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

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.