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 (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 to >90% 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.
Kimball
S R
SR
Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Horetsky
R L
RL
Jagus
R
R
Jefferson
L S
LS
D000818:Animals; D016367:Baculoviridae / Q000502:physiology; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics*; D006418:Heme / Q000494:pharmacology; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000187:drug effects / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000821:virology; D013997:Time Factors; D019892:eIF-2 Kinase / Q000378:metabolism
D017931:DNA Primers; D015852:Eukaryotic Initiation Factor-2; D011994:Recombinant Proteins; D012097:Repressor Proteins; D006418:Heme; D019892:eIF-2 Kinase