Ultrahigh-Throughput Multiplexed Screening of Purified Protein from Cell-Free Expression Using Droplet Microfluidics.

Screening large libraries is essential for protein engineering, but traditional approaches are limited by low throughput and biological variability. Here, we present an ultrahigh-throughput droplet microfluidics platform utilizing cell-free expression to screen libraries of purified proteins. We use bifunctional agarose beads to capture both DNA and encoded proteins, facilitating complete reagent exchange between incompatible reaction steps. This enhances the signal-to-noise ratio compared to live-cell expression systems. We introduce multiplexed screening to increase the loading per drop, enhancing the throughput by ∼100 times to 10 million variants in under 1 h of sorting. We use this platform to engineer Lipase A (BsLipA) for improved thermotolerance. We screen a combinatorial library where 15 residues are saturated and identify multiple variants with 9-12 substitutions and >40 °C improvement in thermotolerance after a single round of mutagenesis. This platform offers a robust, scalable solution for protein engineering.