Korepanova Alla, Gao Fei P, Hua Yuanzi, Qin Huajun, Nakamoto Robert K, Cross Timothy A
Department of Chemistry and Biochemistry, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32306, USA.
Protein Sci. 2005 Jan;14(1):148-58. doi: 10.1110/ps.041022305.
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 tRNA 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.
来自结核分枝杆菌基因组的70种整合膜蛋白已在大肠杆菌中克隆并表达。基于T7启动子的带有六组氨酸亲和标签的载体与带有额外tRNA基因以补充大肠杆菌中使用较少密码子的BL21大肠杆菌菌株相结合最为成功。所表达的蛋白质具有广泛的分子量和跨膜螺旋数量。已在大肠杆菌细胞裂解物的膜和不溶性部分中观察到这些蛋白质的表达,在某些情况下,也在可溶性部分中观察到表达。膜部分中最高的表达水平局限于较窄的分子量范围和相对较少的跨膜螺旋。相比之下,不溶性聚集体中的过表达分布在广泛的分子量和跨膜螺旋数量范围内。