Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
Int J Biol Macromol. 2013 Mar;54:44-50. doi: 10.1016/j.ijbiomac.2012.11.029. Epub 2012 Dec 3.
We have previously found that the N-terminal sequence of the outer membrane protein from moderate halophile is similar to the sequence of the well-known pore forming porin proteins from other Gram-negative bacteria. This highly expressed outer membrane protein was purified from Halomonas sp. 40 and reconstituted into liposome. It showed a permeability activity in the liposome swelling assay. Based on the N-terminal and internal amino acid sequences of this major outer membrane, we have cloned here the porin gene, hopP (halophilic outer membrane protein), from Halomonas sp. 40. The hopP gene encodes the porin precursor comprising 366 amino acid residues that include a 21 amino acid signal peptide. Mature porin (345 amino acids, 37,611 Da) is a highly acidic protein, just as is so for many halophilic proteins and was soluble when expressed in Escherichia coli with N-terminal His-tag. Purified recombinant His-porin was soluble even after heat-treatment at 95 °C for 5 min in the absence of salt. Circular dichroism analysis of His-porin showed conversion into a β-sheet rich structure by the addition of NaCl at 0.9-2.7 M.
我们之前发现中度嗜盐菌的外膜蛋白的 N 端序列与其他革兰氏阴性菌中著名的孔形成孔蛋白的序列相似。这种高度表达的外膜蛋白从 Halomonas sp. 40 中纯化，并重新构成脂质体。它在脂质体肿胀试验中表现出渗透性活性。基于该主要外膜的 N 端和内部氨基酸序列，我们在这里从 Halomonas sp. 40 中克隆了孔蛋白基因 hopP（嗜盐外膜蛋白）。hopP 基因编码包含 366 个氨基酸残基的孔蛋白前体，其中包括 21 个氨基酸的信号肽。成熟的孔蛋白（345 个氨基酸，37611 Da）是一种高度酸性的蛋白质，就像许多嗜盐蛋白一样，在大肠杆菌中表达时带有 N 端 His 标签是可溶性的。即使在没有盐的情况下在 95°C 下加热处理 5 分钟，纯化的重组 His-porin 也是可溶的。His-porin 的圆二色性分析表明，在 0.9-2.7 M 的 NaCl 存在下，其转化为富含β-折叠的结构。
