Keywords

Green Fluorescent Protein Ecgp123 Codon Optimization For Subcellular Localization Of Sulfolobus Icelandic Bacteria

Abstract

[Purpose] To develop eCGP123 (enhanced consensus green protein variant 123) fluorescent protein that can be efficiently expressed in the extreme thermoacidophilic model Crenarchaeae Sulfolobus islandicus and used as an intracellular protein of S. islandicus Positioning tool. [Method] The green fluorescent protein mutant eCGP123 has extremely high thermal stability, acid resistance and reversible fluorescence properties. This study mainly optimizes and synthesizes the eCGP123 gene according to the codon preference of S. islandicus, expresses it in Escherichia coli and studies its protein properties; by fusing proteins with different intracellular localizations at the C terminus of eCGP123. (Including FtsZ derived from E.coli and UpsE, PCNA1 and SiRe_1200 derived from S.islandicus, etc.), construct an expression strain of eCGP123 and its fusion protein, and use laser confocal microscopy to analyze the expression of eCGP123 and its fusion protein in E.coli and S. Subcellular localization in living cells of islandicus. [Results] We confirmed that the codon-optimized eCGP123 expressed and purified in E. coli has the same absorbance value and higher thermal stability as wild-type green fluorescent protein. Cytological analysis shows that the cell division-related proteins FtsZ and SiRe_1200 are mainly located in the middle of dividing cells of E.coli and S.islandicus respectively; the flagellar component protein UpsE is evenly distributed in a punctate shape and may be located on the cell membrane; the DNA replication sliding clamp subunit PCNA1 is distributed in a regional punctate pattern, suggesting the location of DNA replication regions. The subcellular localization of the protein was basically consistent with the expected results. [Conclusion] Green fluorescent protein eCGP123 can be used as a reporter protein in protein localization analysis of S. islandicus cells, and can be used as an important tool for functional gene research in this model strain, but further optimization of conditions is required.

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Original research done by Ni Jinfeng, Shen Yulong, Ma Jiaxing, Huang Qihong

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