Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection.
Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection.
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DOI：
10.1186/1472-6750-7-29
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发表时间：
2007-06-01
影响因子：
3.5
通讯作者：
Yeung AT
中科院分区：
文献类型：
--
作者：
Pimkin M;Caretti E;Canutescu A;Yeung JB;Cohn H;Chen Y;Oleykowski C;Bellacosa A;Yeung AT
未知突变的检测在研究和医学中都很重要。为此，从芹菜中提取的一种错配特异性内切酶CEL I已被建立为高通量项目的有用工具。在此之前，人们只在多种植物中描述了类似CEL i的活性，而不能在细菌中以活性形式表达。我们描述了活性重组植物错配内切酶的表达及其活性的修饰。我们还报道了从菠菜中克隆的一个同源基因，我们将其命名为spi核酸酶。活性的CEL I和SP I核酸酶以c端六组氨酸融合体表达，并从细胞培养基中亲和纯化。两种重组酶在患者源性DNA BRCA1基因突变检测中均有活性。从菠菜中纯化的天然SP核酸酶不能切割单核苷酸取代和含有鸟嘌呤核苷酸的环，但重组SP I核酸酶可以切割这些位点。昆虫细胞表达的CEL I同源物可能与从植物组织中纯化的原生对应物不相同。该表达系统将促进基于CEL i的突变检测技术的进一步发展。
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. 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. 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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影响因子：
3.9
作者：
Bannwarth, S;Procaccio, V;Paquis-Flucklinger, V
通讯作者：
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影响因子：
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