Insertion of carrier proteins into hydrophilic loops of the Escherichia coli lactose permease
Insertion of carrier proteins into hydrophilic loops of the Escherichia coli lactose permease
复制标题
DOI：
10.1016/s0005-2736(02)00398-x
复制
发表时间：
2002-08-19
影响因子：
3.4
通讯作者：
Privé, GG
中科院分区：
文献类型：
--
作者：
Engel, CK;Chen, L;Privé, GG
我们描述了一组融合蛋白的大肠杆菌乳糖（紫胶）通透酶，其中一组五个不同的可溶性“载体”蛋白（细胞色素（b562），黄氧还蛋白，T4溶菌酶，β-内酰胺酶和70 kDa的热休克ATP酶结构域）的设计和表征被系统地插入到选定的循环位置的转运。设计目标是增加通透酶暴露的亲水性表面积，同时使所得融合蛋白的内部柔性最小化，以改善膜蛋白的结晶性质。插入乳糖通透酶亲水环的融合蛋白具有转运乳糖的活性，但只有插入E. coliCytochromer（b562）E.大肠杆菌favodoxin或T4溶菌酶以接近野生型紫胶通透酶的水平表达。用这三种载体测试了八个其他环位置，从而鉴定出另外的具有活性且良好表达的融合蛋白。通过结合从单载体插入的结果，我们已经表达了功能性的“双融合”蛋白，其含有插入在两个不同环位置中的细胞色素（b562）结构域。(C)2002 Elsevier Science B. V.保留所有权利。
We describe the design and characterization of a set of fusion proteins of the Escherichia coli lactose (lac) permease in which a set of five different soluble "carrier" proteins (cytochromer(b562), flavodoxin, T4 lysozyme, beta-lactamase and 70 kDa heat shock ATPase domain) were systematically inserted into selected loop positions of the transporter. The design goal was to increase the exposed hydrophilic surface area of the permease, while minimizing the internal flexibility of the resulting fusion proteins in order to improve the crystallization properties of the membrane protein. Fusion proteins with insertions into the central hydrophilic loop of the lac permease were active in transport lactose, although only the fusion proteins with E. coli cytochromer(b562) E. coli favodoxin or T4 lysozyme were expressed at near wild-type lac permease levels. Eight other loop positions were tested with these three carriers, leading to the identification of additional fusion proteins that were active and well-expressed. By combining the results from the single carrier insertions, we have expressed functional "double fusion" proteins containing cytochromer(b562) domains inserted in two different loop positions. (C) 2002 Elsevier Science B.V. All rights reserved.