Use of nonreducing SDS-PAGE for monitoring renaturation of recombinant protein synthesis initiation factor, eIF-4 alpha.
The purification of biologically active human protein synthesis initiation factor 4 alpha, eIF-4 alpha, overexpressed in Escherichia coli, is complicated by its localization in insoluble inclusion bodies, as well as its possession of four cysteines. Two of these cysteines have been reported to be reduced in the native molecule and two form a disulfide bond. A method is described, using nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis, for monitoring renaturation of the polypeptide during staged dialyses in decreasing urea concentrations. The production of biologically active eIF-4 alpha only occurs when the polypeptide is totally reduced during solubilization of the inclusion bodies in 8 M urea. This requires a minimum dithiothreitol concentration of 50 mM. Conversely, reformation of the disulfide bonds only occurs when the staged dialyses are performed at lower concentrations of sulfhydryl reagents. Once renatured, as described, eIF-4 alpha can be purified by affinity chromatography on m7GTP-Sepharose. Approximately 20 micrograms of biologically active eIF-4 alpha per milliliter of bacterial culture can be obtained. The affinity-purified eIF-4 alpha has activity equivalent to that reported for purified native human eIF-4 alpha, as measured by its activity in a rabbit reticulocyte translation system. The method described is applicable to the purification of other cysteine-containing polypeptides that accumulate to high levels in inclusion bodies.
Center of Marine Biotechnology, Maryland Biotechnology Institute, Baltimore 21202.
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D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism
D010448:Peptide Initiation Factors; D011994:Recombinant Proteins; C075833:eIF-4; D004247:DNA; D004229:Dithiothreitol