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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Christensen, Søren Brøgger Vangheluwe, Peter Eggermont, Jan Wuytack, Frank Nissen, Poul Møller, Jesper V. Van Veldhoven, Paul P. Waelkens, Etienne Vandecaetsbeek, Ilse Liu, Huizhen Raeymaekers, Luc |
| Description | Author Affiliation: Vandecaetsbeek I ( Laboratory of Cellular Transport Systems) |
| 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. |
| ISSN | 00063002 |
| Journal | Biochimica et Biophysica Acta (BBA) - Reviews on Cancer |
| Issue Number | 5 |
| Volume Number | 1813 |
| Language | English |
| Publisher | Elsevier |
| Publisher Date | 2011-05-01 |
| Publisher Place | Netherlands |
| Access Restriction | Open |
| Subject Keyword | Calcium-Transporting ATPases Isolation & Purification Chromatography, Affinity Intracellular Space Enzymology Sarcoplasmic Reticulum Calcium-Transporting ATPases Thapsigargin Metabolism Binding Sites Chemistry Mutant Proteins Protein Structure, Secondary Recombinant Proteins Research Support, Non-U.S. Gov't Biochemistry |
| Content Type | Text |
| Resource Type | Article |
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