Expression of Functional eIF-4Ehuman: Purification, Detailed Characterization, and Its Use in Isolating eIF-4E Binding Proteins

• Published in: Protein expression and purification 1997-02, Vol.9 (1), p.53-60
• Main Authors: Hagedorn, Curt H., Spivak-Kroizman, Taly, Friedland, Diana E., Goss, Dixie J., Xie, Yiping
• Format: Article
• Language: English
• Subjects: Chromatography, Affinity; Escherichia coli - genetics; Eukaryotic Initiation Factor-4E; Gene Expression Regulation; Humans; Peptide Initiation Factors - genetics; Peptide Initiation Factors - isolation & purification; Peptide Initiation Factors - metabolism; Protein Binding; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; RNA Caps - metabolism; RNA-Binding Proteins - genetics; RNA-Binding Proteins - isolation & purification; RNA-Binding Proteins - metabolism; Tumor Cells, Cultured
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1996.0661

Protein–mRNA cap interactions represent a critical point for regulating gene expressionin 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-4Ehumanwas 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 × 105m−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 inEscherichia coliprovides a useful tool for studying eIF-4E/protein and eIF-4E/mRNA cap interactions and their role in regulating mammalian gene expression.