Reconstitution and purification of eukaryotic initiation factor 2B (eIF2B) expressed in Sf21 insect cells

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Abstract

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 reconstituted in vivo by 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 elF2B complex had high GEF activity as assayed by using [3H]GDP-bound to eIF2 as a substrate. Alternatively, eIF2B with high GEF activity was reconstituted in vitro by mixing crude cell lysates containing different eIF2B subunits. The latter results suggest that eIF2B activity in vivo could 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.

Original languageEnglish (US)
Pages (from-to)16-22
Number of pages7
JournalProtein Expression and Purification
Volume13
Issue number1
DOIs
StatePublished - Jan 1 1998

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Eukaryotic Initiation Factor-2B
Sf9 Cells
Insects
Guanosine Triphosphate
Baculoviridae
Eukaryotic Initiation Factors
Guanine Nucleotide Exchange Factors
Peptide Initiation Factors
Protein Subunits
Gel Chromatography
Epitopes
Alleles

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

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title = "Reconstitution and purification of eukaryotic initiation factor 2B (eIF2B) expressed in Sf21 insect cells",
abstract = "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 reconstituted in vivo by 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 elF2B complex had high GEF activity as assayed by using [3H]GDP-bound to eIF2 as a substrate. Alternatively, eIF2B with high GEF activity was reconstituted in vitro by mixing crude cell lysates containing different eIF2B subunits. The latter results suggest that eIF2B activity in vivo could 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.",
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