The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium

Rong Xiao; Stephen Anderson; James Aramini; Rachel Belote; William A. Buchwald; Colleen Ciccosanti; Ken Conover; John K. Everett; Keith Hamilton; Yuanpeng Janet Huang; Haleema Janjua; Mei Jiang; Gregory J. Kornhaber; Dong Yup Lee; Jessica Y. Locke; Li Chung Ma; Melissa Maglaqui; Lei Mao; Saheli Mitra; Dayaban Patel; Paolo Rossi; Seema Sahdev; Seema Sharma; Ritu Shastry; G. V.T. Swapna; Saichu N. Tong; Dongyan Wang; Huang Wang; Li Zhao; Gaetano T. Montelione; Thomas B. Acton

doi:https://doi.org/10.1016/j.jsb.2010.07.011

The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium

Rong Xiao, Stephen Anderson, James Aramini, Rachel Belote, William A. Buchwald, Colleen Ciccosanti, Ken Conover, John K. Everett, Keith Hamilton, Yuanpeng Janet Huang, Haleema Janjua, Mei Jiang, Gregory J. Kornhaber, Dong Yup Lee, Jessica Y. Locke, Li Chung Ma, Melissa Maglaqui, Lei Mao, Saheli Mitra, Dayaban PatelPaolo Rossi, Seema Sahdev, Seema Sharma, Ritu Shastry, G. V.T. Swapna, Saichu N. Tong, Dongyan Wang, Huang Wang, Li Zhao, Gaetano T. Montelione, Thomas B. Acton

Rutgers, The State University

Research output: Contribution to journal › Article › peer-review

96 Scopus citations

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.

Original language	English (US)
Pages (from-to)	21-33
Number of pages	13
Journal	Journal of Structural Biology
Volume	172
Issue number	1
DOIs	https://doi.org/10.1016/j.jsb.2010.07.011
State	Published - Oct 2010

ASJC Scopus subject areas

Structural Biology

Keywords

6X-His tag
Construct optimization
Disorder prediction
HDX-MS
High-throughput protein production
Laboratory Information Management System
Ligation-independent cloning
Multiple Displacement Amplification
NMR
NMR microprobe screening
Parallel protein purification
Protein Structure Initiative
Structural genomics
T7 Escherichia coli expression system
Wheat germ cell-free
X-ray crystallography

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https://doi.org/10.1016/j.jsb.2010.07.011

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Xiao, R., Anderson, S., Aramini, J., Belote, R., Buchwald, W. A., Ciccosanti, C., Conover, K., Everett, J. K., Hamilton, K., Huang, Y. J., Janjua, H., Jiang, M., Kornhaber, G. J., Lee, D. Y., Locke, J. Y., Ma, L. C., Maglaqui, M., Mao, L., Mitra, S., ... Acton, T. B. (2010). The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium. Journal of Structural Biology, 172(1), 21-33. https://doi.org/10.1016/j.jsb.2010.07.011

Xiao, Rong ; Anderson, Stephen ; Aramini, James ; Belote, Rachel ; Buchwald, William A. ; Ciccosanti, Colleen ; Conover, Ken ; Everett, John K. ; Hamilton, Keith ; Huang, Yuanpeng Janet ; Janjua, Haleema ; Jiang, Mei ; Kornhaber, Gregory J. ; Lee, Dong Yup ; Locke, Jessica Y. ; Ma, Li Chung ; Maglaqui, Melissa ; Mao, Lei ; Mitra, Saheli ; Patel, Dayaban ; Rossi, Paolo ; Sahdev, Seema ; Sharma, Seema ; Shastry, Ritu ; Swapna, G. V.T. ; Tong, Saichu N. ; Wang, Dongyan ; Wang, Huang ; Zhao, Li ; Montelione, Gaetano T. ; Acton, Thomas B. / The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium. In: Journal of Structural Biology. 2010 ; Vol. 172, No. 1. pp. 21-33.

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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.",

keywords = "6X-His tag, Construct optimization, Disorder prediction, HDX-MS, High-throughput protein production, Laboratory Information Management System, Ligation-independent cloning, Multiple Displacement Amplification, NMR, NMR microprobe screening, Parallel protein purification, Protein Structure Initiative, Structural genomics, T7 Escherichia coli expression system, Wheat germ cell-free, X-ray crystallography",

author = "Rong Xiao and Stephen Anderson and James Aramini and Rachel Belote and Buchwald, {William A.} and Colleen Ciccosanti and Ken Conover and Everett, {John K.} and Keith Hamilton and Huang, {Yuanpeng Janet} and Haleema Janjua and Mei Jiang and Kornhaber, {Gregory J.} and Lee, {Dong Yup} and Locke, {Jessica Y.} and Ma, {Li Chung} and Melissa Maglaqui and Lei Mao and Saheli Mitra and Dayaban Patel and Paolo Rossi and Seema Sahdev and Seema Sharma and Ritu Shastry and Swapna, {G. V.T.} and Tong, {Saichu N.} and Dongyan Wang and Huang Wang and Li Zhao and Montelione, {Gaetano T.} and Acton, {Thomas B.}",

