Identification and characterisation of PmaCI an endonuclease of novel specificity from Pseudomonas maltophila.
We report the use of MonoQ FPLC (Fast Protein Liquid Chromatography) for the rapid purification of a novel Type II restriction endonuclease PmaCI, from Pseudomonas maltophila, which recognises the sequence 5'-CAC decreases GTG-3'. The resulting enzyme is free of other nucleases to a level suitable for its characterisation by multiple-substrate digestion and DNA sequencing techniques. This method appears to be widely applicable and we have used it for the isolation of restriction endonucleases of comparable purity from a range of other organisms. Also described is a rapid method for screening a library of small inserted regions in recombinant M13 molecules for the presence and subsequent screening of restriction sites of interest.
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004262:DNA Restriction Enzymes / Q000302:isolation & purification* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D011489:Protein Denaturation; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity
D004262:DNA Restriction Enzymes; C048066:endodeoxyribonuclease PmaCI; D015252:Deoxyribonucleases, Type II Site-Specific