Team:Washington

Overview

Stabilizing proteins is an incredibly important and time consuming task in the field of protein engineering. Our team has developed a high throughput method to select for the increased expression and stability of engineered proteins, making them more amenable to large-scale production in Escherichia coli and other downstream applications. Our method involves the insertion of proteins into a Gal4-VP16 transactivator that binds a promoter directly upstream of a GFP gene. This allows for the subsequent selection of mutants associated with higher GFP output, correlating to higher stability, using fluorescence-activated cell sorting. Additionally, our method utilizes degrons to alter the dynamic range of this GFP output. This revolutionary method is a potentially generalizable alternative to current, labor intensive approaches for the selection of stable protein variants. Engineered proteins selected through this method could be produced in bacteria and aid in the development of thermostable, de novo protein therapeutics.