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. 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.
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, Commissariat à l'Energie Atomique Saclay, 91191 Gif-sur-Yvette Cedex, France.
Nielsen
Rikke C
RC
Sørensen
Thomas Lykke-Møller
TL
Møller
Jesper V
JV
le Maire
Marc
M
Nissen
Poul
P
Jaxel
Christine
C
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression / Q000235:genetics*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases