title: A high-throughput microfluidic method for generating and characterizing transcription factor mutant libraries.
abstract: 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.
authors.affiliation: Laboratory of Biological Network Characterization, EPFL Lausanne, Lausanne, Switzerland.
authors.forename: Marcel
authors.initials: M
authors.lastname: Geertz
authors.forename: Sylvie
authors.initials: S
authors.lastname: Rockel
authors.forename: Sebastian J
authors.initials: SJ
authors.lastname: Maerkl
mesh_terms: D000818:Animals; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D015723:Gene Library; D005898:Glass / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D055616:Microtechnology; D050505:Mutant Proteins / Q000235:genetics*; D009154:Mutation; D011327:Printing; D015202:Protein Engineering / Q000295:instrumentation*; D012821:Silanes / Q000737:chemistry; D013499:Surface Properties; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D050505:Mutant Proteins; D012821:Silanes; D014157:Transcription Factors