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. Optimizing the number of galactose induction steps and increasing the amount of Gal4p transcription factor both improved expression. Lowering the temperature from 28 to 18 degrees C during expression enhanced the recovery of detergent-extractible active protein. In the "light membrane fraction," thought to mainly contain internal membranes, we are able to recover about 14-18 mg Ca(2+)-ATPase per liter of yeast culture in a bioreactor. Solubilization of this membrane fraction by n-dodecyl beta-D: -maltopyranoside (DDM) allowed us to recover the largest amount of active protein. The in vivo biotinylated recombinant protein was then bound to a streptavidin-Sepharose resin. Selective elution of the biotinylated SERCA1a was carried out after thrombin action on the resin-bound protein. We were able to obtain 200-500 microg/L of yeast culture of a 50% pure SERCA1a that displays an ATPase activity similar to that of the native rabbit Ca(2+)-ATPase. To succeed in crystallization, an additional size exclusion chromatography step was necessary. This step increases purity to 70%, removes aggregated protein and exchanges DDM for C(12)E(8).
Cardi
Delphine
D
CEA, Institut de Biologie et Technologies de Saclay, University of Paris-Sud, Gif-sur-Yvette, France.
Montigny
Cédric
C
Arnou
Bertrand
B
Jidenko
Marie
M
Marchal
Estelle
E
le Maire
Marc
M
Jaxel
Christine
C
D000818:Animals; D019149:Bioreactors; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D012995:Solubility
D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases