Volume 2, Issue 8 (10-2012)                   NCMBJ 2012, 2(8): 79-87 | Back to browse issues page

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Kaligh S, Bandehpour M, Yassaee V R, Parivar K, Kazemi B. Cloning, Expression and Purification CEL I Endonuclease Enzyme from Celery Plants. NCMBJ 2012; 2 (8) :79-87
URL: http://ncmbjpiau.ir/article-1-259-en.html
Department of Biology, Faculty of Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran , skhalighfard96@gmail.com
Abstract:   (34426 Views)

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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Type of Study: Research Article | Subject: Genetics
Received: 2012/12/22 | Published: 2012/10/15

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