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Ribosome display for selection of active dihydrofolate reductase mutants using immobilized methotrexate on agarose
Fumio Takahashi1, Takashi Ebihara, Masayasu Mie
1Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, 226-8501, Yokohama, Japan.
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.