title: High-level synthesis in Escherichia coli of functional cap-binding eukaryotic initiation factor eIF-4E and affinity purification using a simplified cap-analog resin.
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 micrograms 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.
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Canada.
authors.forename: I
authors.initials: I
authors.lastname: Edery
authors.forename: M
authors.initials: M
authors.lastname: Altmann
authors.forename: N
authors.initials: N
authors.lastname: Sonenberg
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D039561:Eukaryotic Initiation Factor-4E; D005786:Gene Expression Regulation; D010084:Oxidation-Reduction; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000302:isolation & purification; D012314:RNA Cap Analogs; D012315:RNA Caps; D013329:Structure-Activity Relationship
chemical_list: D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D012314:RNA Cap Analogs; D012315:RNA Caps