Linked Life Data

21215281

Source:http://linkedlifedata.com/resource/pubmed/id/21215281

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5
pubmed:dateCreated
2011-4-18
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0006-3002
pubmed:author
pubmed:copyrightInfo
2011 Elsevier B.V. All rights reserved.
pubmed:issnType
Print
pubmed:volume
1813
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1118-27
pubmed:meshHeading
pubmed:year
2011
pubmed:articleTitle
Thapsigargin affinity purification of intracellular P(2A)-type Ca(2+) ATPases.
pubmed: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
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't