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.overexpression ͉ Ca 2ϩ -ATPase ͉ SERCA1a T he major protein component of the sarcoplasmic reticulum (SR) membrane is a Ca 2ϩ -transporting ATPase that is responsible for the cessation of muscle contraction cycle. Since its discovery (1), rabbit SR Ca 2ϩ -ATPase (SERCA1a, sarcoplasmic-endoplasmic reticulum Ca 2ϩ -ATPase isoform 1a) has been a favorite subject for membrane studies, because vesicles predominantly consisting of Ca 2ϩ -ATPase and with intact Ca 2ϩ -transporting properties can be prepared in large yield from skeletal muscle. Moreover, this protein is a paradigm of P-type ATPases, which constitute the major cation transporter family. P-type ATPases are characterized by the formation of a covalent aspartylphosphorylated intermediate during their catalytic cycle. Crystallization attempts of SERCA1a started a long time ago from detergent-solubilized membranes, which provided valuable initial structural information as to how to proceed with crystallization of this difficult-to-handle membrane protein (2, 3). Recently, several 3D structures corresponding to various conformations of the catalytic cycle have been described, all of them obtained from the native protein of SR (4-10). With the object of studying structural and functional properties of mutants of this transporter, we here outline a procedure of purification of Ca 2ϩ -ATPase after heterologous expression in yeast (11). Our aim was to increase the quantity and, most importantly, the quality of our purified protein to be used for 3D crystallization. For this, we have chosen to use a recombinant protein: the SERCA1a fused to a biotin acceptor domain (SERCA1a-BAD). This procedure allows us to specifically purify the fusion protein by affinity chromatography after in vivo biotinylation, which selects for properly folded protein. Finally, we show, with wild-type SERCA1a as an example, that the purified protein can be used for crystallization and functional studies, including transport, and that its structure is isomorphous to the native enzyme from rabbit. MethodsProtein Purification. Expression in yeast was performed as described in ref. 11. Starting from a 4-liter culture, 75 ml ...

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