优化的绿色荧光蛋白在超嗜热嗜酸古菌冰岛硫化叶菌中的表达与应用

Expression and application of an improved green fluorescence protein in the hyperthermophilic acidophile Sulfolobus islandicus

摘要:

[目的] 开发可用于在极端嗜热嗜酸模式泉古菌冰岛硫化叶菌（Sulfolobus islandicus）中进行高效表达的eCGP123（enhanced consensus green protein variant 123）荧光蛋白，并用作S.islandicus的细胞内蛋白定位工具。[方法] 绿色荧光蛋白突变体eCGP123具有极高的热稳定性、耐酸性和可逆的荧光特性等。本研究主要对eCGP123的基因根据S.islandicus密码子偏好性进行优化与合成，在大肠杆菌（Escherichia coli）中表达并研究其蛋白性质；通过在eCGP123的C末端分别融合具有不同细胞内定位的蛋白（包括E.coli来源的FtsZ和S.islandicus来源的UpsE、PCNA1和SiRe_1200等），构建eCGP123及其融合蛋白的表达菌株，用激光共聚焦显微镜分析eCGP123及其融合蛋白在E.coli和S.islandicus活细胞中的亚细胞定位。[结果] 我们确认了在E.coli中表达并纯化密码子优化后的eCGP123具有与野生型绿色荧光蛋白相同的吸光值和较高的热稳定性。细胞学分析显示细胞分裂相关蛋白FtsZ和SiRe_1200分别主要定位于E.coli和S.islandicus分裂细胞的中间；鞭毛组分蛋白UpsE呈点状均匀分布，可能定位于细胞膜上；DNA复制滑动夹亚基PCNA1呈区域性点状分布，暗示了DNA复制区域的位置。蛋白的亚细胞定位与预期结果基本吻合。[结论] 绿色荧光蛋白eCGP123可以作为报告蛋白，应用于S.islandicus细胞的蛋白定位分析中，可作为该模式菌株中功能基因研究的重要工具，但需要进一步优化条件。

Abstract:

[Objective] To explore an efficiently expressed fluorescence protein, enhanced consensus green protein variant 123 (eCGP123), and its application on sub-cellular localization of proteins in the thermophilic acidophile Sulfolobus islandicus.[Methods] eCGP123 exhibits extreme thermostability, acid stability and reversible photo-switching. We optimized the sequence of eCGP123 gene according to S. islandicus codon usage. The protein purified from E. coli was characterized. We fused various proteins with different cellular localizations at C-terminal of eCGP123, including FtsZ from E. coli, and UpsE, PCNA1 and SiRe_1200 from S. islandicus. We constructed eCGP123 and its fusion proteins expression strains and analyzed their sub-cellular localizations in the host cells by laser confocal microscopy.[Results] Consistent with previous results, the optimized eCGP123 expressed from E. coli had the same absorption spectrum as the wild type green fluorescence protein and was thermostable in vitro. We found that the cell division proteins FtsZ and SiRe_1200 mainly localized at the mid-cell of E. coli and S. islandicus, respectively. The pili component protein UpsE evenly distributes in the cells as dots, while DNA replication clamp subunit PCNA1 formed several foci in certain area of a cell indicating the locations of DNA replication.[Conclusion] The optimized eCGP123 can utilized as a protein reporter for analysis of protein sub-cellular localizations in S. islandicus. This will be an important tool for functional studies of genes in the model species. However, more improvements are still needed for the application.