EXPRESSION AND PURIFICATION OF THE ALPHA-SUBUNIT OF EUKARYOTIC INITIATION-FACTOR EIF2 - USE AS A KINASE SUBSTRATE

The alpha-subunit of eukaryotic initiation factor eIF2 (eIF2 alpha) pl ays an important role in the regulation of mRNA translation through mo dulation of the interaction of eIF2 and a second initiation factor, eI F2B. The interaction of the two proteins is regulated in vivo by phosp horylation of eIF2 alpha at Ser(51). In the present study, rat eIF2 al pha was expressed in Sf21 cells using the baculovirus expression syste m, The recombinant protein was purified to >90% homogeneity in a singl e immunoaffinity chromatographic step. The protein was free of endogen ous eIF2 alpha kinase activity and was rapidly phosphorylated by the e IF2 alpha kinases HCR and PKR. A variant of eIF2 alpha in which the ph osphorylation site was changed to Ala was also expressed and purified. The variant eIF2 alpha was not phosphorylated by either HCR or PKR, d emonstrating that the kinases specifically phosphorylate the correct s ite in the recombinant protein even in the absence of the other two su bunits of the protein. In summary, a rapid and inexpensive method for obtaining eIF2 alpha has been developed. Use of the wildtype and varia nt forms of eIF2 alpha to measure eIF2 alpha kinase activity in cell a nd tissue extracts should greatly facilitate examination of the regula tion of mRNA translation under a variety of conditions. (C) 1998 Acade mic Press.