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 recombinant protein was expressed under the control of a galactose-inducible promoter in the yeast Saccharomyces cerevisiae. The biotinylation reaction of the protein was performed directly in vivo in yeast. After solubilization of the yeast light membrane fraction, the biotinylated protein was retained specifically using the strong biotin-avidin interaction. Finally, digestion by the protease thrombin allowed the separation of the Ca2+-ATPase from the biotinylated domain. At this step, Ca2+-ATPase is in a relatively purified form (about 40%). After a size-exclusion HPLC step, the purity of the protein is about 70%, and evaluation of the conformational changes during the catalytic cycle by monitoring the intrinsic fluorescence is demonstrated. The major advantage of this avidin procedure is the particularly good specific ATPase activity as compared with that of a purified His-tagged Ca2+-ATPase.
Jidenko
Marie
M
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, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France.
Lenoir
Guillaume
G
Fuentes
José M
JM
le Maire
Marc
M
Jaxel
Christine
C
D000818:Animals; D001360:Avidin / Q000378:metabolism; D001665:Binding Sites; D019921:Biotinylation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D001360:Avidin; D013917:Thrombin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases