Ultra-high throughput screening for novel protease specificities.

Abstract

The screening of large libraries of enzyme variants remains an essential tool in evolving biocatalysts toward improved properties for applications in medicine, chemistry, and a broad variety of other fields. Over the last decades, the technology for conducting systematic screens of arrayed members of a library of enzyme variants has made great strides in terms of increasing throughput and reducing assay volume. Here, we describe in detail an alternative to arrayed analysis, which is a screen based on density shifts in result of changed enzyme function, which allows highly parallelized screening. Specifically, we link changes in protease substrate specificity in vivo to the production of an alternative reporter protein, catalase. Depending on the catalase expression level, microcolonies of library bacteria with active protease variants contained in polymeric droplets generate an oxygen bubble, which causes a density shift in the droplet and enables it to float.