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Showing 1 results for Hek293t Cell Line
S. Askari, S. Hasannia, R. Hasan Sajedi, V.r. Yassaee,
Volume 10, Issue 3 (9-2019)
Abstract
CEL I endonuclease pertaining to the S1 endonuclease family. The enzyme, with its high specificity, has the ability to identify different types of mutations and base replacement in the DNA molecule, which makes it important in commercial products to use in research and clinical laboratories. Although the enzyme exists in the celery plant, the extraction of the enzyme is a time-consuming process and not economical and the yield of the final product is low. In addition, due to its post-translational modifications to achieve the final active structure, no report has published to indicate the expression of the active form of this enzyme in the bacterial hosts yet. Therefore, one of the production sources of the active form of this enzyme is its cloning and expression in eukaryotic hosts, including yeast and mammalian cell lines. In this study, in order to express CEL I endonuclease, its gene sequence was optimized and synthesized in host eukaryotic HEK293T. CEL I was subcloned by double digest with KpnI and XhoI enzymes in the pBudCE4.1expression vector. The expression construct was transfected into the HEK293T cell line by lipofectamine transfection. Expression of the recombinant protein after transfection into HEK293T cells was confirmed by multiple methods including polyacrylamide gel electrophoresis, ELISA, RT-PCR, and western blot reaction. The analysis of SDS-PAGE and western blot data confirmed the molecular weight of approximately 30kDa. Purification was carried out with the Ni-NTA column and the amount of purified protein was determined to be about 0.2mg/ml. Finally, the activity of endonuclease enzyme was investigated on both normal and mutated heteroduplex DNA amplified by PCR. The results showed that the expression of this protein in HEK293T host had shown sufficient activity.
