Ribosome display for selection of active dihydrofolate reductase mutants using immobilized methotrexate on agarose beads.
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.
Takahashi
Fumio
F
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, 226-8501, Yokohama, Japan.
Ebihara
Takashi
T
Mie
Masayasu
M
Yanagida
Yasuko
Y
Endo
Yaeta
Y
Kobatake
Eiry
E
Aizawa
Masuo
M
D002474:Cell-Free System; D005784:Gene Amplification; D008727:Methotrexate / Q000737:chemistry*; D009154:Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D012685:Sepharose; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate