Division of Bioscience, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Okayama, 700-8530, Japan.
Extremophiles. 2011 May;15(3):403-10. doi: 10.1007/s00792-011-0371-6. Epub 2011 Apr 7.
An OmpA family protein (FopA) previously reported as one of the major outer membrane proteins of an acidophilic iron-oxidizing bacterium Acidithiobacillus ferrooxidans was characterized with emphasis on the modification by heat and the interaction with peptidoglycan. A 30-kDa band corresponding to the FopA protein was detected in outer membrane proteins extracted at 75°C or heated to 100°C for 10 min prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the band was not detected in outer membrane proteins extracted at ≤40°C and without boiling prior to electrophoresis. By Western blot analysis using the polyclonal antibody against the recombinant FopA, FopA was detected as bands with apparent molecular masses of 30 and 90 kDa, suggesting that FopA existed as an oligomeric form in the outer membrane of A. ferrooxidans. Although the fopA gene with a sequence encoding the signal peptide was successfully expressed in the outer membrane of Escherichia coli, the recombinant FopA existed as a monomer in the outer membrane of E. coli. FopA was detected in peptidoglycan-associated proteins from A. ferrooxidans. The recombinant FopA also showed the peptidoglycan-binding activity.
先前有报道称，一种嗜酸氧化亚铁硫杆菌的主要外膜蛋白之一是 OmpA 家族蛋白（FopA），本研究重点对其热修饰和与肽聚糖的相互作用进行了研究。在进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）之前，将外膜蛋白在 75°C 下提取或加热至 100°C 10 min，可检测到对应于 FopA 蛋白的 30 kDa 条带。然而，在≤40°C 下提取的外膜蛋白和电泳前未煮沸的外膜蛋白中未检测到该条带。使用针对重组 FopA 的多克隆抗体进行 Western blot 分析，检测到 FopA 存在明显的 30 和 90 kDa 大小的条带，表明 FopA 以寡聚体形式存在于 A. ferrooxidans 的外膜中。尽管带有编码信号肽序列的 fopA 基因在外膜中成功表达，但重组 FopA 在外膜中以单体形式存在。FopA 存在于 A. ferrooxidans 的肽聚糖相关蛋白中。重组 FopA 也显示出肽聚糖结合活性。
