Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia;
Acta Naturae. 2009 Apr;1(1):91-5.
The effective expression of recombinant membrane proteins in E.coli depends upon the targeting and insertion of proteins into the cellular membrane, as well as on those proteins adopting the correct spatial structure. A significant technological problem involves the design of approaches for detecting the location of target proteins within a host cell. Using a hybrid potassium channel KcsA-Kv1.3 as a model, we developed a technological scheme which is suitable for the study of membrane localization in E.coli cells of recombinant proteins containing voltage-gated eukaryotic potassium channels as the functional active site. The scheme involves both biochemical and fluorescent methods for detecting target proteins in the cytoplasmic membrane of E.coli, as well as the study of the ligand-binding activity of membrane-embedded proteins.
重组膜蛋白在大肠杆菌中的有效表达取决于蛋白质靶向和插入细胞膜，以及蛋白质采用正确的空间结构。一个重大的技术问题涉及设计方法来检测靶蛋白在宿主细胞内的位置。我们使用混合钾通道 KcsA-Kv1.3 作为模型，开发了一种技术方案，该方案适用于研究含有电压门控真核钾通道作为功能活性位点的重组蛋白在大肠杆菌细胞中的膜定位。该方案涉及用于检测大肠杆菌细胞质膜中靶蛋白的生化和荧光方法，以及研究膜嵌入蛋白的配体结合活性。
