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

The alpha-subunit of eukaryotic initiation factor eIF2 (eIF2 alpha) 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 eIF2 alpha at Ser(51). In the present study, rat eIF2 alpha 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 eIF2 alpha kinase activity and was rapidly phosphorylated by the eIF2 alpha kinases HCR and PKR. A variant of eIF2 alpha in which the phosphorylation site was changed to Ala was also expressed and purified. The variant eIF2 alpha 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 eIF2 alpha has been developed. Use of the wildtype and variant forms of eIF2 alpha to measure eIF2 alpha kinase activity in cell and tissue extracts should greatly facilitate examination of the regulation of mRNA translation under a variety of conditions. (C) 1998 Academic Press.