title: Extremely thermostable elongation factor G from Aquifex aeolicus: cloning, expression, purification, and characterization in a heterologous translation system.
abstract: The fus gene of the translation factor G (EF-G) from the hyperthermophilic bacterium Aquifex aeolicus was cloned under control of a phage promoter and overexpressed in Escherichia coli with the T7 RNA polymerase system. A heat denaturation step at 95 degrees C was used to purify the protein from the cell extract. This approach simplified the chromatographic procedures and decreased the protein loss since most of Escherichia coli proteins were denatured and precipitated. Ten milligrams of the highly purified protein was isolated from 4 liters of induced culture. The overproduced EF-G was active in ribosome-dependent GTP hydrolysis and a poly(U)-directed polyphenylalanine translation system with E. coli 70S ribosomes. The method presented here might facilitate functional and structural studies of important components of the protein biosynthesis system.
authors.affiliation: Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142292, Russia.
authors.forename: K A
authors.initials: KA
authors.lastname: Martemyanov
authors.forename: A
authors.initials: A
authors.lastname: Liljas
authors.forename: A T
authors.initials: AT
authors.lastname: Gudkov
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002457:Cell Extracts; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016946:Gram-Negative Aerobic Rods and Cocci / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D020653:Peptide Elongation Factor G / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D014176:Protein Biosynthesis; D011489:Protein Denaturation
chemical_list: D001426:Bacterial Proteins; D002457:Cell Extracts; D020653:Peptide Elongation Factor G; D010455:Peptides; C012001:polyphenylalanine