title: Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering.
abstract: The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3-4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF-DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering.
authors.affiliation: Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Blackburn
authors.affiliation: Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Petrova
authors.affiliation: Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Bruno E
authors.initials: BE
authors.lastname: Correia
authors.affiliation: Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland sebastian.maerkl@epfl.ch.
authors.forename: Sebastian J
authors.initials: SJ
authors.lastname: Maerkl
mesh_terms: D005813:Genes, Synthetic; D056656:Lab-On-A-Chip Devices; D015202:Protein Engineering / Q000379:methods*; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D014157:Transcription Factors