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Gene. 1988 Dec 30;74(2):517-25.

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

Author information

1
Department of Biochemistry, McGill University, Montreal, Canada.

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.

PMID:
3246354
DOI:
10.1016/0378-1119(88)90184-9
[Indexed for MEDLINE]

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