(164i) Modulated Cell-Free Protein Synthesis System for Biomanufacturing of Multiple Disulfide Bond Containing Therapeutic Proteins

The cell-free system (CFS) activates complex biological processes without using intact living cells. Unlike the living system, CFS utilizes the cell extract retaining cellular components that allows direct access to biocatalytic enzymes, easy substrate addition and product removal, and rapid sampling. The distinctive perspective of the CFS brings the technological advantages over traditional fermentation methods such as decoupling of protein synthesis from cell growth and reproduction, which allows engineers to solely utilize the catalysts from cells, enabling the study of the protein synthesis process by examining the role of the supplements such as chaperones, elongation factors, ribosomes in the CFS. In this work, we studied E. coli crude cell extracts that facilitate multiple disulfide bonds containing therapeutic protein production. We established a strategy for modulating multiple cell extracts harboring periplasmic chaperone proteins and assembled them in optimal conditions. To achieve the overarching goal, we first utilized Gaussia luciferase for tuning the modulated CFS and then applied the strategy for the biomanufacturing of different human therapeutic proteins containing multiple disulfide bonds. We expect that the modulated cell-free biomanufacturing platform for disulfide bond-containing protein will promote high-yielding therapeutic protein and increase opportunities for discovering new therapeutic proteins.