title: Expression of functional eIF-4Ehuman: purification, detailed characterization, and its use in isolating eIF-4E binding proteins.
abstract: Protein-mRNA cap interactions represent a critical point for regulating gene expression in vivo. For example, a rapid stimulation of gene expression at the mRNA level is mediated by insulin regulating the availability of functional cap-binding protein (eIF-4E). In addition, several viruses modify cap binding proteins to regulate host vs viral gene expression. However, little is known about the molecular details of eIF-4E interactions with m7GTP mRNA caps, with regulatory proteins (e.g., eIF-4E binding proteins), and with proteins within the eIF-4F complex. To study these protein-mRNA and protein-protein interactions in mammalian systems we have constructed a T7 polymerase-driven expression vector containing the coding sequence for human eIF-4E. Recombinant eIF-4Ehuman was purified in a functional state by m7GTP affinity chromatography and Mono Q FPLC. This recombinant protein has biological and physical characteristics that are similar or identical to native eIF-4E. Fluorescence titration studies determined the equilibrium constant for recombinant eIF-4E/m7GTP binding to be 10.1 +/- 0.3 x 10(5) M(-1). To isolate eIF-4E binding proteins, recombinant eIF-4E was linked to agarose beads and incubated with cell lysates. Several proteins were isolated, including a 220-kDa protein that was confirmed to be the p220 subunit of eIF-4F by its proteolysis during incubation with lysates 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 regulating mammalian gene expression.
authors.affiliation: Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA. changedo@bimcore.emory.edu
authors.forename: C H
authors.initials: CH
authors.lastname: Hagedorn
authors.forename: T
authors.initials: T
authors.lastname: Spivak-Kroizman
authors.forename: D E
authors.initials: DE
authors.lastname: Friedland
authors.forename: D J
authors.initials: DJ
authors.lastname: Goss
authors.forename: Y
authors.initials: Y
authors.lastname: Xie
mesh_terms: D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D039561:Eukaryotic Initiation Factor-4E; D005786:Gene Expression Regulation; D006801:Humans; D010448:Peptide Initiation Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D012315:RNA Caps / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D012315:RNA Caps; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins