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

Abstract

In this study, elongation factor Tu (EF-Tu) from Geobacillus anatolicus was cloned, expressed and purifed as a His-tagged recombinant protein in Escherichia coli. At first, the nucleic acid sequence of Geobacillus anatolicus tuf gene was determined by using sequence data obtained from the bacteria phylogenetically related to Geobacillus anatolicus. The tuf gene and its neighbouring genes of the tuf gene sequences were used to design appropriate oligonucleotide primers and these primers were used to amplify the chromosomal region carrying the tuf gene of Geobacillus anatolicus. The sequence information from this fragment was then used to design primers to sequence and clone the complete tuf gene into an appropriate expression vector. This vector was used to express and purify the protein in high quantities in Escherichia coli. Six additional histidine residues were inserted into the recombinant protein of Geobacillus anatolicus at its Cterminal region to purify the protein using Ni-affinity chromotography to homogeneity. It was found that recombinant Geobacillus anatolicus EF-Tu is fully competent in forming a binary complex with Geobacillus anatolicus EF-Ts. It was determined that it has a significantly higher mobility rate in non-denaturing PAGE as compared to Escherichia coli EF-Tu, not explainable with molecular mass difference between these proteins. The mobility of the Geobacillus anatolicus EF-Tu.EF-Ts complex does not differ from the mobility of the Geobacillus anatolicus EF-Ts, indicating a large conformational change of EF-Tu upon binding to EF-Ts. The recombinant Geobacillus anatolicus EF-Tu can form a ternary complex with GTP and Phe-tRNAPhe. Geobacillus anatolicus retained its GDP binding activity fully after incubation at 60°C for 10 min, and it still has approximately %20 of its activity after incubating for 10 min at 80°C.