Cycling probe technology with RNase H attached to an oligonucleotide.
A streptavidin-RNase H gene fusion was constructed by cloning the Thermus thermophilus RNase H coding sequence in the streptavidin expression vector pTSA18F. The gene was expressed in Escherichia coli, and the resulting fusion protein was purified to apparent homogeneity. The fusion protein was shown to have a molecular weight of 128 kDa and to consist of four subunits. Furthermore, heat treatment of the fusion enzyme showed that it was stable as a tetramer at 65 degrees C. The fusion enzyme was shown to have both biotin binding and RNase H catalytic properties. Using cycling probe technology (CPT), the fusion enzyme was compared to the native RNase H with a biotinylated probe at different ratios of probe:enzyme and varying amounts of synthetic target DNA. At a ratio of 1:1, the fusion enzyme was active in CPT, but the native enzyme was not; both enzymes were active at a 1:5000 ratio of probe:enzyme. The fusion enzyme was further tested using biotinylated and non-biotinylated probes and was shown to be active at a 1:1 ratio with the biotinylated probe but not with the non-biotinylated probe. These experiments show that through binding of the streptavidin-RNase H fusion enzyme to the biotinylated probe, the efficiency of the cycling probe reaction is enhanced.
Bekkaoui
F
F
ID Biomedical, Burnaby, BC, Canada. fouzib@idbiomed.com
Poisson
I
I
Crosby
W
W
Cloney
L
L
Duck
P
P
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*; D016914:Ribonuclease H / Q000235:genetics*; D017421:Sequence Analysis; D019809:Streptavidin; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D001710:Biotin; D019809:Streptavidin; D016914:Ribonuclease H