Cell-free protein synthesis for producing 'difficult-to-express' proteins

Biomanufacturing requires the stable production of active biological molecules including proteins. However, a significant percentage of genes cannot be expressed efficiently in a cell-based system due to cellular homeostasis. Upon overproduction, proteins often form inclusion bodies by becoming insoluble, lose biological activities due to improper folding, or are easily degraded by proteases. Many molecular tools and protocols have been established to improve the production yield of 'difficult-to-express' proteins in cell based systems; however, in vivo approaches have a substantial limitation in that the host strains should be maintained healthy for stable protein production and folding. Cell-free protein synthesis (CFPS) offers superior advantages in synthesizing 'difficult-to-express' proteins due to reaction environment openness and no cell viability constraints. In CFPS systems, active protein production can be maximized by optimizing the reaction environment with selective supplements of positive effectors and the elimination of negative effectors. This review summarizes current strategies for the high-quality production of difficult-to-express proteins including toxic cancer therapeutics, antimicrobial peptides/proteins, toxins, vaccines, antibodies, membrane proteins, and proteins containing nonstandard amino acids. (C) 2018 Elsevier B.V. All rights reserved.