Bacterial elongation factors EF-Tu, their mutants, chimeric forms, and domains: isolation and purification.
Prokaryotic elongation factors EF-Tu form a family of homologous, three-domain molecular switches catalyzing the binding of aminoacyl-tRNAs to ribosomes during the process of mRNA translation. They are GTP-binding proteins, or GTPases. Binding of GTP or GDP regulates their conformation and thus their activity. Because of their particular structure and regulation, various activities (also outside of the translation system) and a relative abundance they represent attractive tools for studies of many basic but still not fully understood mechanisms both of the translation process, the structure-function relationships in EF-Tu molecules themselves and proteins and energy transduction mechanisms in general. The review critically summarizes procedures for the isolation and purification of native and engineered eubacterial elongation factors EF-Tu and their mutants on a large as well as small scale. Current protocols for the purification of both native and polyHis-tagged or glutathione-S-transferase (GST)-tagged EF-Tu proteins and their variants using conventional procedures and the Ni-NTA-Agarose or Glutathione Sepharose are presented.
Jonák
J
J
Department of Gene Expression, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 37 Prague 6, Czech Republic. jjon@img.cas.cz
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002845:Chromatography / Q000295:instrumentation / Q000379:methods; D008958:Models, Molecular; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D010444:Peptide Elongation Factor Tu