Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex.
Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex.
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DOI：
10.1016/j.pep.2008.08.002
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发表时间：
2008-12
影响因子：
1.6
通讯作者：
Fox BG
中科院分区：
文献类型：
--
作者：
Goren MA;Fox BG
用小麦胚芽提取液在单层脂质体存在下进行了人硬脂酰辅酶A去饱和酶的体外翻译，观察到表达的完整膜蛋白几乎完全转移到脂质体中。此外，去饱和酶和人细胞色素b5的共翻译导致两种膜蛋白都转移到脂质体中。通过密度梯度离心法进行简单的一步纯化，可得到经毛细管电泳法鉴定为高纯度去饱和酶的蛋白脂质体。在体外重组非血红素铁和血红素活性中心后，通过将硬脂酰辅酶A转化为油酰辅酶A来证明重组酶复合体的功能。这种简单的翻译方法避免了使用洗涤剂或其他脂类来稳定和分离具有催化活性的整体膜酶。讨论了无细胞翻译在组装和纯化其他完整膜蛋白复合体方面的适用性。
A wheat germ cell-free extract was used to perform in vitro translation of human stearoyl-CoA desaturase in the presence of unilamelar liposomes, and near complete transfer of the expressed integral membrane protein into the liposome was observed. Moreover, co-translation of the desaturase along with human cytochrome b5 led to transfer of both membrane proteins into the liposomes. A simple, single step purification via centrifugation in a density gradient yielded proteoliposomes with the desaturase in high purity as judged by capillary electrophoresis. After in vitro reconstitution of the non-heme iron and heme active sites, the function of the reconstituted enzyme complex was demonstrated by conversion of stearoyl-CoA to oleoyl-CoA. This simple translation approach obviates the use of detergents or other lipids to stabilize and isolate a catalytically active integral membrane enzyme. The applicability of cell-free translation to the assembly and purification of other integral membrane protein complexes is discussed.
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