note = "Funding Information: We thank Profs. C. Arrowsmith, J. Hunt, M. Gerstein, M. Inouye, J. Marcotrigiano, and L. Tong, along with all the members of the NESG Consortium, for valuable advice in the development of the NESG Protein Sample Production Platform. This work was supported by a grant from the National Institute of General Medical Sciences Protein Structure Initiative U54-GM074958 (to G.T.M.).",

year = "2010",

month = oct,

doi = "https://doi.org/10.1016/j.jsb.2010.07.011",

language = "English (US)",

volume = "172",

pages = "21--33",

journal = "Journal of Structural Biology",

issn = "1047-8477",

publisher = "Academic Press Inc.",

number = "1",

}

Xiao, R, Anderson, S, Aramini, J, Belote, R, Buchwald, WA, Ciccosanti, C, Conover, K, Everett, JK, Hamilton, K, Huang, YJ, Janjua, H, Jiang, M, Kornhaber, GJ, Lee, DY, Locke, JY, Ma, LC, Maglaqui, M, Mao, L, Mitra, S, Patel, D, Rossi, P, Sahdev, S, Sharma, S, Shastry, R, Swapna, GVT, Tong, SN, Wang, D, Wang, H, Zhao, L, Montelione, GT & Acton, TB 2010, 'The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium', Journal of Structural Biology, vol. 172, no. 1, pp. 21-33. https://doi.org/10.1016/j.jsb.2010.07.011

The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium. / Xiao, Rong; Anderson, Stephen; Aramini, James; Belote, Rachel; Buchwald, William A.; Ciccosanti, Colleen; Conover, Ken; Everett, John K.; Hamilton, Keith; Huang, Yuanpeng Janet; Janjua, Haleema; Jiang, Mei; Kornhaber, Gregory J.; Lee, Dong Yup; Locke, Jessica Y.; Ma, Li Chung; Maglaqui, Melissa; Mao, Lei; Mitra, Saheli; Patel, Dayaban; Rossi, Paolo; Sahdev, Seema; Sharma, Seema; Shastry, Ritu; Swapna, G. V.T.; Tong, Saichu N.; Wang, Dongyan; Wang, Huang; Zhao, Li; Montelione, Gaetano T.; Acton, Thomas B.

In: Journal of Structural Biology, Vol. 172, No. 1, 10.2010, p. 21-33.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium

AU - Xiao, Rong

AU - Anderson, Stephen

AU - Aramini, James

AU - Belote, Rachel

AU - Buchwald, William A.

AU - Ciccosanti, Colleen

AU - Conover, Ken

AU - Everett, John K.

AU - Hamilton, Keith

AU - Huang, Yuanpeng Janet

AU - Janjua, Haleema

AU - Jiang, Mei

AU - Kornhaber, Gregory J.

AU - Lee, Dong Yup

AU - Locke, Jessica Y.

AU - Ma, Li Chung

AU - Maglaqui, Melissa

AU - Mao, Lei

AU - Mitra, Saheli

AU - Patel, Dayaban

AU - Rossi, Paolo

AU - Sahdev, Seema

AU - Sharma, Seema

AU - Shastry, Ritu

AU - Swapna, G. V.T.

AU - Tong, Saichu N.

AU - Wang, Dongyan

AU - Wang, Huang

AU - Zhao, Li

AU - Montelione, Gaetano T.

AU - Acton, Thomas B.

N1 - Funding Information: We thank Profs. C. Arrowsmith, J. Hunt, M. Gerstein, M. Inouye, J. Marcotrigiano, and L. Tong, along with all the members of the NESG Consortium, for valuable advice in the development of the NESG Protein Sample Production Platform. This work was supported by a grant from the National Institute of General Medical Sciences Protein Structure Initiative U54-GM074958 (to G.T.M.).

PY - 2010/10

Y1 - 2010/10

N2 - 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.

AB - 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.

KW - 6X-His tag

KW - Construct optimization

KW - Disorder prediction

KW - HDX-MS

KW - High-throughput protein production

KW - Laboratory Information Management System

KW - Ligation-independent cloning

KW - Multiple Displacement Amplification

KW - NMR

KW - NMR microprobe screening

KW - Parallel protein purification

KW - Protein Structure Initiative

KW - Structural genomics

KW - T7 Escherichia coli expression system

KW - Wheat germ cell-free

KW - X-ray crystallography

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U2 - https://doi.org/10.1016/j.jsb.2010.07.011

DO - https://doi.org/10.1016/j.jsb.2010.07.011

M3 - Article

C2 - 20688167

VL - 172

SP - 21

EP - 33

JO - Journal of Structural Biology

JF - Journal of Structural Biology

SN - 1047-8477

IS - 1

ER -

The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium

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ASJC Scopus subject areas

Keywords

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