Thapsigargin affinity purification of intracellular P2A-type Ca2+ ATPases

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Ilse Vandecaetsbeek, Søren Brøgger Christensen, Huizhen Liu, Paul P van Veldhoven, Etienne Waelkens, Jan Eggermont, Luc Raeymaekers, Jesper V Møller, Poul Nissen, Frank Wuytack, Peter Vangheluwe

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.
OriginalsprogEngelsk
TidsskriftBBA General Subjects
Vol/bind1813
Udgave nummer5
Sider (fra-til)1118-1127
ISSN0304-4165
DOI
StatusUdgivet - 1 maj 2011

Bibliografisk note

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

ID: 33247074