Jidenko Marie, Nielsen Rikke C, Sørensen Thomas Lykke-Møller, Møller Jesper V, le Maire Marc, Nissen Poul, Jaxel Christine
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.
Proc Natl Acad Sci U S A. 2005 Aug 16;102(33):11687-91. doi: 10.1073/pnas.0503986102. Epub 2005 Aug 8.
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.
来自兔子的Ca2+-ATP酶SERCA1a（肌浆网-内质网Ca2+-ATP酶同工型1a）已在酿酒酵母中过表达。基于表达宿主中的天然生物素化，通过抗生物素蛋白琼脂糖亲和层析对该膜蛋白进行纯化，随后进行高效液相色谱凝胶过滤。过表达蛋白的功能和结构特性均验证了该方法。因此，在蛋白脂质体中重构后，钙依赖性ATP酶活性和钙转运基本保持完整。此外，重组蛋白结晶的形式与来自兔子的天然SERCA1a蛋白同晶型，且衍射特性相似。这代表了源自异源表达系统的哺乳动物膜蛋白的成功结晶，为研究SERCA1a的突变形式开辟了道路。
