Applied molecular evolution of O-6-benzylguanine-resistant DNA alkyltransferases in human hematopoietic cells

Applied molecular evolution is a rapidly developing technology that can be used to create and identify novel enzymes that nature has not selected, An important application of this technology is the creation of highly drug-res istant enzymes for cancer gene therapy. Seventeen O-6-alkylguanine-DNA alky ltransferase (ACT) mutants highly resistant to O-6-benzylguanine (BG) were identified previously by screening 8 million variants, using genetic comple mentation in Escherichia coil. To examine the potential of these mutants fo r use in humans, the sublibrary of AGT clones was introduced to human hemat opoietic cells and stringently selected for resistance to killing by the co mbination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea, This competitive analysis between the mutants in human cells revealed three AGT mutants that conferred remarkable resistance to the combination of BG and 1,3-bis(2-chl oroethyl)-1-nitrosourea, Of these, one was recovered significantly more fre quently than the others. Upon further analysis, this mutant displayed a lev el of BG resistance in human hematopoietic cells greater than that of any p reviously reported mutant.