Cell-free synthesis system suitable for disulfide-containing proteins

Abstract

Many important therapeutic targets are secreted proteins with multiple disulfide bonds, such as antibodies, cytokines, hormones, and proteases. The preparation of these proteins for structural and functional analyses using cell-based expression systems still suffers from several issues, such as inefficiency, low yield, and difficulty in stable-isotope labeling. The cell-free (or in vitro) protein synthesis system has become a useful protein production method. The openness of the cell-free system allows direct control of the reaction environment to promote protein folding, making it well suited for the synthesis of disulfide-containing proteins. In this study, we developed the Escherichia coli (E. coli) cell lysate-based cell-free synthesis system for disulfide-containing proteins, which can produce sufficient amounts of functional proteins for NMR analyses. Disulfide bond formation was facilitated by the use of glutathione buffer. In addition, disulfide isomerase, DsbC, catalyzed the efficient shuffling of incorrectly formed disulfide bonds during the protein synthesis reaction. We successfully synthesized milligram quantities of functional 15N-labeled higher eukaryotic proteins, bovine pancreatic trypsin inhibitor (BPTI) and human lysozyme C (LYZ). The NMR spectra and functional analyses indicated that the synthesized proteins are both catalytically functional and properly folded. Thus, the cell-free system is useful for the synthesis of disulfide-containing proteins for structural and functional analyses.