Takahashi Fumio, Ebihara Takashi, Mie Masayasu, Yanagida Yasuko, Endo Yaeta, Kobatake Eiry, Aizawa Masuo
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, 226-8501, Yokohama, Japan.
FEBS Lett. 2002 Mar 6;514(1):106-10. doi: 10.1016/s0014-5793(02)02334-7.
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.
