Normalized screening of protein engineering libraries by split-GFP crude cell extract quantification

Abstract

The different expression level and solubility showed by each protein variant represents an important challenge during screening campaigns: Usually, the total activity measurement constitutes the only criterion for identifying improved variants. This hampers the chances of finding interesting mutants, especially if the aim is to improve activity: On the one hand, interesting but poorly soluble variants will remain undetectable. On the other hand, a mutation might not increase activity, but improve expression level or solubility. The split-GFP technology offers an affordable and technically simple manner for overcoming that constraints, making protein library screening more efficient through the normalization of the detected enzymatic activities in relation to the quantified protein contents responsible for them.