High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics

Won Bae Jeon; David J. Aceti; Craig A. Bingman; Frank C. Vojtik; Andrew C. Olson; Jason M. Ellefson; Janet E. McCombs; Hassan K. Sreenath; Paul G. Blommel; Kory D. Seder; Brendan T. Burns; Holalkere V. Geetha; Amy C. Harms; Grzegorz Sabat; Michael R. Sussman; Brian G. Fox; George N. Phillips

doi:10.1007/s10969-005-1908-7

High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics

Won Bae Jeon
, David J. Aceti
, Craig A. Bingman
, Frank C. Vojtik
, Andrew C. Olson
, Jason M. Ellefson
, Janet E. McCombs
, Hassan K. Sreenath
, Paul G. Blommel
, Kory D. Seder
, Brendan T. Burns
, Holalkere V. Geetha
, Amy C. Harms
, Grzegorz Sabat
, Michael R. Sussman
, Brian G. Fox
, George N. Phillips

Division of Biotechnology

University of Wisconsin-Madison

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

61 Scopus citations

Abstract

The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)₆-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated ÄKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)₆-MBP tags by TEV protease, (His)₆-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and ¹⁵N and ¹³C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.

Original language	English
Pages (from-to)	143-147
Number of pages	5
Journal	Journal of Structural and Functional Genomics
Volume	6
Issue number	2-3
DOIs	https://doi.org/10.1007/s10969-005-1908-7
State	Published - Sep 2005

Bibliographical note

Funding Information:
This work was supported by the National Institutes of Health, Protein Structure Initiative Grant P50 GM-64598.

Keywords

Arabidopsis proteins
High-throughput
His-tag
Nickel affinity purification
Structural genomics

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10.1007/s10969-005-1908-7

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Jeon, W. B., Aceti, D. J., Bingman, C. A., Vojtik, F. C., Olson, A. C., Ellefson, J. M., McCombs, J. E., Sreenath, H. K., Blommel, P. G., Seder, K. D., Burns, B. T., Geetha, H. V., Harms, A. C., Sabat, G., Sussman, M. R., Fox, B. G., & Phillips, G. N. (2005). High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics. Journal of Structural and Functional Genomics, 6(2-3), 143-147. https://doi.org/10.1007/s10969-005-1908-7

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title = "High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics",

abstract = "The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)6-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated {\"A}KTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)6-MBP tags by TEV protease, (His)6-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and 15N and 13C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.",

keywords = "Arabidopsis proteins, High-throughput, His-tag, Nickel affinity purification, Structural genomics",

author = "Jeon, \{Won Bae\} and Aceti, \{David J.\} and Bingman, \{Craig A.\} and Vojtik, \{Frank C.\} and Olson, \{Andrew C.\} and Ellefson, \{Jason M.\} and McCombs, \{Janet E.\} and Sreenath, \{Hassan K.\} and Blommel, \{Paul G.\} and Seder, \{Kory D.\} and Burns, \{Brendan T.\} and Geetha, \{Holalkere V.\} and Harms, \{Amy C.\} and Grzegorz Sabat and Sussman, \{Michael R.\} and Fox, \{Brian G.\} and Phillips, \{George N.\}",

note = "Funding Information: This work was supported by the National Institutes of Health, Protein Structure Initiative Grant P50 GM-64598.",

year = "2005",

month = sep,

doi = "10.1007/s10969-005-1908-7",

language = "English",

volume = "6",

pages = "143--147",

journal = "Journal of Structural and Functional Genomics",

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Jeon, WB, Aceti, DJ, Bingman, CA, Vojtik, FC, Olson, AC, Ellefson, JM, McCombs, JE, Sreenath, HK, Blommel, PG, Seder, KD, Burns, BT, Geetha, HV, Harms, AC, Sabat, G, Sussman, MR, Fox, BG & Phillips, GN 2005, 'High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics', Journal of Structural and Functional Genomics, vol. 6, no. 2-3, pp. 143-147. https://doi.org/10.1007/s10969-005-1908-7

TY - JOUR

T1 - High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics

AU - Jeon, Won Bae

AU - Aceti, David J.

AU - Bingman, Craig A.

AU - Vojtik, Frank C.

AU - Olson, Andrew C.

AU - Ellefson, Jason M.

AU - McCombs, Janet E.

AU - Sreenath, Hassan K.

AU - Blommel, Paul G.

AU - Seder, Kory D.

AU - Burns, Brendan T.

AU - Geetha, Holalkere V.

AU - Harms, Amy C.

AU - Sabat, Grzegorz

AU - Sussman, Michael R.

AU - Fox, Brian G.

AU - Phillips, George N.

N1 - Funding Information: This work was supported by the National Institutes of Health, Protein Structure Initiative Grant P50 GM-64598.

PY - 2005/9

Y1 - 2005/9

N2 - The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)6-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated ÄKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)6-MBP tags by TEV protease, (His)6-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and 15N and 13C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.

AB - The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)6-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated ÄKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)6-MBP tags by TEV protease, (His)6-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and 15N and 13C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.

KW - Arabidopsis proteins

KW - High-throughput

KW - His-tag

KW - Nickel affinity purification

KW - Structural genomics

UR - https://www.scopus.com/pages/publications/20944444192

U2 - 10.1007/s10969-005-1908-7

DO - 10.1007/s10969-005-1908-7

M3 - Article

C2 - 16211511

AN - SCOPUS:20944444192

SN - 1345-711X

VL - 6

SP - 143

EP - 147

JO - Journal of Structural and Functional Genomics

JF - Journal of Structural and Functional Genomics

IS - 2-3

ER -

High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics

Abstract

Bibliographical note

Keywords

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