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 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.Keywords
This publication has 41 references indexed in Scilit:
- Crystals of Native and Modified Bovine Rhodopsins and Their Heavy Atom DerivativesJournal of Molecular Biology, 2004
- Roles of Conserved P Domain Residues and Mg2+ in ATP Binding in the Ground and Ca2+-activated States of Sarcoplasmic Reticulum Ca2+-ATPasePublished by Elsevier BV ,2004
- Purified Human MDR 1 Modulates Membrane Potential in Reconstituted ProteoliposomesBiochemistry, 2003
- Heterologous expression and purification systems for structural proteomics of mammalian membrane proteinsComparative and Functional Genomics, 2002
- In Vitro Biotinylation Provides Quantitative Recovery of Highly Purified Active Lactose Permease in a Single StepBiochemistry, 1998
- Rapid purification of a functionally active plant sucrose carrier from transgenic yeast using a bacterial biotin acceptor domainFEBS Letters, 1995
- Improved methods for building protein models in electron density maps and the location of errors in these modelsActa Crystallographica Section A Foundations of Crystallography, 1991
- Interaction of magnesium and inorganic phosphate with calcium-deprived sarcoplasmic reticulum adenosine triphosphatase as reflected by organic solvent induced perturbationBiochemistry, 1985
- Reconstitution of sarcoplasmic reticulum Ca2+-ATPase with excess lipid. Dispersion of the pump unitsBiochimica et Biophysica Acta (BBA) - Biomembranes, 1983
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970