Reconstitution and Purification of Eukaryotic Initiation Factor 2B (eIF2B) Expressed in Sf21 Insect Cells

Eukaryotic initiation factor eIF2B plays a key role in the regulation of protein synthesis through its ability to catalyze the exchange of GDP bound to a second initiation factor, eIF2, for free GTP. In contrast to other GDP–GTP exchange factors (GEFs), which are often single subunit proteins, eIF2B...

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Bibliographic Details
Published in:Protein expression and purification 1998-06, Vol.13 (1), p.16-22
Main Authors: Fabian, John R., Kimball, Scot R., Jefferson, Leonard S.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Baculoviridae - genetics
Base Sequence
Cell Line
Cloning, Molecular
DNA Primers
Eukaryotic Initiation Factor-2 - genetics
Eukaryotic Initiation Factor-2 - metabolism
Eukaryotic Initiation Factor-2B
Guanine Nucleotide Exchange Factors
Proteins - genetics
Proteins - isolation & purification
Proteins - metabolism
Rats
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Spodoptera
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Summary:Eukaryotic initiation factor eIF2B plays a key role in the regulation of protein synthesis through its ability to catalyze the exchange of GDP bound to a second initiation factor, eIF2, for free GTP. In contrast to other GDP–GTP exchange factors (GEFs), which are often single subunit proteins, eIF2B consists of five dissimilar subunits. In the studies reported here the baculovirus expression vector system (BEVS) was used to express FLAG epitope tagged alleles for the α, β, γ, δ, and ϵ subunits of rat eIF2B in Sf21 cells. The eIF2B holoprotein was reconstitutedin vivoby coexpression of all five subunits in Sf21 cells and was subsequently purified to greater than 98% homogeneity using a two-step procedure involving an anti-FLAG immunoaffinity column followed by gel filtration chromatography. The purified five-subunit eIF2B complex had high GEF activity as assayed by using [3H]GDP-bound to eIF2 as a substrate. Alternatively, eIF2B with high GEF activity was reconstitutedin vitroby mixing crude cell lysates containing different eIF2B subunits. The latter results suggest that eIF2B activityin vivocould involve alterations in the concentration and/or the availability of individual subunits for holoprotein assembly. Overall, the results show the utility of the baculovirus–insect cell system for the expression, assembly, and purification of active recombinant multisubunit factors.
ISSN:1046-5928
1096-0279
DOI:10.1006/prep.1998.0860