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 language | English (US) |
|---|---|
| Pages (from-to) | 16-22 |
| Number of pages | 7 |
| Journal | Protein Expression and Purification |
| Volume | 13 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 1 1998 |
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All Science Journal Classification (ASJC) codes
- Biotechnology
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Reconstitution and purification of eukaryotic initiation factor 2B (eIF2B) expressed in Sf21 insect cells. / Fabian, John R.; Kimball, Scot; Jefferson, Leonard "Jim".
In: Protein Expression and Purification, Vol. 13, No. 1, 01.01.1998, p. 16-22.Research output: Contribution to journal › Article
TY - JOUR
T1 - Reconstitution and purification of eukaryotic initiation factor 2B (eIF2B) expressed in Sf21 insect cells
AU - Fabian, John R.
AU - Kimball, Scot
AU - Jefferson, Leonard "Jim"
PY - 1998/1/1
Y1 - 1998/1/1
N2 - 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.
AB - 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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UR - http://www.scopus.com/inward/citedby.url?scp=0032103789&partnerID=8YFLogxK
U2 - 10.1006/prep.1998.0860
DO - 10.1006/prep.1998.0860
M3 - Article
C2 - 9631509
AN - SCOPUS:0032103789
VL - 13
SP - 16
EP - 22
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
IS - 1
ER -