Sequence-specific DNA affinity chromatography: application of a group-specific adsorbent for the isolation of restriction endonucleases.
The use of sequence-specific DNA affinity adsorbents for the isolation of restriction endonucleases EcoRI and SphI to near homogeneity has been reported. However, the high cost of these adsorbents is a limiting factor for their wider application. This paper reports the application of sequence-specific DNA affinity ligands containing recognition sequences for 34 restriction endonucleases as group-specific ligands in the isolation of restriction endonucleases. Crude samples of six restriction endonucleases, namely BshFI, BamHI, SmaI, SacII, PvuII and SalI, were shown to bind to these adsorbents and could be eluted at different KCl concentrations. High purification factors and recoveries were obtained. Restriction endonuclease BshFI, an isoschizomer of HaeIII, from the microorganism Bacillus sphaericus was purified to near homogeneity employing a two-step procedure which involves DNA-cellulose chromatography and oligonucleotide-ligand affinity chromatography. The enzyme exists as a monomer with an apparent relative molecular mass of 34,000 as determined by both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and size-exclusion chromatography.
Pozidis
C
C
Institute of Molecular Biology and Biotechnology, Enzyme Technology Division, Crete, Greece.
Vlatakis
G
G
Bouriotis
V
V
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D004262:DNA Restriction Enzymes / Q000302:isolation & purification* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D004247:DNA; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases