Affinity partitioning of restriction endonucleases. Application to the purification of EcoR I and EcoR V.
Partitioning of restriction endonucleases between two liquid aqueous phases can be strongly influenced by group-specific ligands included in the two-phase system. Three restriction endonucleases, namely EcoR I, EcoR V and BamH I, were partitioned within an aqueous dextran-polyethylene glycol (PEG) system. The enzymes could be extracted into the upper PEG phase by using either triazine dyes or herring DNA as affinity ligands. The influence of the endogenous bacterial nucleic acids, concentration of polymerbound dye and concentration of sodium chloride on the system were examined. A partial purification of EcoR I (up to 52-fold) and EcoR V (up to 37-fold) was achieved using a combination of affinity partitioning and ion-exchange chromatography, providing an extremely fast and economical method for the isolation of restriction endonucleases free from contaminating nuclease activities.
Vlatakis
G
G
Enzyme Technology Division, Institute of Molecular Biology and Biotechnology, Crete, Greece.
Bouriotis
V
V
D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D015245:Deoxyribonuclease BamHI / Q000302:isolation & purification; D015246:Deoxyribonuclease EcoRI / Q000302:isolation & purification* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D012965:Sodium Chloride; D013056:Spectrophotometry, Ultraviolet
D012965:Sodium Chloride; D004247:DNA; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases