Purification of elongation factors EF-Tu and EF-G from Escherichia coli by covalent chromatography on thiol-sepharose.
The elongation factors EF-Tu and EF-G of Escherichia coli are involved in the transport of aminoacyl-tRNA to ribosomes and the translocation of ribosomes on mRNA, respectively. Both possess cysteine residues that are important for activity. We took advantage of this property to design a purification protocol based on thiol-Sepharose chromatography, a method involving thiol-disulfide interchange between protein thiol groups and the glutathione-2-pyridyl-disulfide conjugate of the affinity resin. Bacterial cells were lysed by a lysozyme-EDTA method, and the lysate supernatant was purified by chromatography on, first, DEAE-Sephacel and, then thiol-Sepharose. Both elongation factors were purified in a single procedure, since DEAE-Sephacel fractions containing both factors were loaded on the thiol-Sepharose column. Thiol-Sepharose chromatography efficiently separates each elongation factor from all contaminating proteins. The purified elongation factors were characterized by SDS-PAGE, protein sequencing, and biological activity. The specific reactivities of the elongation factors with thiol-Sepharose allow their efficient purification and suggest that they possess hitherto undiscovered properties connected with their reactive thiols.
Biochimie génétique, Institut Jacques Monod, Université Paris 7, 2 place Jussieu, Paris Cedex 05, 75251, France.
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Caldas
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El Yaagoubi
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Kohiyama
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Richarme
D000595:Amino Acid Sequence; D002847:Chromatography, Agarose / Q000379:methods*; D002848:Chromatography, DEAE-Cellulose; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000737:chemistry / Q000302:isolation & purification; D006153:Guanosine Diphosphate / Q000378:metabolism; D008970:Molecular Weight; D020653:Peptide Elongation Factor G; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives
D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D006153:Guanosine Diphosphate; C037851:thiol-sepharose; D012685:Sepharose; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu