title: Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection.
abstract: BACKGROUND
  The detection of unknown mutations is important in research and medicine. For this purpose, a mismatch-specific endonuclease CEL I from celery has been established as a useful tool in high throughput projects. Previously, CEL I-like activities were described only in a variety of plants and could not be expressed in an active form in bacteria.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Basic Science, Fox Chase Cancer Center, Philadelphia, PA, USA. MA_Pimkin@fccc.edu <MA_Pimkin@fccc.edu>
authors.forename: Maxim
authors.initials: M
authors.lastname: Pimkin
authors.forename: Elena
authors.initials: E
authors.lastname: Caretti
authors.forename: Adrian
authors.initials: A
authors.lastname: Canutescu
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Yeung
authors.forename: Heather
authors.initials: H
authors.lastname: Cohn
authors.forename: Yibai
authors.initials: Y
authors.lastname: Chen
authors.forename: Catherine
authors.initials: C
authors.lastname: Oleykowski
authors.forename: Alfonso
authors.initials: A
authors.lastname: Bellacosa
authors.forename: Anthony T
authors.initials: AT
authors.lastname: Yeung
mesh_terms: D000077102:Apium / Q000201:enzymology* / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics; D018724:Spinacia oleracea / Q000201:enzymology* / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D004720:Endonucleases; C406922:CEL I nuclease