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Poul Nissen

Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae

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DOI

  • Marie Jidenko, Unité de Recherche Associée 2096 of the Centre National de la Recherche Scientifique and Service de Biophysique des Fonctions Membranaires, Département de Biologie Joliot Curie, Commissariat à l'Energie Atomique Saclay, 91191 Gif-sur-Yvette Cedex, France.
  • ,
  • Rikke C Nielsen
  • ,
  • Thomas Lykke-Møller Sørensen
  • Jesper V Møller
  • Marc le Maire
  • ,
  • Poul Nissen
  • Christine Jaxel

The Ca2+-ATPase SERCA1a (sarcoplasmic-endoplasmic reticulum Ca2+-ATPase isoform 1a) from rabbit has been overexpressed in Saccharomyces cerevisiae. This membrane protein was purified by avidin agarose affinity chromatography based on natural biotinylation in the expression host, followed by HPLC gel filtration. Both the functional and structural properties of the overexpressed protein validate the method. Thus, calcium-dependent ATPase activity and calcium transport are essentially intact after reconstitution in proteoliposomes. Moreover, the recombinant protein crystallizes in a form that is isomorphous to the native SERCA1a protein from rabbit, and the diffraction properties are similar. This represents a successful crystallization of a mammalian membrane protein derived from a heterologous expression system, and it opens the way for the study of mutant forms of SERCA1a.

Original languageEnglish
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue33
Pages (from-to)11687-91
Number of pages5
ISSN0027-8424
DOIs
Publication statusPublished - 16 Aug 2005

    Research areas

  • Animals, Calcium-Transporting ATPases/chemistry, Chromatography, Gel, Crystallization, Crystallography, X-Ray, Gene Expression/genetics, Models, Molecular, Protein Structure, Tertiary, Rabbits, Saccharomyces cerevisiae/genetics, Sarcoplasmic Reticulum Calcium-Transporting ATPases

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