Efficient expression of codon-adapted affinity tagged super folder green fluorescent protein for synchronous protein localization and affinity purification studies in Tetrahymena thermophila

WOS: 000354430200001
PubMed ID: 25887423
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 x 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 x His-TtsfGFP to assess the affinity/localization dual tag feature. Fluorescence microscopy of the 6 x 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 x 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.
Anadolu University Scientific Research Projects Commission (AUBAP) [1109F147]; TUBITAK (The Scientific and Research Council of Turkey) [113Z036, 112T422]
We first thank Aaron Turkewitz for providing vector pVGF. We owe special thanks to Nurcin Kucukoglu for her help in the localization experiments and Serkan Dereli for his help in the digital preparation of the figures. This study was supported by the Anadolu University Scientific Research Projects Commission (AUBAP) under Grant No. 1109F147 and TUBITAK (The Scientific and Research Council of Turkey) under Grant Nos. 113Z036 and 112T422 offered to Muhittin Arslanyolu.