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.
journal_name
BMC Biotechnoljournal_title
BMC biotechnologyauthors
Pimkin M,Caretti E,Canutescu A,Yeung JB,Cohn H,Chen Y,Oleykowski C,Bellacosa A,Yeung ATdoi
10.1186/1472-6750-7-29subject
Has Abstractpub_date
2007-06-01 00:00:00pages
29issn
1472-6750pii
1472-6750-7-29journal_volume
7pub_type
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journal_title:BMC biotechnology
pub_type: 杂志文章
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journal_title:BMC biotechnology
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pub_type: 杂志文章
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更新日期:2016-05-27 00:00:00
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journal_title:BMC biotechnology
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更新日期:2013-12-20 00:00:00
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journal_title:BMC biotechnology
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更新日期:2011-06-02 00:00:00
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journal_title:BMC biotechnology
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