Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering

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.