Purification of elongation factors EF-Tu and EF-G from Escherichia coli by covalent chromatography on thiol-sepharose

被引:9
作者
Caldas, TD [1 ]
El Yaagoubi, A [1 ]
Kohiyama, M [1 ]
Richarme, G [1 ]
机构
[1] Univ Paris 07, Inst Jacques Monod, F-75251 Paris 05, France
关键词
D O I
10.1006/prep.1998.0922
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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, (C) 1998 Academic Press.
引用
收藏
页码:65 / 70
页数:6
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