Overexpression in Escherichia coli, purification and characterization of Thermoanaerobacter tengcongensis elongation factor G with multiple rare codons.

The fusA gene encoding a thermophilic protein EF-G with multiple rare condons was cloned from Thermoanaerobacter tengcongensis (TteEF-G) and overexpressed in Escherichia coli by cotransfering a RIG plasmid to overcome the potential codon-bias problem originated from Arg, Ile and Gly. The recombinant protein was identified by MALDI-TOF-MS for molecular mass with approximation of 76 kDa and by trypsin digestion coupled LC-MS/MS for peptide sequence coverage of 61.3%. The in vivo complementary assay indicates that TteEF-G could significantly rescue the E. coli LJ14 (frr(ts)) at the non-permission temperature of 42 degrees C in the bi-transformant of TteRRF and TteEF-G. This study indicated that coexpression of rare codons' cognate tRNA is a useful method for protein overexpression in E. coli.