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Use of nonreducing SDS-PAGE for monitoring renaturation of recombinant protein synthesis initiation factor, eIF-4 alpha.

Abstract：

: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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Stern BD,Wilson M,Jagus R

doi

10.1006/prep.1993.1041

subject

Has Abstract

pub_date

1993-08-01 00:00:00

pages

320-7

issue

4

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(83)71041-7

journal_volume

4

pub_type

杂志文章

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