Open AccessIdentification and characterisation ofPmaCl an endonuclease of novel specificity fromPseudomonas maltophila
Author(s) -
Jeremy N.B. Walker,
P.D.G. Dean,
J. R. Saunders
Publication year - 1986
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/14.3.1293
Subject(s) - biology , restriction enzyme , recombinant dna , endonuclease , fast protein liquid chromatography , microbiology and biotechnology , dna , enzyme , restriction digest , restriction map , ecori , biochemistry , restriction fragment , genetics , base sequence , gene
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.