Enhance Methanol Tolerability of M. Extorquens AM1 By Adaptive Evolution

Authors: 
Cui, L. Y., Tsinghua University
Liang, W. F., Tsinghua University
Zhang, C., Tsinghua University
Xing, X. H., Tsinghua University
Zhu, W. L., Tsinghua University
Chinese Hamster Ovary (CHO) cells are routinely optimized to stably express monoclonal antibodies (mAbs) at high titers. However, CHO cell technology can be unwieldy for rapid, high-throughput production and screening of candidate therapeutic proteins. Here, we have developed a cell-free protein biomanufacturing platform utilizing a commercially available CHO extract for the cell-free synthesis of mAbs. Our specific aims were to: (1) optimize cell-free reaction parameters to maximize protein yields, (2) screen additives to enhance disulfide bond formation for intact mAb production, and (3) establish the extract as a predictable tool for ranking yields of candidate antibodies. Optimization of the reaction conditions increased protein yields 8-fold to 700 mg/L using green fluorescent protein (GFP) as a reporter. Additives such as oxidized glutathione established an oxidizing cell-free environment for disulfide bond formation, and chaperones with disulfide isomerase activity further increased intact mAb yields 2-fold. Lastly, light and heavy chain expression was optimized to minimize aggregation products. Using our optimized platform, we demonstrate for the first time the cell-free synthesis of active, intact mAb using CHO extract. Additionally, the cell-free system demonstrated the capacity to rank known high and low producing mAb sequences in the same order as seen from CHO cell-based platforms. Unlike stable or transient transfection-based screening, which requires 8-14 days for setup and execution, results using our CHO-based CFPS system are attained within 2 days. Further development of the customizable cell-free platform would provide a tool for rapid, high-throughput ranking of mAb producers and has implications for the synthesis of difficult-to-express proteins.