title: Overexpression in Escherichia coli, purification and characterization of Thermoanaerobacter tengcongensis elongation factor G with multiple rare codons.
abstract: 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.
authors.affiliation: Center of System Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Liqiang
authors.initials: L
authors.lastname: Zhang
authors.forename: Peng
authors.initials: P
authors.lastname: Xue
authors.forename: Hongjie
authors.initials: H
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D020653:Peptide Elongation Factor G / Q000096:biosynthesis* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D046968:Thermoanaerobacter / Q000737:chemistry*
chemical_list: D020653:Peptide Elongation Factor G