Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli
Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli
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DOI：
10.1110/ps.041022305
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发表时间：
2005-01-01
期刊：
影响因子：
8
通讯作者：
Cross, TA
中科院分区：
文献类型：
--
作者：
Korepanova, A;Gao, FP;Cross, TA
从结核分枝杆菌基因组中克隆并在大肠杆菌中表达了70个完整的膜蛋白。结合T7启动子载体与hexa-His亲和标签和BL21大肠杆菌菌株与额外的IRNA基因来补充稀疏使用的大肠杆菌密码子是最成功的。所表达的蛋白具有广泛的分子量和跨膜螺旋数。这些蛋白的表达已在大肠杆菌细胞裂解物的膜和不溶性部分中观察到，在某些情况下，在可溶性部分中也观察到。在膜组分中的最高表达水平被限制在较窄的分子量范围和相对较少的跨膜螺旋。相反，在不溶性聚集体中，过表达分布在广泛的分子量和跨膜螺旋数量范围内。
Seventy integral membrane proteins from the Mycobacterium tuberculosis genome have been cloned and expressed in Escherichia coli. A combination of T7 promoter-based vectors with hexa-His affinity tags and BL21 E. coli strains with additional IRNA genes to supplement sparsely used E. coli codons have been most successful. The expressed proteins have a wide range of molecular weights and number of transmembrane helices. Expression of these proteins has been observed in the membrane and insoluble fraction of E. coli cell lysates and, in some cases, in the soluble fraction. The highest expression levels in the membrane fraction were restricted to a narrow range of molecular weights and relatively few transmembrane helices. In contrast, overexpression in insoluble aggregates was distributed over a broad range of molecular weights and number of transmembrane helices.