Thapsigargin affinity purification of intracellular P2A-type Ca2+ ATPases
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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.
I: BBA General Subjects, Bind 1813, Nr. 5, 01.05.2011, s. 1118-1127.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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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 - Former Faculty of Pharmaceutical Sciences
U2 - 10.1016/j.bbamcr.2010.12.020
DO - 10.1016/j.bbamcr.2010.12.020
M3 - Journal article
C2 - 21215281
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