Cloning, expression and purification of recombinant elongation factor Ts from hyperthermophilic bacteria Geobacillus Anatolicus

Abstract

Elongation factor EF-Ts from Geobacillus anatolicus was cloned, expressed and purifed as a His-tagged recombinant protein in Escherichia coli. The first step in this study was the determination of the nucleic acid sequence of the tsf gene of Geobacillus anatolicus. In order to achive this first goal, sequence data for the tsf gene and the neighbouring genes of the the bacteria phylogenetically related to Geobacillus anatolicus were used to design the appropriate oligonucleotide primers to amplify the chromosomal region carrying the tsf gene. This sequence information was then used to design primers to clone the complete tsf gene including 6 additional histidine residues at its C-terminal into an appropriate expression vector. This vector was used to express and purify the protein in high quantities in Escherichia coli. Ni-affinity chromotography was then used to purify the recombinant protein to homogeneity. Geobacillus anatolicus EF-Ts was found out to be fully active in binding Escherichia coli EF-Tu.