; Hunt, John F. ; Montelione, Gaetano T. / Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium. In: Methods in Enzymology. 2005 ; Vol. 394. pp. 210-243.
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AU - Acton, Thomas B.

AU - Gunsalus, Kristin C.

AU - Xiao, Rong

AU - Ma, Li Chung

AU - Aramini, James

AU - Baran, Michael C.

AU - Chiang, Yi Wen

AU - Climent, Teresa

AU - Cooper, Bonnie

AU - Denissova, Natalia G.

AU - Douglas, Shawn M.

AU - Everett, John K.

AU - Ho, Chi Kent

AU - Macapagal, Daphne

AU - Rajan, Paranji K.

AU - Shastry, Ritu

AU - Shih, Liang Yu

AU - Swapna, G. V T

AU - Wilson, Michael

AU - Wu, Margaret

AU - Gerstein, Mark

AU - Inouye, Masayori

AU - Hunt, John F.

AU - Montelione, Gaetano T.

PY - 2005

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