Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae
- 8 August 2005
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 102 (33) , 11687-11691
- https://doi.org/10.1073/pnas.0503986102
Abstract
The Ca 2+ -ATPase SERCA1a (sarcoplasmic–endoplasmic reticulum Ca 2+ -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.Keywords
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