A high-throughput microfluidic method for generating and characterizing transcription factor mutant libraries

Methods Mol Biol. 2012:813:107-23. doi: 10.1007/978-1-61779-412-4_6.

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.

MeSH terms

  • Animals
  • DNA, Complementary / biosynthesis
  • DNA, Complementary / genetics
  • Gene Library*
  • Glass / chemistry
  • Microfluidic Analytical Techniques / methods*
  • Microtechnology
  • Mutant Proteins / genetics*
  • Mutation*
  • Printing
  • Protein Engineering / instrumentation*
  • Silanes / chemistry
  • Surface Properties
  • Transcription Factors / genetics*

Substances

  • DNA, Complementary
  • Mutant Proteins
  • Silanes
  • Transcription Factors