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Pimkin M , Caretti E , Canutescu A , Yeung JB , Cohn H , Chen Y , Oleykowski C , Bellacosa A , Yeung AT
Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection
BMC Biotechnol. 2007 ;7 :29
PMID: 17543120    PMCID: PMC1896157   
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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. CONCLUSION: 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.
Notes
Pimkin, Maxim Caretti, Elena Canutescu, Adrian Yeung, Jeffrey B Cohn, Heather Chen, Yibai Oleykowski, Catherine Bellacosa, Alfonso Yeung, Anthony T CA06927/CA/NCI NIH HHS/United States CA71426/CA/NCI NIH HHS/United States RR05539/RR/NCRR NIH HHS/United States Research Support, N.I.H., Extramural Research Support, U.S. Gov't, Non-P.H.S. England BMC biotechnology BMC Biotechnol. 2007 Jun 1;7:29.