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

BMC Biotechnol 2007 Jun 1;7:29. Epub 2007 Jun 1.

Basic Science, Fox Chase Cancer Center, Philadelphia, PA, USA.

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.

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Source
http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472
Publisher Site
http://dx.doi.org/10.1186/1472-6750-7-29DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1896157PMC
June 2007
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