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

Ghosh, Phalguni and Cheng, Jilin and Chou, Tsui-Fen and Jia, Yan and Avdulov, Svetlana and Bitterman, Peter B. and Polunovsky, Vitaly A. and Wagner, Carston R. (2008) Expression, purification and characterization of recombinant mouse translation initiation factor eIF4E as a dihydrofolate reductase (DHFR) fusion protein. Protein Expression and Purification, 60 (2). pp. 132-139. ISSN 1046-5928. PMCID PMC2617730. doi:10.1016/j.pep.2008.03.024. https://resolver.caltech.edu/CaltechAUTHORS:20220323-565905000

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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 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 μM. Nevertheless increased concentrations of eIF4E and DHFR–eIF4E significantly inhibited translation in a dose dependent manner by a maximum at 2 μM 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.pep.2008.03.024DOIArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/pmc2617730/PubMed CentralArticle
ORCID:
AuthorORCID
Chou, Tsui-Fen0000-0003-2410-2186
Bitterman, Peter B.0000-0002-7995-7117
Wagner, Carston R.0000-0001-7927-719X
Additional Information:© 2008 Elsevier. Received 13 December 2007, Revised 14 March 2008, Available online 31 March 2008. This work was partially supported by Grants from NIH-HL076779 (PBB), HL073719 (PBB), NCI U01-CA091220 (VAP) and an AHC Faculty Development Award (CRW) Technical assistance by Cindy Choy is gratefully acknowledged.
Funders:
Funding AgencyGrant Number
NIHHL076779
NIHHL073719
NIHU01-CA091220
University of MinnesotaUNSPECIFIED
Subject Keywords:Translational initiation factor; eIF4E; Fluorescence quenching; DHFR
Issue or Number:2
PubMed Central ID:PMC2617730
DOI:10.1016/j.pep.2008.03.024
Record Number:CaltechAUTHORS:20220323-565905000
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20220323-565905000
Official Citation:Phalguni Ghosh, Jilin Cheng, Tsui-Fen Chou, Yan Jia, Svetlana Avdulov, Peter B. Bitterman, Vitaly A. Polunovsky, Carston R. Wagner, Expression, purification and characterization of recombinant mouse translation initiation factor eIF4E as a dihydrofolate reductase (DHFR) fusion protein, Protein Expression and Purification, Volume 60, Issue 2, 2008, Pages 132-139, ISSN 1046-5928, https://doi.org/10.1016/j.pep.2008.03.024.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:114051
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:25 Mar 2022 19:47
Last Modified:25 Mar 2022 19:47

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