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

Access to Document

10.1007/s10969-005-1908-7

Cite this

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

@article{4c9a04e458cd4b84b8bf5dbe31d54c06,

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",

issn = "1345-711X",

publisher = "Springer Netherlands",

number = "2-3",

}

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 - http://www.scopus.com/inward/record.url?scp=20944444192&partnerID=8YFLogxK

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

Access to Document

Other files and links

Fingerprint

Cite this