Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection.
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.
Pimkin
Maxim
M
Basic Science, Fox Chase Cancer Center, Philadelphia, PA, USA. MA_Pimkin@fccc.edu <MA_Pimkin@fccc.edu>
Caretti
Elena
E
Canutescu
Adrian
A
Yeung
Jeffrey B
JB
Cohn
Heather
H
Chen
Yibai
Y
Oleykowski
Catherine
C
Bellacosa
Alfonso
A
Yeung
Anthony T
AT
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*
D011994:Recombinant Proteins; D004720:Endonucleases; C406922:CEL I nuclease