Expression, purification and characterization of recombinant mouse translation initiation factor eIF4E as a dihydrofolate reductase (DHFR) fusion protein.
One of the earliest steps in translation initiation is recognition of the mRNA cap structure (m7GpppX) by the initiation factor eIF4E. Studies of interactions between purified eIF4E and its binding partners provide important information for understanding mechanisms underlying translational control in normal and cancer cells. Numerous impediments of the available methods used for eIF4E purification led us to develop a novel methodology for obtaining fractions of eIF4E free from undesired by-products. Herein we report methods for bacterial expression of eIF4E tagged with mutant dihydrofolate reductase (DHFR) followed by isolation and purification of the DHFR-eIF4E protein by using affinity and anion exchange chromatography. Fluorescence quenching experiments indicated the cap-analog, 7MeGTP, bound to DHFR-eIF4E and eIF4E with a dissociation constant (K(d)) of 6+/-5 and 10+/-3 nM, respectively. Recombinant eIF4E and DHFR-eIF4E were both shown to significantly enhance in vitro translation in dose dependent manner by 75% at 0.5 microM. Nevertheless increased concentrations of eIF4E and DHFR-eIF4E significantly inhibited translation in a dose dependent manner by a maximum at 2 microM of 60% and 90%, respectively. Thus, we have demonstrated that we have developed an expression system for fully functional recombinant eIF4E. We have also shown that the fusion protein DHFR-eIF4E is functional and thus may be useful for cell based affinity tag studies with fluorescently labeled trimethoprim analogs.
Ghosh
Phalguni
P
Department of Medicinal Chemistry, University of Minnesota, 8-174 Weaver Densford Hall, 308 Harvard Street S.E., Minneapolis, MN 55455, USA.
Cheng
Jilin
J
Chou
Tsui-Fen
TF
Jia
Yan
Y
Avdulov
Svetlana
S
Bitterman
Peter B
PB
Polunovsky
Vitaly A
VA
Wagner
Carston R
CR
D000818:Animals; D001483:Base Sequence; D002853:Chromatography, Liquid / Q000379:methods; D017931:DNA Primers; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics*; D005453:Fluorescence; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D017931:DNA Primers; D039561:Eukaryotic Initiation Factor-4E; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase