Cloning, experimental and bioinformatic characterization of glutathion S-transferase zeta (GST-Z) gene in Tetrahymena thermophila

Enzymes of the Glutathione-S-transferase (GST) help to detoxificate effects of endogen and exogenous toxic compounds in living organisms. Results of the Tetrahymena genome project showed that T. thermophila has 19 different GST genes that represent members only from four subgroups as theta, omega, mu, zeta. mRNA sequences of TtGSTz gene (847 bp) was determined by using genomic sequence (77.m00138) from genome project, EST sequences of cDNAs from EST projects and experimental result of 3?RACE. The region of TtGSTz cDNA gene between start and stop codon was cloned and sequenced to confirm the existence of the gene in T. thermophila SB210. Comparison of genomic and cDNA sequence of TtGSTz gene by PCR and agarose gel analysis showed that there are totally 122 bp long intron in the genome copy. Multiple alignment of TtGSTz amino acid sequence with other GST zeta homologs showed the existence of conserved characteristic motif (SSCX[WH]RVIAL) of zeta subgroup in TtGSTz. Change in expression of TtGSTz mRNA level under the hydrogen peroxide and cold (4ºC) stresses with time intervals was analyzed with semi-quantitative RT-PCR method. After seven necessary multiple point mutations (TAA > CAA or TAG > CAG) on mRNA protein coding region of TtGSTz gene, it was transferred to pET16b expression plasmid and recombinant 6XHis-TtGSTz protein was possible to produce in E. coli BL21-DE3. Recombinant 6XHis-TtGSTz protein (24 kDa) was purified not only by using nickel agarose but also by gluthathion-sepharose 4B. Analysis of recombinant 6XHis-TtGSTz by SDS-PAGE showed that the protein was successfully produced by both purification methods. Anallyzes of purified 6xHis-TtGSTz by using anti-His antibody in the Western blot showed that the recombinant protein was succesfully produced.