Active site-directed double mutants of dihydrofolate reductase

Emine A. Ercikan-Abali, Shin Mineishi, Youzhi Tong, Saori Nakahara, Mark C. Waltham, Debabrata Banerjee, Wen Chen, Michel Sadelain, Joseph R. Bertino

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Variants of dihydrofolate reductase (DHFR), which confer resistance to antifulates, 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 Leu22 and Phe31 (i.e., Phe22-Ser31, Tyr22- Ser31, Phe22-Gly31, and Tyr22-Gly31). 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 (> 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 fur one of the double mutants that provided high-level methotrexate resistance following retrovirus-mediated gene transfer in NIH3T3 cells.

Original languageEnglish
Pages (from-to)4142-4145
Number of pages4
JournalCancer Research
Issue number18
Publication statusPublished - 15 Sept 1996
Externally publishedYes

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