Time-efficient and Semi-automated Production and Screening of Proteins

Fast, flexible and non-randomized modification, production and screening of proteins in fully automated system are of high interest in biological research and applications. The conventional methods for protein engineering and screening, especially for mutations of multiple residues. are time consuming and often unreliable. We demonstrate here a new, fast and flexible protein production and screening method which combines linear expression template (LET) based cell free protein synthesis (CFPS) with specific screening methods. This approach is demonstrated using green fluorescence protein, phosphoserine aminotransferase (serC) and aspartokinase III (AKIII) as model systems. The results show that mutants with changes in different protein properties upon multiple point mutations can be produced and screened within 6 to 15 h. This method can be used further to generate mutants of enzymes and multi-enzyme complexes and be implemented within the workflow of a feedback-guided protein optimization and screening system.