Expression and purification of the subunits of human translational initiation factor 2 (eIF2): phosphorylation of eIF2 alpha and beta.
Eukaryotic initiation factor 2 (eIF2) is a GDP-binding protein with three subunits: alpha, beta, and gamma. It delivers initiator tRNA (Met-tRNAi) to 40S ribosomes in a GTP-dependent manner. The factor regulates the translation of messenger RNAs through the phosphorylation of serine 51 residue in the small or alpha-subunit of eIF2 (eIF2alpha) and modulation of its interaction with a rate-limiting heteropentameric protein eIF2B. To understand the structural, functional, and regulatory roles of each of these subunits in the various activities of phosphorylated and unphosphorylated eIF2, such, as its ability to interact with GTP, Met-tRNAi, 40S ribosomes and with various proteins, we have for the first time over expressed all the three subunits of human eIF2 independently, and, also together in Sf9 cells using pFast Bac HT vector of baculovirus expression system. The expression of all subunits increased with increase in infection time up to 72 h. We have also over expressed three mutant forms of eIF2alpha viz, S51A, S51D, and S48A in which the serine at 51 or 48 position is replaced by an alanine or aspartic acid with 6x histidine tag at the N-terminus. Further, any of the two subunits or all the three subunits of eIF2 were coexpressed by multiple infection of cells with recombinant viruses. Purified alpha (wt and mutants) and beta subunits were found suitable to serve as substrates for different kinases. The recombinant subunits of eIF2alpha and beta-subunits were also phosphorylated in cultured insect cells. Phosphorylation of eIF2alpha in vitro was not significantly different in the presence and absence of the other subunits.
Suragani
Rajasekhar N V S
RN
Department of Biochemistry, University of Hyderabad, Hyderabad 500 046, India.
Ghosh
Sudip
S
Ehtesham
Nasreen Z
NZ
Ramaiah
Kolluru V A
KV
D019943:Amino Acid Substitution / Q000235:genetics; D003001:Cloning, Molecular; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D020717:Eukaryotic Initiation Factor-2B; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D021122:Protein Subunits / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
D015852:Eukaryotic Initiation Factor-2; D020717:Eukaryotic Initiation Factor-2B; D021122:Protein Subunits