Neophytou Irene, Harvey Richard, Lawrence Jayne, Marsh Phil, Panaretou Barry, Barlow David
Pharmaceutical Sciences Division, King's College London, Franklin Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK.
Appl Microbiol Biotechnol. 2007 Nov;77(2):375-81. doi: 10.1007/s00253-007-1174-7. Epub 2007 Sep 9.
A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous membrane-protein expression is demonstrated by the expression and insertion into the E. coli cell membrane of the human membrane proteins: occludin, claudin 4, duodenal ferric reductase and a J-type inwardly rectifying potassium channel. The proteins are produced with C-terminal hexahistidine tags (to permit purification of the expressed fusion proteins using immobilized metal affinity chromatography) and a peptidase cleavage site (to allow recovery of the unfused eukaryotic protein).
描述了一种融合蛋白表达系统，该系统可用于在大肠杆菌（E. coli）中生产真核整合膜蛋白。真核膜蛋白靶点与高表达的大肠杆菌内膜蛋白GlpF（甘油传导通道蛋白）的C末端融合。该系统对异源膜蛋白表达的通用效用通过人膜蛋白occludin、claudin 4、十二指肠铁还原酶和一种J型内向整流钾通道在大肠杆菌细胞膜中的表达和插入得到证明。这些蛋白通过C末端六组氨酸标签（用于使用固定化金属亲和色谱法纯化表达的融合蛋白）和肽酶切割位点（用于回收未融合的真核蛋白）进行生产。
