Development of a flow cytometric method to evaluate the impact of N-terminal sequences on protein stability

Protein library generation coupled with high-throughput analysis has become an increasingly mainstream method to study the contribution of N-terminal regions to protein stability. Given this trend, the establishment of experimental best practices would benefit the field. In this chapter, we present a method for the generation and screening of N-terminal libraries using a flow cytometric method that pairs Fluorescence-Activated Cell Sorting (FACS) with Next Generation Sequencing (NGS). We demonstrate the ability of our workflow to identify a stabilizing effect imparted by acidic residues in the third position of an N-degron within the model microbe Escherichia coli. To conclude, we comment on the applications and generalizability of this technology for screening clustered mutagenized regions.