High-performance liquid chromatography for the purification of restriction endonucleases, application to BanII, SacI, and SphI.
Conventional fractionation methods are time consuming, thus they prolong the time required to process low-stability restriction enzymes. We now report a rapid and effective two-step chromatographic method that affords high purity endonucleases in a short time. Accordingly, an inexpensive chromatographic adsorbent such as phosphocellulose or dyed agarose in the first step is coupled to a high-performance ion exchanger, namely, MonoQ, in the second step. The purification schemes reported here are now in routine use to prepare high-purity BanII, SacI, and SphI as judged by the "overdigestion" and "cut-ligate-recut" stringent quality tests.
D001407:Bacillus / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004262:DNA Restriction Enzymes / Q000302:isolation & purification*; D015252:Deoxyribonucleases, Type II Site-Specific; D005069:Evaluation Studies as Topic; D013302:Streptomyces / Q000201:enzymology
D004262:DNA Restriction Enzymes; C046184:endodeoxyribonuclease PaeI; C024408:endodeoxyribonuclease SacI; D015252:Deoxyribonucleases, Type II Site-Specific; C051481:GRGCYC-specific type II deoxyribonucleases