Armero Gimenez, J. | Search bioRxiv

bioRxiv2025

Le click c'est chic: a plug-and-play virus-like particle vaccination platform enabled by non-canonical amino acid incorporation and click chemistry in the tobacco BY-2 cell-free protein synthesis system

Non-canonical amino acids (ncaas) can provide recombinant proteins with novel exciting functionalities beyond the limits of nature, such as orthogonal reaction groups. Notably, ncaa introduction can be used in vaccinology to enhance the adaptability and immunogenicity of putative vaccine candidates. Cell-free protein synthesis (CFPS) represents the most promising methodology to introduce ncaa into recombinant proteins of interest. However, traditionally used prokaryotic CFPS systems show limitations to produce complex proteins requiring post-translational modifications, whilst eukaryotic CFPS systems have historically been difficult to scale and show low protein yields. In this work, we establish the site-specific introduction of ncaas into complex proteins with the high-yielding and scalable eukaryotic tobacco BY-2 CFPS system (BYL), commercialised as ALiCE(R). The tyrosine transferase from Escherichia coli (eTyrT) was tested for amber suppression-mediated ncaa incorporation in BYL. eTyrT showed high incorporation yields of up to 2mg/ml recombinant protein for the azido-tyrosine and alkyne-tyrosine ncaas, with linear scalability up to 10ml without any losses in protein yield. 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). BYL efficiently produced the alkyne-modified RBD and azido-modified HBc VLPs, and their conjugation via copper-catalysed azide-alkyne cycloaddition (CuAAC) led to structurally intact, RBD-coated particles. VLP-RBD conjugates could efficiently hemagglutinate chicken erythrocytes where the individual proteins could not, proving both the sialic-acid binding activity of the RBD and its multivalent presentation by the HBc VLP. Finally, when used to vaccinate mice the conjugated RBD-VLPs showed a greater protection against live influenza challenge than free RBD. 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.

molecular biology↗

bioRxiv2022

ALiCE: A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system

Eukaryotic cell-free protein synthesis (CFPS) systems have the potential to simplify and speed up the expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and the inability to scale such systems have so far prevented their widespread adoption in protein research and manufacturing. Here, we present a detailed demonstration for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in under 48 hours, complete with native disulfide bonds and N-glycosylation. An optimised version of the technology is commercialised as ALiCE(R), engineered for high yields of up to 3 mg/mL. Recent advances in the scaling of BYL production methodologies have allowed scaling of the CFPS reaction. We show simple, linear scale-up of batch mode reporter proten expression from a 100 L microtiter plate format to 10 mL and 100 mL volumes in standard Erlenmeyer flasks, culminating in preliminary data from 1 L reactions in a CELL-tainer(R) CT20 rocking motion bioreactor. As such, these works represent the first published example of a eukaryotic CFPS reaction scaled past the 10 mL level by several orders of magnitude. We show the ability of BYL to produce the simple reporter protein eYFP and large, multimeric virus-like particles directly in the cytosolic fraction. Complex proteins are processed using the native microsomes of BYL and functional expression of multiple classes of complex, difficult-to-express proteins is demonstrated, specifically: a dimeric, glycoprotein enzyme, glucose oxidase; the monoclonal antibody adalimumab; the SARS-Cov-2 receptor-binding domain; human epidermal growth factor; and a G protein-coupled receptor membrane protein, cannabinoid receptor type 2. Functional binding and activity are shown using a combination of surface plasmon resonance techniques, a serology-based ELISA method and a G protein activation assay. Finally, in-depth post-translational modification (PTM) characterisation of purified proteins through disulfide bond and N-glycan analysis is also revealed - previously difficult in the eukaryotic CFPS space due to limitations in reaction volumes and yields. Taken together, BYL provides a real opportunity for screening of complex proteins at the microscale with subsequent amplification to manufacturing-ready levels using off-the-shelf protocols. This end-to-end platform suggests the potential to significantly reduce cost and the time-to-market for high value proteins and biologics.

biochemistry↗