Senaratne R H, Mobasheri H, Papavinasasundaram K G, Jenner P, Lea E J, Draper P
National Institute for Medical Research, Mill Hill, London NW7 1AA, England.
J Bacteriol. 1998 Jul;180(14):3541-7. doi: 10.1128/JB.180.14.3541-3547.1998.
An open reading frame in the genomic database of Mycobacterium tuberculosis H37Rv was identified as having homology with an outer membrane protein. We found that the gene specified a protein belonging to the OmpA family, which includes some porins of gram-negative organisms. The gene was amplified by PCR and cloned into Escherichia coli. Overexpression of the gene was toxic to the host, but limited amounts could be purified from cells before growth ceased. A truncated gene devoid of the code for a presumed signal sequence was well expressed, but the protein had no pore-forming activity in the liposome swelling assay. However, the intact protein, OmpATb, behaved as a porin of low specific activity, with a pore diameter of 1.4 to 1.8 nm, and was also active in planar lipid bilayers, showing a single-channel conductance of 700 pS. The protein had a molecular mass of about 38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal rabbit antiserum raised to the truncated protein recognized a protein of similar molecular mass in detergent extracts of broken M. tuberculosis cells. Reverse transcription-PCR confirmed that the gene for OmpATb was expressed in M. tuberculosis cells growing in culture. Comparison of the purified protein with that in the detergent-extracted preparation using liposomes and planar lipid bilayers showed that the two materials had similar pore-forming properties. OmpATb is different from either of the mycobacterial porins described so far. This is the first report of a porin-like molecule from M. tuberculosis; the porin is likely to be important in controlling the access of hydrophilic molecules to the bacterial cell.
在结核分枝杆菌H37Rv的基因组数据库中，一个开放阅读框被鉴定为与一种外膜蛋白具有同源性。我们发现该基因编码一种属于OmpA家族的蛋白质，该家族包括一些革兰氏阴性菌的孔蛋白。通过PCR扩增该基因并克隆到大肠杆菌中。该基因的过表达对宿主有毒性，但在生长停止前可从细胞中纯化出少量蛋白。一个缺失假定信号序列编码的截短基因表达良好，但该蛋白在脂质体肿胀试验中没有成孔活性。然而，完整的蛋白OmpATb表现为一种低比活性的孔蛋白，孔径为1.4至1.8纳米，并且在平面脂质双分子层中也有活性，显示单通道电导为700 pS。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中，该蛋白的分子量约为38 kDa。用截短蛋白制备的兔多克隆抗血清可识别破碎的结核分枝杆菌细胞去污剂提取物中分子量相似的一种蛋白。逆转录-PCR证实OmpATb基因在培养的结核分枝杆菌细胞中表达。使用脂质体和平面脂质双分子层对纯化蛋白与去污剂提取物中的蛋白进行比较，结果表明这两种物质具有相似的成孔特性。OmpATb与迄今为止描述的任何一种分枝杆菌孔蛋白都不同。这是关于结核分枝杆菌中一种类似孔蛋白分子的首次报道；该孔蛋白可能在控制亲水分子进入细菌细胞方面发挥重要作用。
