Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection

We describe expression of active recombinant plant mismatch endonucleases and modification of their activities. We also report the cloning of a CEL I ortholog from Spinacia oleracea (spinach) which we termed SP I nuclease. Active CEL I and SP I nucleases were expressed as C-terminal hexahistidine fusions and affinity purified from the cell culture media. Both recombinant enzymes were active in mutation detection in BRCA1 gene of patient-derived DNA. Native SP nuclease purified from spinach is unable to incise at single-nucleotide substitutions and loops containing a guanine nucleotide, but the recombinant SP I nuclease can cut at these sites.The insect cell-expressed CEL I orthologs may not be identical to their native counterparts purified from plant tissues. The present expression system should facilitate further development of CEL I-based mutation detection technologies.Nucleases of the S1 family are widely used as tools for probing single-stranded regions of DNA and RNA [1-3] as well as for the removal of single-stranded regions from dsDNA [3,4]. One class of plant homologs of S1, represented by CEL I from celery, are particularly capable of efficient cutting at single base substitutions and loops [5-7]. Several CEL I-based mutation detection techniques have been developed [8-12]. They are relatively simple yet highly reliable and capable of detecting a mutation in pools of several DNA samples. Adaptation of this approach to the Tilling method of recovering chemically derived mutations at target regions [10,13] has allowed CEL I to contribute to many plant genetics programs [14-17], as well as zebrafish [18,19], drosophila [20], and mouse ES cells research [21]. Moreover, it is beginning to be successfully applied to programs of disease mutation detection [6,22-27]. A CEL I ortholog, CEL II nuclease, is the principal component of the SURVEYOR Mutation Detection Kits (Transgenomic, Inc.) [7].The P1 nuclease of Penicillium citrinum is a close ortholog of the S1 nu