Ribosome display for selection of active dihydrofolate reductase mutants using immobilized methotrexate on agarose beads

FEBS Letters
Fumio TakahashiMasuo Aizawa

Abstract

Ribosome display was applied to the selection of an enzyme. As a model, we selected and amplified the dihydrofolate reductase (DHFR) gene by ribosome display utilizing a wheat germ cell-free protein synthesis system based on binding affinity to its substrate analog, methotrexate, immobilized on agarose beads. After three rounds of selection, the DHFR gene could be effectively selected and preferentially amplified from a small proportion in a mixture also containing competitive genes. Active enzymes were expressed and amplified and by sequence analysis, four mutants of DHFR were identified. These mutants showed as much activity as the wild-type enzyme.

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Citations

Sep 11, 2007·Molecular Biotechnology·Jong-Myung KimMyung-Shin Lee
Sep 2, 2003·Trends in Biotechnology·Ana Fernandez-GacioJacques Fastrez
Mar 1, 2007·Nature Methods·Christian ZahndAndreas Plückthun
Jun 12, 2008·Biochemical and Biophysical Research Communications·Masayasu MieEiry Kobatake
Mar 27, 2007·Biotechnology Advances·Claudia SheedyJ Christopher Hall
Apr 24, 2007·The FEBS Journal·Ykelien L BoersmaWim J Quax
May 23, 2006·Biochemical and Biophysical Research Communications·Hiroshi IharaEiry Kobatake
Jul 21, 2004·Journal of Immunological Methods·Dasa Lipovsek, Andreas Plückthun
Feb 16, 2005·Expert Opinion on Biological Therapy·Maria A T Groves, Jane K Osbourn
Mar 29, 2003·Expert Opinion on Biological Therapy·Christelle Souriau, Peter J Hudson
Jan 27, 2006·Expert Opinion on Biological Therapy·Achim RotheBarbara E Power
Apr 22, 2014·Biochimica Et Biophysica Acta·Takashi KanamoriTakuya Ueda
Dec 1, 2006·Biochemical and Biophysical Research Communications·Tomoaki MatsuuraTetsuya Yomo
Jul 8, 2005·Expert Review of Proteomics·Mingyue He, Farid Khan
Aug 3, 2004·Biotechnology and Bioengineering·Michael C Jewett, James R Swartz

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