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Heterologous Expression of Codon-Optimized Azurin Transferred by Magnetofection Method in MCF-10A Cells

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Abstract

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.

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Data availability

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

The authors kindly acknowledge TÜBİTAK (The Scientific and Technological Research Council of Turkey) (Project Number: 118Z150) for the financial support and DAYTAM (East Anatolia High Technology Application and Research Center, Atatürk University, Erzurum, Turkey) for the equipment support. M.A. and K.S. are also thankful for The Council of Higher Education (CoHE, 100/2000) Ph.D. Scholarship Program, Turkey.

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Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Graduate School of Natural and Applied Sciences, Atatürk University, Erzurum, Turkey

    Saadet Kalakenger, Seyda Yildiz Arslan & Melek Acar

  2. Department of Nanoscience and Nanoengineering, Graduate School of Natural and Applied Sciences, Atatürk University, Erzurum, Turkey

    Fatma Turhan, Kubra Solak & Ahmet Mavi

  3. Department of Mathematics and Science Education, Education Faculty of Kazım Karabekir, Atatürk University, Erzurum, Turkey

    Ahmet Mavi

  4. Department of Molecular Biology and Genetics, Faculty of Science, Atatürk University, Erzurum, Turkey

    Yagmur Unver

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  1. Saadet Kalakenger
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Kalakenger, S., Yildiz Arslan, S., Turhan, F. et al. Heterologous Expression of Codon-Optimized Azurin Transferred by Magnetofection Method in MCF-10A Cells. Mol Biotechnol 66, 1434–1445 (2024). https://doi.org/10.1007/s12033-023-00798-9

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