Active site-directed double mutants of dihydrofolate reductase
Ercikan-Abali, E.A.; Mineishi, S.; Tong, Y.; Nakahara, S.; Waltham, M.C.; Banerjee, D.; Chen, W.; Sadelain, M.; Bertino, J.R.
Cancer Research 56(18): 4142-4145
1996
ISSN/ISBN: 0008-5472 PMID: 8797582 Document Number: 458333
Variants of dihydrofolate reductase (DHFR), which confer resistance to antifolates, are used as dominant selectable markers in vitro and in vivo and may be useful in the context of gene therapy. To identify improved mutant human DHFRs with increased catalytic efficiency and decreased binding to methotrexate, we constructed by site-directed mutagenesis four variants with substitutions at both Leu-22 and Phe-31 (i.e., Phe-22-Ser-31, Tyr-22-Ser-31,Phe-22-Gly-31, and Tyr-22-Gly-31). Antifolate resistance has been observed previously when individual changes are made at these active-site residues. Substrate and antifolate binding properties of these "double" mutants revealed that each have greatly diminished affinity for antifolates ( gt 10,000-fold) yet only slightly reduced substrate affinity. Comparison of in vitro measured properties with those of single-residue variants indicates that double mutants are indeed significantly superior. This was verified for one of the double mutants that provided high-level methotrexate resistance following retrovirus-mediated gene transfer in NIH3T3 cells.