A High-Throughput Microfluidic Method for Generating and Characterizing Transcription Factor Mutant Libraries
Characterizing libraries of mutant proteins is a challenging task, but can lead to detailed functional insights on a specific protein, and general insights for families of proteins such as transcription factors. Challenges in mutant protein screening consist in synthesizing the necessary expression-ready DNA constructs and transforming them into a suitable host for protein expression. Protein purification and characterization are also non-trivial tasks that are not easily scalable to hundreds or thousands of protein variants. Here we describe a method based on a high-throughput microfluidic platform to screen and characterize the binding profile of hundreds of transcription factor variants. DNA constructs are synthesized by a rapid two-step PCR approach without the need of cloning or transformation steps. All transcription factor mutants are expressed on-chip followed by characterization of their binding specificities against 64 different DNA target sequences. The current microfluidic platform can synthesize and characterize up to 2,400 protein–DNA pairs in parallel. The platform method is also generally applicable, allowing high-throughput functional studies of proteins.
Key wordsMicroarrays Transcription factor binding sites High-throughput Binding affinities DNA array Protein array Surface chemistry Two-step PCR Microfluidics
- 3.Jolma, A., Kivioja, T., Toivonen, J., Cheng, L., Wei, G., Enge, M., Taipale, M., Vaquerizas, J. M., Yan, J., Sillanpää, M. J., Bonke, M., Palin, K., Talukder, S., Hughes, T. R., Luscombe, N. M., Ukkonen, E., and Taipale, J. (2010) Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities, Genome Res 20, 861–873.PubMedCrossRefGoogle Scholar