title: Efficient expression of codon-adapted affinity tagged super folder green fluorescent protein for synchronous protein localization and affinity purification studies in Tetrahymena thermophila.
abstract: BACKGROUND
  A superior Green Fluorescent Protein (GFP) mutant, known as superfolder GFP (sfGFP), is more soluble, faster folding, and is the brightest of the known GFP mutants. This study aimed to create a codon-adapted sfGFP tag (TtsfGFP) for simultaneous protein localization and affinity purification in Tetrahymena thermophila.
  
  
  RESULTS
  In vivo fluorescence spectroscopic analyses of clones carrying a codon-adapted and 6 × His tagged TtsfGFP cassette showed approximately 2-4-fold increased fluorescence emission compared with the control groups at 3 h. Fluorescence microscopy also revealed that TtsfGFP reached its emission maxima at 100 min, which was much earlier than controls expressing EGFP and sfGFP (240 min). A T. thermophila ATP-dependent DNA ligase domain containing hypothetical gene (H) was cloned into the 3' end of 6 × His-TtsfGFP to assess the affinity/localization dual tag feature. Fluorescence microscopy of the 6 × His-TtsfGFP-H clone confirmed its localization in the macro- and micronucleus of vegetative T. thermophila. Simultaneous affinity purification of TtsfGFP and TtsfGFP-H with Ni-NTA beads was feasible, as shown by Ni-NTA purified proteins analysis by SDS-PAGE and western blotting.
  
  
  CONCLUSIONS
  We successfully codon adapted the N-terminal 6 × His-TtsfGFP tag and showed that it could be used for protein localization and affinity purification simultaneously in T. thermophila. We believe that this dual tag will advance T. thermophila studies by providing strong visual traceability of the target protein in vivo and in vitro during recombinant production of heterologous and homologous proteins.
authors.affiliation: Graduate School of Science, Department of Advance Technologies, Biotechnology Program, Anadolu University, Yunusemre Campus, Eskisehir, Turkey. ylmzgurkan@gmail.com.
authors.forename: Gürkan
authors.initials: G
authors.lastname: Yilmaz
authors.affiliation: Department of Biology, Faculty of Science, Anadolu University, Yunusemre Campus, Eskisehir, Turkey. marslanyolu@anadolu.edu.tr.
authors.forename: Muhittin
authors.initials: M
authors.lastname: Arslanyolu
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003062:Codon / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D016808:Tetrahymena thermophila / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D003062:Codon; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins