Heterologous expression and affinity purification of eukaryotic membrane proteins in view of functional and structural studies: The example of the sarcoplasmic reticulum Ca(2+)-ATPase.

Heterologous SERCA1a Ca(2+)-ATPase (sarco-endoplasmic reticulum Ca(2+)-adenosine triphosphatase isoform 1a) from rabbit was expressed in yeast Saccharomyces cerevisiae as a fusion protein, with a biotin acceptor domain (BAD) linked to the SERCA C-terminus by a thrombin cleavage site. Thanks to the pYeDP60 vector, the recombinant protein was expressed under the control of a galactose-inducible promoter. Biotinylation of the protein occurred directly in yeast.

Expression in yeast and purification of a membrane protein, SERCA1a, using a biotinylated acceptor domain.

We have recently described the final steps leading to the crystallization of a mammalian membrane protein, the rabbit sarcoplasmic reticulum Ca2+-ATPase, after heterologous expression. Here, we detail the initial steps leading to this new purification method. A biotin acceptor domain was fused at the C-terminal part of Ca2+-ATPase and a thrombin site was inserted between both coding regions.

The ABC transporter BmrA from Bacillus subtilis is a functional dimer when in a detergent-solubilized state.

BmrA from Bacillus subtilis is a half-size ABC (ATP-binding cassette) transporter involved in multidrug resistance. Although its supramolecular organization has been investigated after reconstitution in a lipid bilayer environment, and shows a dimeric and possibly a tetrameric form, the precise quaternary structure in a detergent-solubilized state has never been addressed. In the present study, BmrA was purified from Escherichia coli membranes using an optimized purification protocol and different detergents.

Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae.

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.

Organic solvent extracted EmrE solubilized in dodecyl maltoside is monomeric and binds drug ligand.

Ethidium multidrug resistance protein (EmrE) is a member of the small multidrug resistance family of proteins and is responsible for resistance to a diverse group of lipophilic cations. To examine the multimeric state(s), size-exclusion HPLC and sedimentation velocity experiments were performed with EmrE solubilized in N-dodecyl-beta-d-maltopyranoside (DM) detergent. EmrE was purified from Escherichia coli membranes using organic extraction with a 3:1 chloroform:methanol solvent followed by LH-20 chromatography and the recovered pure protein was re-solubilized in a buffer containing 2% DM.