Noncanonical amino acids enable plug and play vaccine platform in the ALiCE cell free system.

Non-canonical amino acids (ncaas) are increasingly used in vaccinology to improve vaccine adaptability and immunogenicity. Cell-free protein synthesis (CFPS) offers a promising route for site-specific ncaa incorporation, but conventional prokaryotic CFPS systems show limitations to produce complex proteins requiring post-translational modifications while eukaryotic systems have historically been difficult to scale and show low protein yields. Here we establish efficient site-specific introduction of ncaas into complex proteins with the high-yielding and scalable eukaryotic tobacco BY-2 CFPS system (BYL), commercialized as ALiCE®. ncaa incorporation yields reached up to 2 mg/ml with linear scalability up to 10 ml. We applied ncaa incorporation in BYL to enable click chemistry bioconjugation of the receptor binding domain (RBD) of influenza hemagglutinin to pre-assembled hepatitis B core (HBc) virus-like particles (VLPs). The resulting VLP-RBD conjugates exhibited hemagglutination activity, unlike the individual components, and protected mice from weight loss after influenza challenge. This research thus enables ncaa introduction for recombinant proteins produced in BYL, constructing a novel plug-and-play vaccine platform and further expanding the capabilities of BYL to produce vaccine candidates and other proteins of interest.