Heterologous Expression of Codon-Optimized Azurin Transferred by Magnetofection Method in MCF-10A Cells
Kalakenger, Saadet, Arslan, Seyda Yildiz, Turhan, Fatma, Acar, Melek, Solak, Kubra, Mavi, Ahmet, Unver, Yagmur
Transfection efficiency of the immortalized human breast epithelial cell line MCF-10A remains an issue that needs to be resolved. In this study, it was aimed to deliver a recombinant DNA (pCMV-Azu-GFP) to the MCF-10A cells by the magnetofection method using magnetic nanoparticles (MNPs) and a simple magnet to accelerate the DNA delivery. Surface positively modified silica-coated iron oxide MNPs (MSNP-NH2) were produced and characterized via TEM, FTIR, and DLS analyses. The recombinant DNA (rDNA) was obtained by the integration of codon-optimized azurin to produce a fusion protein. Then, rDNA cloned in Escherichia coli cells was validated by sequence analysis. The electrostatically conjugated rDNA on MSNP-NH2 with an enhancer polyethyleneimine (PEI) was studied by agarose gel electrophoresis and the optimum conditions were determined to apply to the cell. A dose-dependent statistical difference was observed on treated cells based on the MTS test. The expression of the fusion protein after magnetofection was determined using laser scanning confocal microscope imaging and western blot analysis. It was observed that the azurin gene could be transferred to MCF-10A cells by magnetofection. Thus, when the azurin gene is used as a breast cancer treatment agent, it can be expressed in healthy cells without toxic effects.
- Journal
- MOLECULAR BIOTECHNOLOGY
- Year
- 2024
- Volume
- 66
- Issue
- 6
- Pages
- 1434-1445
- ISSN
- 1073-6085
- eISSN
- 1559-0305
- DOI
- 10.1007/s12033-023-00798-9
- WoS UID
- WOS:001018124400001