Pimkin Maxim, Caretti Elena, Canutescu Adrian, Yeung Jeffrey B, Cohn Heather, Chen Yibai, Oleykowski Catherine, Bellacosa Alfonso, Yeung Anthony T
Basic Science, Fox Chase Cancer Center, Philadelphia, PA, USA.
BMC Biotechnol. 2007 Jun 1;7:29. doi: 10.1186/1472-6750-7-29.
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.
未知突变的检测在研究和医学中至关重要。为此，来自芹菜的错配特异性核酸内切酶CEL I已成为高通量项目中的一种有用工具。此前，类似CEL I的活性仅在多种植物中被描述，且无法在细菌中以活性形式表达。
我们描述了活性重组植物错配核酸内切酶的表达及其活性的修饰。我们还报告了从菠菜（Spinacia oleracea）中克隆出的一种CEL I直系同源物，我们将其命名为SP I核酸酶。活性CEL I和SP I核酸酶以C端六组氨酸融合蛋白的形式表达，并从细胞培养基中进行亲和纯化。这两种重组酶在检测患者来源DNA的BRCA1基因中的突变时均具有活性。从菠菜中纯化的天然SP核酸酶无法在单核苷酸取代处以及含有鸟嘌呤核苷酸的环处切割，但重组SP I核酸酶可以在这些位点切割。
昆虫细胞表达的CEL I直系同源物可能与从植物组织中纯化的天然对应物不同。目前的表达系统应有助于基于CEL I的突变检测技术的进一步发展。
