title: In vivo selection of active deoxyribonucleoside kinase by a mutagenic nucleoside analog.
abstract: A novel in vivo selection method for active deoxyribonucleoside kinase proteins is described here. A pool of randomly mutated genes for deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) was prepared and inserted into an expression vector. Enzymatically active mutants were selected by repeated cycles, including (i) introduction into Escherichia coli, (ii) treatment of the E. coli pool with a mutagenic deoxyribonucleoside (2-hydroxy-dA), and (iii) selection of antibiotic-resistant colonies resulting from mutations by phosphorylated 2-hydroxy-dA and the subsequent isolation of the plasmid DNAs. The ratio of the resistant colonies increased by two orders of magnitude from the first cycle to the fifth cycle, and then reached a plateau. Fifteen Dm-dNK mutants selected after the seventh and eighth evolution cycles were actually active in vivo. Moreover, one of the mutant proteins was as active as the wild-type protein in vitro. These results indicate that this novel in vivo evolution method was useful and that similar strategies would be applicable to other deoxyribonucleoside kinases. In addition, the distribution of mutated amino acids suggests important residues/regions in the Dm-dNK protein.
authors.affiliation: Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan; Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, Japan; Graduate School of Science and Engineering, Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan. Electronic address: hirokam@hiroshima-u.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kamiya
authors.affiliation: Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
authors.forename: Mana
authors.initials: M
authors.lastname: Ito
authors.affiliation: Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Nishi
authors.affiliation: Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
authors.forename: Hideyoshi
authors.initials: H
authors.lastname: Harashima
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives*; D000818:Animals; D003839:Deoxyadenosines / Q000737:chemistry / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009153:Mutagens / Q000737:chemistry / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D061987:Sf9 Cells
chemical_list: D003839:Deoxyadenosines; D029721:Drosophila Proteins; D009153:Mutagens; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases; D000241:Adenosine
keywords: 2-Hydroxy-2′-deoxyadenosine; Active mutants; Drosophila melanogaster deoxyribonucleoside kinase; In vivo selection; Random mutant pool