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

10Citations
Citations of this article
46Readers
Mendeley users who have this article in their library.

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. © 2007 Pimkin et al; licensee BioMed Central Ltd.

Cite

CITATION STYLE

APA

Pimkin, M., Caretti, E., Canutescu, A., Yeung, J. B., Cohn, H., Chen, Y., … Yeung, A. T. (2007). Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection. BMC Biotechnology, 7. https://doi.org/10.1186/1472-6750-7-29

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free