Cell-free protein synthesis: applications come of age

Abstract

Cell-free protein synthesis has emerged as a powerful technology platform to help satisfy the growing demand for simple and efficient protein production. While used for decades as a foundational research tool for understanding transcription and translation, recent advances have made possible cost-effective microscale to manufacturing scale synthesis of complex proteins. Protein yields exceed grams protein produced per liter reaction volume, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and reaction scale has reached the 100-liter milestone. These advances have inspired new applications in the synthesis of protein libraries for functional genomics and structural biology, the production of personalized medicines, and the expression of virus-like particles, among others. In the coming years, cell-free protein synthesis promises new industrial processes where short protein production timelines are crucial as well as innovative approaches to a wide range of applications.

Figure 1

Cell-free protein synthesis systems exploit crude cell extracts to produce valuable therapeutics and vaccines, among other products.

Figure 2

Cartoon comparison of in vivo recombinant DNA protein expression with cell-free protein synthesis (CFPS). CFPS systems provide a more rapid process/product development timeline. Example proteins shown include a virus-like particle (VLP), single-chain antibody variable fragment (scFv), and a membrane bound protein (MBP).

Figure 3

Historical trends of cell-free protein synthesis systems. Blue squares = E. coli extract (ECE), red circles = wheat germ extract (WGE), purple triangles = insect cell extract (ICE), and green diamonds = rabbit reticulocyte lysate (RRL). (A) Cell-free protein synthesis yields for a batch reaction. (B) “Effective” cell-free protein synthesis yields for a fed-batch or continuous exchange cell-free (CECF) reaction based on the total volume of reaction and feeding solutions. (C) Reaction length for active protein synthesis in a batch cell-free reaction. (D) Rate of protein synthesis during a cell-free reaction. (E) Protein yield per dollar of NXPs (e.g., ATP, ADP, AMP, GTP, etc.) and energy source, which are the dominant substrate costs of CFPS reactions. (F) Scale of cell-free reaction volumes. Cited references can be found in Supplementary Table 1.

Figure 4

Timeline: Synthetic biology milestones in the production of complex therapeutic proteins. Abbreviations: scFv: single-chain antibody variable fragment, vtPA: variant of human tissue-type plasminogen activator, GM-CSF: granulocyte macrophage colony stimulating factor, IGF-I: insulin-like growth factor I, cIFN-α: human consensus interferon-alpha, rhGM-CSF: human granulocyte macrophage colony-stimulating factor. Cited references can be found in Supplementary Table 2.