# Integrating Gene Synthesis and Microfluidic Protein Analysis for Rapid Protein Engineering

## TL;DR

This paper introduces a fast method to design and test proteins, enabling rapid engineering of zinc-finger transcription factors for DNA binding.

## Contribution

APE-MITOMI integrates gene synthesis and microfluidic analysis, bypassing traditional cloning and cell-based steps for rapid protein characterization.

## Key findings

- Over 440 zinc-finger transcription factors were synthesized and characterized in 3-4 days.
- Engineering precise DNA binding energy landscapes for ZF TFs remains challenging despite sequence-specific design.
- ZF-DNA affinity can be engineered independently of sequence specificity and explained by in silico modeling.

## 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 (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 440 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.