TAILIEUCHUNG - Characterization of affinity tag features of recombinant Tetrahymena thermophila glutathione-S-transferase zeta for Tetrahymena protein expression vectors
Glutathione S-transferase (GST) is the one of most widely used affinity tags in biotechnology applications. The present study named a GST gene as the TtGSTz1 gene, characterized with the conserved glutathione binding motif. | Turk J Biol 36 (2012) 513-526 © TÜBİTAK doi: Characterization of affinity tag features of recombinant Tetrahymena thermophila glutathione-S-transferase zeta for Tetrahymena protein expression vectors Cem ÖZİÇ1, Muhittin ARSLANYOLU2 1 Department of Biology, Faculty of Sciences, Kafkas University, 36100 Kars - TURKEY 2 Department of Biology, Faculty of Sciences, Anadolu University, Yunusemre Campus, 26470 Eskisehir - TURKEY Received: ● Accepted: Abstract: Glutathione S-transferase (GST) is the one of most widely used affinity tags in biotechnology applications. The present study named a GST gene as the TtGSTz1 gene, characterized with the conserved glutathione binding motif (SSTSWRVRIAL) under a GST zeta subfamily from Tetrahymena thermophila. Phylogenetic analysis of the TtGstz1p protein sequence, with its orthologs from different GST classes, showed that it is a member of the unicellular GSTz monophyletic clade, which is positioned close to the bacterial GSTz clade. Seven codons of the TtGSTz1 gene were first engineered by introducing silent mutations (TAA > CAA or TAG > CAG) to the code for glutamine instead of stop signals in E. coli. The recombinant TtGSTz1 gene, with its 6XHis tag, was expressed using the pET16b expression plasmid and E. coli BL21(DE3). SDS-PAGE analysis of the 6XHis-TtGstz1p protein confirmed its expression and purification by nickel agarose and glutathione-sepharose 4B. Additionally, both anti-His and anti-GST antibodies recognized the purified 6XHis-TtGSTz in the western blot analysis. In conclusion, the results presented here suggest that the 6XHisTtGstz1p fusion protein could be used as a dual tag in Tetrahymena expression vectors with a sequential 2-step protein purification procedure. Key words: GSTzeta, Tetrahymena thermophila, recombinant, E. coli, protein expression, tag Introduction The characterization of gene function at the protein level requires the production and purification of
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