In Vitro Compartmentalisation of Cell-Free Protein Synthesis Reaction for Protein Directed Evolution

Directed evolution is a technique that allows improvements on a selected feature through cycles of diversification, selection and amplification. This work seeks to establish a platform for protein directed evolution with the aim of performing early screenings to biopharmaceutical candidates in an ultra-high-throughput fashion for improved soluble expression and stability. The platform works using semipermeable gel-shell beads (GSBs)¹ containing an E. coli CFPS reaction mix and a single DNA molecule encoding a library member. These GSBs can be sorted by fluorescent properties using a conventional FACS machine. Protein fluorescent labelling is enabled by the semi-permeability of the GSBs membrane and the incorporation of non-standard amino acids (nsAA) for selective conjugation. We propose different selection strategies based on different fluorescent markers. The present work shows CFPS in GSBs analysed by flow cytometry and nsAA incorporation and labelling in bulk. Our projections indicate we should be able to sort ~10⁶ variants per selection round. We believe these results are promising for the development of the designed platform which can impact positively in the early stages of biopharmaceuticals pipelines selecting variants with improved physico-chemical properties.

1. Fischlechner, M. et al. Evolution of enzyme catalysts caged in biomimetic gel-shell beads. Nat. Chem. 6, 791–6 (2014).