Thapsigargin affinity purification of intracellular P2A-type Ca2+ ATPases

Research output: Contribution to journalJournal articleResearchpeer-review

Standard

Thapsigargin affinity purification of intracellular P2A-type Ca2+ ATPases. / Vandecaetsbeek, Ilse; Christensen, Søren Brøgger; Liu, Huizhen; van Veldhoven, Paul P; Waelkens, Etienne; Eggermont, Jan; Raeymaekers, Luc; Møller, Jesper V; Nissen, Poul; Wuytack, Frank; Vangheluwe, Peter.

In: BBA General Subjects, Vol. 1813, No. 5, 01.05.2011, p. 1118-1127.

Research output: Contribution to journalJournal articleResearchpeer-review

Harvard

Vandecaetsbeek, I, Christensen, SB, Liu, H, van Veldhoven, PP, Waelkens, E, Eggermont, J, Raeymaekers, L, Møller, JV, Nissen, P, Wuytack, F & Vangheluwe, P 2011, 'Thapsigargin affinity purification of intracellular P2A-type Ca2+ ATPases', BBA General Subjects, vol. 1813, no. 5, pp. 1118-1127. https://doi.org/10.1016/j.bbamcr.2010.12.020

APA

Vandecaetsbeek, I., Christensen, S. B., Liu, H., van Veldhoven, P. P., Waelkens, E., Eggermont, J., ... Vangheluwe, P. (2011). Thapsigargin affinity purification of intracellular P2A-type Ca2+ ATPases. BBA General Subjects, 1813(5), 1118-1127. https://doi.org/10.1016/j.bbamcr.2010.12.020

Vancouver

Vandecaetsbeek I, Christensen SB, Liu H, van Veldhoven PP, Waelkens E, Eggermont J et al. Thapsigargin affinity purification of intracellular P2A-type Ca2+ ATPases. BBA General Subjects. 2011 May 1;1813(5):1118-1127. https://doi.org/10.1016/j.bbamcr.2010.12.020

Author

Vandecaetsbeek, Ilse ; Christensen, Søren Brøgger ; Liu, Huizhen ; van Veldhoven, Paul P ; Waelkens, Etienne ; Eggermont, Jan ; Raeymaekers, Luc ; Møller, Jesper V ; Nissen, Poul ; Wuytack, Frank ; Vangheluwe, Peter. / Thapsigargin affinity purification of intracellular P2A-type Ca2+ ATPases. In: BBA General Subjects. 2011 ; Vol. 1813, No. 5. pp. 1118-1127.

Bibtex

@article{42e4e21b250747b897064be160b68f34,
title = "Thapsigargin affinity purification of intracellular P2A-type Ca2+ ATPases",
abstract = "The ubiquitous sarco(endo)plasmic reticulum (SR/ER) Ca(2+) ATPase (SERCA2b) and secretory-pathway Ca(2+) ATPase (SPCA1a) belong both to the P(2A)-type ATPase subgroup of Ca(2+) transporters and play a crucial role in the Ca(2+) homeostasis of respectively the ER and Golgi apparatus. They are ubiquitously expressed, but their low abundance precludes purification for crystallization. We have developed a new strategy for purification of recombinant hSERCA2b and hSPCA1a that is based on overexpression in yeast followed by a two-step affinity chromatography method biasing towards properly folded protein. In a first step, these proteins were purified with the aid of an analogue of the SERCA inhibitor thapsigargin (Tg) coupled to a matrix. Wild-type (WT) hSERCA2b bound efficiently to the gel, but its elution was hampered by the high affinity of SERCA2b for Tg. Therefore, a mutant was generated carrying minor modifications in the Tg-binding site showing a lower affinity for Tg. In a second step, reactive dye chromatography was performed to further purify and concentrate the properly folded pumps and to exchange the detergent to one more suitable for crystallization. A similar strategy was successfully applied to purify WT SPCA1a. This study shows that it is possible to purify functionally active intracellular Ca(2+) ATPases using successive thapsigargin and reactive dye affinity chromatography for future structural studies. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.",
keywords = "The Faculty of Pharmaceutical Sciences",
author = "Ilse Vandecaetsbeek and Christensen, {S{\o}ren Br{\o}gger} and Huizhen Liu and {van Veldhoven}, {Paul P} and Etienne Waelkens and Jan Eggermont and Luc Raeymaekers and M{\o}ller, {Jesper V} and Poul Nissen and Frank Wuytack and Peter Vangheluwe",
note = "Keywords: SERCA2b, SPCA1a, Ca(2+) pumps, endoplasmic reticulum, golgi, chromatography",
year = "2011",
month = "5",
day = "1",
doi = "10.1016/j.bbamcr.2010.12.020",
language = "English",
volume = "1813",
pages = "1118--1127",
journal = "B B A - General Subjects",
issn = "0304-4165",
publisher = "Elsevier",
number = "5",

