Cloning and expression of P-Brutia GST-zeta in E-coli

Abstract

GlutathioneS-Transferases (GSTs, EC.2.5.1.18) are complex andwidespread enzyme superfamily that detoxify endobiotics and xen-obiotics, and forming generally water soluble and less toxic gluta-thione(GSH)S-conjugate. Since 70’s plant GSTs have beenintensively studied, as their role discovered in herbicide detoxifica-tion. But, there is only very limited number of studies consideringthe molecular characterization of GSTs in gymnosperms, especiallynot inPinus brutia,Ten (Kizilc ̧am) which is the most importantforest tree in the north-eastern Mediterranean area, including Tur-key. Valuing this and considering the role of GST’s under stressconditions, it was decided to examine the cloning and expressionof GST-Zeta isoenzymes inE.coli. After purification of recombin-ant protein the effect of different substrates on expressed proteinwould also be investigated. In this study, the total RNA was isola-ted fromPinus brutianeedles and subsequently cDNA was pre-pared with RT-PCR strategy.PbGST-Zeta gene was amplified bygene specific primers designed to open reading frame of thesequence ofPbGST-Zeta gene and then cloned into pGEM-Teasyvector. DNA sequencing was performed to confirm the sequence inpGEM-T-easy vector, and found as composed of 226 amino acids.Afterwords, thePbGST-Zeta was subcloned into expression vectorpET21a(+). IPTG induced expression conditions ofPbGST-Zetawere optimized inE. coli(BL21 Codon Plus).