EXPRESSION OF FUNCTIONAL EIF-4E(HUMAN) - PURIFICATION, DETAILED CHARACTERIZATION, AND ITS USE IN ISOLATING EIF-4E BINDING-PROTEINS

Protein-mRNA cap interactions represent a critical point for regulatin g gene expression in vivo. For example, a rapid stimulation of gene ex pression at the mRNA level is mediated by insulin regulating the avail ability of functional cap binding protein (eIF-4F). In addition, sever al viruses modify cap binding proteins to regulate host vs viral gene expression. However, little is known about the molecular details of eI F-4E interactions with m(7)GTP mRNA caps, with regulatory proteins (e. g., eIF-4E binding proteins), and with proteins within the eIF-4F comp lex. To study these protein-mRNA and protein-protein interactions in m ammalian systems we have constructed a T7 polymerase-driven expression vector containing the coding sequence for human eIF-4E. Recombinant e IF-4E(human) was purified in a functional state by m(7)GTP affinity ch romatography and Mono Q FPLC. This recombinant protein has biological and physical characteristics that are similar or identical to native e IF-4E. Fluorescence titration studies determined the equilibrium const ant for recombinant eIF-4E/m(7)GTP binding to be 10.1(-1)+/-0.3x10(5) M(-1). To isolate eIF-4E binding proteins, recombinant eIF-4E was link ed to agarose beads and incubated with cell lysates. Several proteins were isolated, including a 220-kDa protein that was confirmed to be th e p220 subunit of eIF-4F by its proteolysis during incubation with lys ates of poliovirus-infected cells. We conclude that recombinant eIF-4E produced in Escherichia coli provides a useful tool for studying eIF- 4E/protein and eIF-4E/mRNA cap interactions and their role in regulati ng mammalian gene expression. (C) 1997 Academic Press.