PMID:9758752: Difference between revisions

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<span style = 'font-size: large'> '''Caldas, TD, El Yaagoubi, A, Kohiyama, M and Richarme, G'''  (1998) Purification of elongation factors EF-Tu and EF-G from Escherichia coli by covalent chromatography on thiol-sepharose.''Protein Expr. Purif.'' '''14''':65-70</span>
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|{{TableHeadingColor}} |'''Abstract''' || 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.
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'''Caldas, TD, El Yaagoubi, A, Kohiyama, M and Richarme, G'''  (1998) Purification of elongation factors EF-Tu and EF-G from Escherichia coli by covalent chromatography on thiol-sepharose. ''Protein Expr. Purif.'' '''14''':65-70
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!align=left  |Abstract
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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.
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[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9758752 PubMed]
Online version:[http://dx.doi.org/10.1006/prep.1998.0922 10.1006/prep.1998.0922]
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|{{TableHeadingColor}} nowrap |'''Links''' ||  [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9758752 PubMed]
!align=left  |Keywords
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Amino Acid Sequence; Chromatography, Agarose/methods; Chromatography, DEAE-Cellulose; Escherichia coli/genetics; Escherichia coli/metabolism; GTP Phosphohydrolase-Linked Elongation Factors/chemistry; GTP Phosphohydrolase-Linked Elongation Factors/isolation & purification; Guanosine Diphosphate/metabolism; Molecular Weight; Peptide Elongation Factor G; Peptide Elongation Factor Tu/genetics; Peptide Elongation Factor Tu/isolation & purification; Peptide Elongation Factor Tu/metabolism; Peptide Elongation Factors/genetics; Peptide Elongation Factors/isolation & purification; Peptide Elongation Factors/metabolism; Sepharose/analogs & derivatives
 
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Latest revision as of 05:05, 15 September 2013

Citation

Caldas, TD, El Yaagoubi, A, Kohiyama, M and Richarme, G (1998) Purification of elongation factors EF-Tu and EF-G from Escherichia coli by covalent chromatography on thiol-sepharose. Protein Expr. Purif. 14:65-70

Abstract

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.

Links

PubMed Online version:10.1006/prep.1998.0922

Keywords

Amino Acid Sequence; Chromatography, Agarose/methods; Chromatography, DEAE-Cellulose; Escherichia coli/genetics; Escherichia coli/metabolism; GTP Phosphohydrolase-Linked Elongation Factors/chemistry; GTP Phosphohydrolase-Linked Elongation Factors/isolation & purification; Guanosine Diphosphate/metabolism; Molecular Weight; Peptide Elongation Factor G; Peptide Elongation Factor Tu/genetics; Peptide Elongation Factor Tu/isolation & purification; Peptide Elongation Factor Tu/metabolism; Peptide Elongation Factors/genetics; Peptide Elongation Factors/isolation & purification; Peptide Elongation Factors/metabolism; Sepharose/analogs & derivatives

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