Constructive approach for synthesis of a functional IgG using a reconstituted cell-free protein synthesis system

• Published in: Scientific reports Vol. 9; no. 1; p. 671
• Main Authors: Murakami, Satoshi, Matsumoto, Rena, Kanamori, Takashi
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 24.01.2019; Nature Publishing Group
• Subjects: 631/337/470; 631/337/574; 82/1; 82/80; Cell culture; Cofactors; DNA biosynthesis; DnaK protein; ErbB-2 protein; High-throughput screening; Humanities and Social Sciences; Immunoglobulin G; Monoclonal antibodies; multidisciplinary; Protein biosynthesis; Protein synthesis; Proteins; Science; Science (multidisciplinary); Trastuzumab
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-018-36691-8

IgG is an indispensable biological experimental tool as well as a widely-used therapeutic protein. However, cell culture-based expression of monoclonal IgG is costly and time-consuming, making this process difficult to use for high-throughput screening in early-stage evaluation of biologics. With the goal of establishing a fast, simple, and robust high-throughput expression system for IgG, we implemented the synthesis of functional aglycosylated IgG by constructive approach based on a reconstituted prokaryotic cell-free protein synthesis system (PURE system). Optimization of the PURE system revealed that the following factors and reaction conditions were needed for IgG synthesis: (1) inclusion of the disulfide bond isomerase DsbC, (2) adjustment of the GSH/GSSG ratio, (3) inclusion of the molecular chaperone DnaK and its cofactors, and (4) use of an extended incubation time. Synthesis temperature and template DNA ratio (light chain-/heavy chain-encoding) also had been optimized for each IgG. Under optimal conditions, peak production of the anti-HER2 antibody trastuzumab reached 124 µg/mL. Furthermore, the active forms of other IgGs, including IgG1, IgG2, and IgG4 subclasses, also were synthesized. These results provide basic information for the development of novel high-throughput expression and functional screening systems for IgG, as well as useful information for understanding the IgG synthesis process.