Dye-ligand chromatography for the resolution and purification of restriction endonucleases.
The resolution of restriction endonucleases from the same microorganism is conventionally achieved by lengthy fractionation protocols. We now report effective single-step procedures that exploit dye-ligand chromatography for the resolution and purification of restriction enzymes. After suitable initial screening, we demonstrated that resolution of two restriction activities can be achieved in one chromatographic step, and further purification can subsequently be effected using selected dye-adsorbents. Accordingly, we resolved in one step, Hpa I from Hpa II, Hind II from Hind III, and Sac I from Sac II. Furthermore, a three-step chromatographic procedure has been developed to purify EcoRV suitable for commercial exploitation, as judged by the "overdigestion" and "cut-ligate-recut" quality control tests.
Vlatakis
G
G
Research Center of Crete, Institute of Molecular Biology and Biotechnology, Iraklio, Greece.
Skarpelis
G
G
Stratidaki
I
I
Bouriotis
V
V
Clonis
Y D
YD
D001419:Bacteria / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods; D004396:Coloring Agents; D004262:DNA Restriction Enzymes / Q000302:isolation & purification*; D015252:Deoxyribonucleases, Type II Site-Specific; D008024:Ligands
D004396:Coloring Agents; D008024:Ligands; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases