Expression and purification of the α-subunit of eukaryotic initiation factor eIF2: Use as a kinase substrate

Scot Kimball, Rick L. Horetsky, Rosemary Jagus, Leonard "Jim" Jefferson

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)415-419
Number of pages5
JournalProtein Expression and Purification
Volume12
Issue number3
DOIs
StatePublished - Jan 1 1998

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Eukaryotic Initiation Factors
Phosphotransferases
Protein Biosynthesis
Recombinant Proteins
Phosphorylation
Sf9 Cells
Peptide Initiation Factors
Tissue Extracts
Baculoviridae
Protein Subunits
Cell Extracts
Proteins

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

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abstract = "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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Expression and purification of the α-subunit of eukaryotic initiation factor eIF2 : Use as a kinase substrate. / Kimball, Scot; Horetsky, Rick L.; Jagus, Rosemary; Jefferson, Leonard "Jim".

In: Protein Expression and Purification, Vol. 12, No. 3, 01.01.1998, p. 415-419.

Research output: Contribution to journalArticle

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