title: Thapsigargin affinity purification of intracellular P(2A)-type Ca(2+) 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.
authors.affiliation: Laboratory of Cellular Transport Systems; Department of Molecular Cell Biology, Campus Gasthuisberg O.&N.1, K.U. Leuven, Herestraat 49, bus 802, B-3000 Leuven, Belgium. llse.Vandecaetsbeek@med.kuleuven.be
authors.forename: Ilse
authors.initials: I
authors.lastname: Vandecaetsbeek
authors.forename: Søren Brøgger
authors.initials: SB
authors.lastname: Christensen
authors.forename: Huizhen
authors.initials: H
authors.lastname: Liu
authors.forename: Paul P
authors.initials: PP
authors.lastname: Van Veldhoven
authors.forename: Etienne
authors.initials: E
authors.lastname: Waelkens
authors.forename: Jan
authors.initials: J
authors.lastname: Eggermont
authors.forename: Luc
authors.initials: L
authors.lastname: Raeymaekers
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Møller
authors.forename: Poul
authors.initials: P
authors.lastname: Nissen
authors.forename: Frank
authors.initials: F
authors.lastname: Wuytack
authors.forename: Peter
authors.initials: P
authors.lastname: Vangheluwe
mesh_terms: D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D042541:Intracellular Space / Q000201:enzymology*; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000302:isolation & purification; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000302:isolation & purification*; D019284:Thapsigargin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D019284:Thapsigargin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human; C403624:ATP2C1 protein, human; D000252:Calcium-Transporting ATPases