High-level synthesis in Escherichia coli of functional cap-binding eukaryotic initiation factor eIF-4E and affinity purification using a simplified cap-analog resin

Isaac Edery, Michael Altmann, Nahum Sonenberg

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77 Scopus citations

Abstract

Numerous studies have established the important role that eukaryotic initiation factor-4E (eIF-4E) plays during protein biosynthesis. However, biochemical characterization of eIF-4E has proved difficult, mainly because of its low abundance in cells. To facilitate studies on eIF-4E, we have overexpressed Saccharomyces cerevisiae eIF-4E in Escherichia coli. The isolation of eIF-4E was simplified by using a cap-analog affinity matrix (agarose resin) that is considerably less demanding to prepare than those previously reported. We describe a simple and rapid purification scheme that can yield 2-5 μg of a homogenous and active preparation of eIF-4E from 1 ml of E. coli culture. E. coli-expressed eIF-4E is active as determined by its ability to bind the cap structure. The results demonstrate that the cap-binding activity of eIF-4E is not dependent on the presence of other proteins that are present at low levels in eIF-4E preparations isolated from eukaryotic cells.

Original languageEnglish (US)
Pages (from-to)517-525
Number of pages9
JournalGene
Volume74
Issue number2
DOIs
StatePublished - Dec 30 1988
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Genetics

Keywords

  • Saccharomyces cerevisiae
  • Translational initiation
  • overexpression
  • recombinant DNA

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