Expression and Purification of the α-Subunit of Eukaryotic Initiation Factor eIF2: Use as a Kinase Substrate
| Autor: | Rosemary Jagus, Scot R. Kimball, Leonard S. Jefferson, Rick L. Horetsky |
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| Rok vydání: | 1998 |
| Předmět: |
Time Factors
Eukaryotic Initiation Factor-2 Heme Spodoptera Polymerase Chain Reaction Chromatography Affinity Cell Line eIF-2 Kinase Eukaryotic initiation factor Animals Initiation factor Phosphorylation DNA Primers eIF2 Base Sequence biology EIF4A1 Protein kinase R Recombinant Proteins Eukaryotic translation initiation factor 4 gamma Rats Repressor Proteins EIF4EBP1 Gene Expression Regulation Biochemistry eIF2B biology.protein Electrophoresis Polyacrylamide Gel Baculoviridae Biotechnology |
| Zdroj: | Protein Expression and Purification. 12:415-419 |
| ISSN: | 1046-5928 |
| DOI: | 10.1006/prep.1998.0863 |
| Popis: | The alpha-subunit of eukaryotic initiation factor eIF2 (eIF2alpha) 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 eIF2alpha at Ser51. In the present study, rat eIF2alpha was expressed in Sf21 cells using the baculovirus expression system. The recombinant protein was purified to90% homogeneity in a single immunoaffinity chromatographic step. The protein was free of endogenous eIF2alpha kinase activity and was rapidly phosphorylated by the eIF2alpha kinases HCR and PKR. A variant of eIF2alpha in which the phosphorylation site was changed to Ala was also expressed and purified. The variant eIF2alpha 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 eIF2alpha has been developed. Use of the wildtype and variant forms of eIF2alpha to measure eIF2alpha kinase activity in cell and tissue extracts should greatly facilitate examination of the regulation of mRNA translation under a variety of conditions. |
| Databáze: | OpenAIRE |
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