In Vitro Protein Synthesis in Semipermeable Artificial Cells

A novel cell free protein synthesis (CFPS) system utilizing layer-by-layer (LbL) polymer assembly was developed to reduce the operational cost of conventional CFPS. This yielded an encapsulated cell system, dubbed eCells, that successfully performs in vitro CFPS and allows cost-effective incorporation of noncanonical amino acids into proteins. The use of eCells in CFPS circumvents the need for traditional cell lysate preparation and purification of amino acyl-tRNA synthetases (aaRS) while still retaining the small scale of an in vitro reaction. eCells were found to be 55% as productive as standard dialysis CFPS at 13% of the cost. The reaction was shown to be scalable over a large range of reaction volumes, and the crowding environment in eCells confers a stabilizing effect on marginally stable proteins, such as the pyrrolysl tRNA synthetase (PylRS), providing a means for their application in in vitro protein expression. Photocaged-cysteine (PCC) and N-e-(tert-butoxycarbonyl)-L-lysine (Boc-lysine) were incorporated into Peptidylprolyl cis-trans isomerase B (PpiB) using small amounts of ncAA with an adequate yield of protein. Fluorescent activated cell sorting (FACS) was used to demonstrate the partition of the lysate within the eCells in contrast to standard one pot cell lysate-based methods.

Automated summary

A novel cell free protein synthesis system called eCells was developed to incorporate noncanonical amino acids into proteins cost-effectively. eCells show 55% productivity of standard CFPS at only 13% of the cost, with the ability to stabilize marginally stable proteins and scale to large reaction volumes. The use of eCells provides a new pathway for in vitro protein expression, demonstrated by the successful incorporation of noncanonical amino acids into proteins.