Abstract
Zinc finger proteins are the most common among families of DNA-binding transcription factors. Designer transcription factors generated by the fusion of engineered zinc finger DNA-binding domains (ZF-DBDs) to effector domains have been valuable tools for the modulation of gene expression and for targeted genome editing. However, ZF-DBDs without effector domains have also been shown to effectively modulate gene expression by competing with sequence-specific DNA-binding transcription factors. Here, we describe the methodology and provide a detailed workflow for the cloning, expression, purification, and direct cell delivery of engineered ZF-DBDs. Using this protocol, ZF-DBDs can be generated with high efficiency in less than 2 weeks. We also describe a nonradioactive method for measuring DNA binding affinity of the purified ZF-DBD proteins as well as a method for direct delivery of the purified ZF-DBDs to mammalian cells.
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Acknowledgment
This work was supported by grants from the National Institutes of Health to J.B. (NIH, R01 DK083389, and R01 DK 052356). We are very grateful to Blanca Ostmark and Nancy Nabilsi for their help.
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Hossain, M.A. et al. (2017). Engineered Zinc Finger DNA-Binding Domains: Synthesis, Assessment of DNA-Binding Affinity, and Direct Protein Delivery to Mammalian Cells. In: Kaufmann, M., Klinger, C., Savelsbergh, A. (eds) Functional Genomics. Methods in Molecular Biology, vol 1654. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7231-9_27
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DOI: https://doi.org/10.1007/978-1-4939-7231-9_27
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Online ISBN: 978-1-4939-7231-9
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