TY - JOUR
T1 - Thapsigargin affinity purification of intracellular P(2A)-type Ca(2+) 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 - 2011 Elsevier B.V. All rights reserved.
PY - 2011
Y1 - 2011
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 - Binding Sites
KW - Calcium-Transporting ATPases
KW - Chromatography, Affinity
KW - Humans
KW - Intracellular Space
KW - Mutant Proteins
KW - Protein Structure, Secondary
KW - Recombinant Proteins
KW - Sarcoplasmic Reticulum Calcium-Transporting ATPases
KW - Thapsigargin
U2 - 10.1016/j.bbamcr.2010.12.020
DO - 10.1016/j.bbamcr.2010.12.020
M3 - Journal article
C2 - 21215281
SN - 0304-4165
VL - 1813
SP - 1118
EP - 1127
JO - BBA General Subjects
JF - BBA General Subjects
IS - 5
ER -