TY - JOUR
T1 - Expression and purification of the α-subunit of eukaryotic initiation factor eIF2
T2 - Use as a kinase substrate
AU - Kimball, Scot R.
AU - Horetsky, Rick L.
AU - Jagus, Rosemary
AU - Jefferson, Leonard S.
N1 - Funding Information:
The authors acknowledge the excellent technical assistance of Lynne Hugendubler and Joan McGwire. This work was supported in part by Grants DK13499 and DK15658 from the National Institutes of Health (to L.S.J.) and Grant MCB9317264 from the National Science Foundation (to R.J.).
PY - 1998/4
Y1 - 1998/4
N2 - The α-subunit of eukaryotic initiation factor eIF2 (eIF2α) plays an important role in the regulation of mRNA translation through modulation of the interaction of eIF2 and a second initiation factor, eIF2B. The interaction of the two proteins is regulated in vivo by phosphorylation of eIF2α at Ser51. In the present study, rat eIF2α was expressed in Sf21 cells using the baculovirus expression system. The recombinant protein was purified to >90% homogeneity in a single immunoaffinity chromatographic step. The protein was free of endogenous eIF2α kinase activity and was rapidly phosphorylated by the eIF2α kinases HCR and PKR. A variant of eIF2α in which the phosphorylation site was changed to Ala was also expressed and purified. The variant eIF2α was not phosphorylated by either HCR or PKR, demonstrating that the kinases specifically phosphorylate the correct site in the recombinant protein even in the absence of the other two subunits of the protein. In summary, a rapid and inexpensive method for obtaining eIF2α has been developed. Use of the wildtype and variant forms of eIF2α to measure eIF2α kinase activity in cell and tissue extracts should greatly facilitate examination of the regulation of mRNA translation under a variety of conditions.
AB - The α-subunit of eukaryotic initiation factor eIF2 (eIF2α) plays an important role in the regulation of mRNA translation through modulation of the interaction of eIF2 and a second initiation factor, eIF2B. The interaction of the two proteins is regulated in vivo by phosphorylation of eIF2α at Ser51. In the present study, rat eIF2α was expressed in Sf21 cells using the baculovirus expression system. The recombinant protein was purified to >90% homogeneity in a single immunoaffinity chromatographic step. The protein was free of endogenous eIF2α kinase activity and was rapidly phosphorylated by the eIF2α kinases HCR and PKR. A variant of eIF2α in which the phosphorylation site was changed to Ala was also expressed and purified. The variant eIF2α was not phosphorylated by either HCR or PKR, demonstrating that the kinases specifically phosphorylate the correct site in the recombinant protein even in the absence of the other two subunits of the protein. In summary, a rapid and inexpensive method for obtaining eIF2α has been developed. Use of the wildtype and variant forms of eIF2α to measure eIF2α kinase activity in cell and tissue extracts should greatly facilitate examination of the regulation of mRNA translation under a variety of conditions.
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U2 - 10.1006/prep.1998.0863
DO - 10.1006/prep.1998.0863
M3 - Article
C2 - 9535710
AN - SCOPUS:0032052990
SN - 1046-5928
VL - 12
SP - 415
EP - 419
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 3
ER -