Journal
FEBS LETTERS
Volume 514, Issue 1, Pages 106-110Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/S0014-5793(02)02334-7
Keywords
ribosome display; dihydrofolate reductase; in vitro protein synthesis; active enzyme mutant
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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. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
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