Aims . CEL I, is isolated from celery and the first eukaryotic nuclease is known to have cleavage DNA with high specificity at sites of the two DNA strands in a heteroduplex DNA molecule . The aim of this study was cloning, expression and purification CEL I endonuclease and a simple method for the detection of mutation s.
Material & Methods . CEL I mRNA was used for RT-PCR. CEL I cDNA was synthesized and cloned into T-vector pTZ57R, then CEL I subcloned into pET32a expression vector. Recombinant plasmid was transformed into BL2I bacterial cells. Expression of recombinant plasmid was analyzed by western blot technique. Protein was purified by affinity chromatography . In first study, Recombinant enzyme was active in mutation detection in Product PCR normal and mutant BRCA1 gene.
Results. The recombinant ( pET32a /CEL I) was successfully expressed in BL2I cells. Western blot analysis showed that the successfully expressed CEL I in the cells transfected . The first study showed that recombinant enzyme is kno wn to nick mismatch sites of the two DNA strands in a heteroduplex DNA molecule.
Conclusion. The expression of CEL I in BL2I cells was strong. In vitro, purification protein was successful. Recombinant nucleases CEL I known to nick mismatch sites in a heteroduplex DNA molecule.
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