Figure 1.

Schematic representation of the workflow. (A) Cell free protein synthesis (CFPS) of the protein of interest (POI) fused with an C-terminal split intein, typically 4–6 hours. The CFPS matrix is depicted in beige; (B) Split intein mediated capture, for 3 hours. The capture peptide is either in solution or immobilized to the surface (the dashed line depicts both scenarios). In orange the ‘Tag’ can be any affinity tag. The red bar is a photo-cleavable amino acid, while the blue bar is an unnatural amino acid linked to a reactive moiety. At this stage the intein will splice itself out spontaneously, without co-factors^46,47; (C) After the intein reaction the CFPS matrix and side-products are washed away in under half an hour, and the final buffer is added (light blue); (D) after light triggered release (3 hours) POI is released into its final formulation. Bioconjugation can be performed now, later (for example in-vivo) or also at the end of step C before light treatment.