Expression, purification and characterization of recombinant mouse translation initiation factor eIF4E as a dihydrofolate reductase (DHFR) fusion protein

Abstract

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 byproducts. 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 analogue, 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 uM. Nevertheless increased concentrations of eIF4E and DHFR-eIF4E significantly inhibited translation in a dose dependent manner by a maximum at 2 uM 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.

Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress

“…We therefore analyzed eIF4E binding to RNAs with different first nucleotides. Most previous studies of eIF4E binding affinity used cap analogs such as m7GTP or m7GpppG and reported Kd values ranging from low μM to few nM ( Ueda et al, 1991 ; Carberry et al, 1992 ; Minich et al, 1994 ; Sha et al, 1995 ; Hagedorn et al, 1997 ; Miyoshi et al, 1999 ; Wieczorek et al, 1999 ; Hsu et al, 2000 ; von Der Haar et al, 2000 ; Niedzwiecka et al, 2002 ; Scheper et al, 2002 ; Ghosh et al, 2008 ; Thillier et al, 2012 ). We chemically synthesized three capped RNA oligos as previously described ( Lavergne et al, 2008 ; Thillier et al, 2012 ), 10 nt long each that differ in their first nucleotide.…”

Design, synthesis and evaluation of analogs of initiation factor 4E (eIF4E) cap-binding antagonist Bn7-GMP

“…[26] This cap-free eIF4E had the advantage of avoiding the contamination of eIF4E purified from cap-affinity chromatography with cap-analog[15]. Generally, DHFR-eIF4E was overexpressed as a fusion protein in BL21 DE3 E. coli cells.…”

Steric and not structure-specific factors dictate the endocytic mechanism of glycosylphosphatidylinositol-anchored proteins

“…Transferrin and streptavidin (Sigma-Aldrich) and rabbit polyclonal anti-DNP IgG (Invitrogen) were labeled with Alexa Fluor 488– or Alexa Fluor 555–succinimidyl ester (Invitrogen) as per the manufacturer’s instructions. E. coli DHFR, expressed from a plasmid constructed by inserting the complete coding sequence of the protein between the NdeI and BamHI sites of pMAL-p4X (New England Biolabs, Inc.), was purified from E. coli by affinity chromatography on methotrexate-Sepharose ( Ghosh et al, 2008 ) and labeled with Alexa Fluor 555–succinimidyl ester. Fluorescein-labeled folate was originally obtained as a gift from S. Mayor (National Centre for Biological Sciences, Bangalore, India) and later synthesized by the method of McAlinden et al (1991) , as was its rhodamine-labeled analogue.…”