Expression of Nicotiana glutinosa ribonucleases in Escherichia coli.
We previously isolated from Nicotiana glutinosa leaves three distinct cDNA clones, NGR1, NGR2, and NGR3, encoding a wound-inducible RNase NW, and putative RNases NGR2 and NGR3, respectively. In this study, we produced RNases NW and NGR3 in Escherichia coli and purified them to homogeneity. RNase NGR3 had non-absolute specificity toward polynucleotides, although RNase NW preferentially cleaved polyinosinic acid (Poly I). Both RNases NW and NGR3 were more active toward diribonucleoside monophosphates ApG, CpU, and GpU. Furthermore, kinetic parameters for RNase NW (Km, 0.778 mM and kcat, 1938 min(-1)) and RNase NGR3 (Km, 0.548 mM and kcat, 408 min(-1)) were calculated using GpU as a substrate.
Hino
Madoka
M
Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Kawano
Shin
S
Kimura
Makoto
M
D003001:Cloning, Molecular / Q000379:methods; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D016601:RNA-Binding Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
C082119:NGR1 protein, S cerevisiae; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D004722:Endoribonucleases; D012260:Ribonucleases; C114175:wound-inducible ribonuclease, Nicotiana