Recombinant protein production of codon adapted superfolder GFP (sfGFP) in Tetrahymena thermophila and its fluorescence characterization

Different mutants of Aequorea victoria Green Fluorescent Protein (Wild Type GFP) such as EGFP have been used without a codon adaptation as a tag for gene localization in Tetrahymena thermophila. The aim of this study is to create a codon adaptive mutant version of Super Folder GFP (sfGFP) gene for Tetrahymena with an affinity purification feature, its recombinant protein production in T.thermophila and comparative characterization with EGFP for experimental use. sfGFP protein coding region was codon-adapted with 21.3 % silent mutations to Tetrahymena and 6XHis tag coding sequence was also added to its 5? end for affinity purification (6XHis-TtsfGFP). The EGFP gene in Tetrahymena pVGF expression vector was replaced into PmeI-ApaI restriction site with 6XHis-TtsfGFP gene to construct MTT1-6XHis-TtsfGFP-rpl29Term protein expression cassette. Non-codon adapted sfGFP was similarly cloned as a control group. All vectors were transformed to conjugant Tetrahymena cells with electroporation. Results of in vivo fluorescence analyses at 3rd hours after the CdCl2 induction of TtsfGFP, sfGFP and EGFP carrying clones showed that TtsfGFP clones have ~2.2-4 fold more fluorescence emission against control groups. Fluorescent microscopic results also showed that TtsfGFP reach to max emission at 100 min, which is much earlier than EGFP and sfGFP clones at ~240 min. A Tetrahymena hypothetical gene (H) was cloned with XhoI-ApaI site to be in the C-Terminal of 6XHis-TtsfGFP of pVTtsfGFP vector and microscopic analysis revealed the fusion protein localizes in macronucleus and micronucleus in Tetrahymena thermophila. Affinitic purifiability of TtsfGFP and TtsfGFP-H with Ni-NTA was confirmed by SDS PAGE and Western blotting experiments. Findings of this study suggest that 6XHis-TtsfGFP tag can be use as a protein localization and affinity purification tag for recombinant protein expression in Tetrahymena thermophila.