}

RIS

TY - JOUR

T1 - Thapsigargin affinity purification of intracellular P2A-type Ca2+ ATPases

AU - Vandecaetsbeek, Ilse

AU - Christensen, Søren Brøgger

AU - Liu, Huizhen

AU - van Veldhoven, Paul P

AU - Waelkens, Etienne

AU - Eggermont, Jan

AU - Raeymaekers, Luc

AU - Møller, Jesper V

AU - Nissen, Poul

AU - Wuytack, Frank

AU - Vangheluwe, Peter

N1 - Keywords: SERCA2b, SPCA1a, Ca(2+) pumps, endoplasmic reticulum, golgi, chromatography

PY - 2011/5/1

Y1 - 2011/5/1

N2 - The ubiquitous sarco(endo)plasmic reticulum (SR/ER) Ca(2+) ATPase (SERCA2b) and secretory-pathway Ca(2+) ATPase (SPCA1a) belong both to the P(2A)-type ATPase subgroup of Ca(2+) transporters and play a crucial role in the Ca(2+) homeostasis of respectively the ER and Golgi apparatus. They are ubiquitously expressed, but their low abundance precludes purification for crystallization. We have developed a new strategy for purification of recombinant hSERCA2b and hSPCA1a that is based on overexpression in yeast followed by a two-step affinity chromatography method biasing towards properly folded protein. In a first step, these proteins were purified with the aid of an analogue of the SERCA inhibitor thapsigargin (Tg) coupled to a matrix. Wild-type (WT) hSERCA2b bound efficiently to the gel, but its elution was hampered by the high affinity of SERCA2b for Tg. Therefore, a mutant was generated carrying minor modifications in the Tg-binding site showing a lower affinity for Tg. In a second step, reactive dye chromatography was performed to further purify and concentrate the properly folded pumps and to exchange the detergent to one more suitable for crystallization. A similar strategy was successfully applied to purify WT SPCA1a. This study shows that it is possible to purify functionally active intracellular Ca(2+) ATPases using successive thapsigargin and reactive dye affinity chromatography for future structural studies. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

AB - The ubiquitous sarco(endo)plasmic reticulum (SR/ER) Ca(2+) ATPase (SERCA2b) and secretory-pathway Ca(2+) ATPase (SPCA1a) belong both to the P(2A)-type ATPase subgroup of Ca(2+) transporters and play a crucial role in the Ca(2+) homeostasis of respectively the ER and Golgi apparatus. They are ubiquitously expressed, but their low abundance precludes purification for crystallization. We have developed a new strategy for purification of recombinant hSERCA2b and hSPCA1a that is based on overexpression in yeast followed by a two-step affinity chromatography method biasing towards properly folded protein. In a first step, these proteins were purified with the aid of an analogue of the SERCA inhibitor thapsigargin (Tg) coupled to a matrix. Wild-type (WT) hSERCA2b bound efficiently to the gel, but its elution was hampered by the high affinity of SERCA2b for Tg. Therefore, a mutant was generated carrying minor modifications in the Tg-binding site showing a lower affinity for Tg. In a second step, reactive dye chromatography was performed to further purify and concentrate the properly folded pumps and to exchange the detergent to one more suitable for crystallization. A similar strategy was successfully applied to purify WT SPCA1a. This study shows that it is possible to purify functionally active intracellular Ca(2+) ATPases using successive thapsigargin and reactive dye affinity chromatography for future structural studies. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

KW - The Faculty of Pharmaceutical Sciences

U2 - 10.1016/j.bbamcr.2010.12.020

DO - 10.1016/j.bbamcr.2010.12.020

M3 - Journal article

VL - 1813

SP - 1118

EP - 1127

JO - B B A - General Subjects

JF - B B A - General Subjects

SN - 0304-4165

IS - 5

ER -

ID: 33247074