Rapid in Vitro Screening of Proteins with Non-Natural Amino Acids

The incorporation of non-natural amino acids (NNAA) can introduce novel functional groups into proteins with site-specific precision and control, with significant applications in basic science and protein engineering. However, NNAA can decrease protein expression and functional activity depending on the location within the protein – a process that is not yet completely understood and difficult to predict. Therefore, practical applications often necessitate a time-consuming optimization of NNAA location by individual expressions, purification and evaluations for many variant proteins. Such optimization could be accelerated by a high-throughput approach. Here, we introduce a highly customizable in vitro system using PCR products as expression templates for rapidly expressing and screening NNAA-containing proteins without cloning or purification procedures. We utilize this technology to simultaneously screen 24 different T4-Lysozyme mutants with different NNAA incorporation sites in a matter of few days, and further explore the effects of NNAA location on expression and activity.