(511a) Expression of Vaccine Targets in a Cell-Free Protein Synthesis System Based on Pichia Pastoris

Cell-free protein synthesis (CFPS) has been established as an alternative approach to living cells for the production of target proteins. CFPS is highly effective for producing toxic or difficult-to-express proteins like membrane proteins. It allows expression in a completely controlled reaction environment with manipulation at every step of protein expression process. CFPS also allows the expression of proteins at point-of-care and on demand. CFPS based on eukaryotic lysates are better suited for the production of proteins requiring post translational modification because they inherently contain all the required machinery. On example is glycoproteins, which are widely used as biologics and therapeutic products. Pichia pastoris CFPS can be a potential alternative for the successful expression of important glycoproteins due to its high protein synthesis efficiency and the existence of engineered strains with humanized glycosylation machinery. Proteins expressed in Pichia pastoris CFPS can be used as vaccine targets or biotherapeutics.

We have developed a method for preparation of Pichia lysate enriched with active microsomes and used that lysate for efficient glycosylation during CFPS. During lysate preparation, organelles like the Endoplasmic reticulum and Golgi body are converted to microsomal vesicles and these endogenous structures retain functionality for post translational modifications (eg: Glycosylation). Nano flow cytometry analysis and fluorescent microscopy were used to characterise and quantify microsomal content of lysates prepared using different centrifugal forces (Fig 1). We also established methods for harvesting of microsomes from the cell-free reaction and extraction of the glycoproteins from microsomal vesicles using SARS-CoV-2 S1 RBD glycoprotein as a model vaccine (Fig 2). PNGase digestion assay, Lectin Western Blotting and Mass Spectrometry were carried out to confirm the presence of glycans on the model protein. Finally, to improve the cost effectiveness of our CFPS, we also established the use of alternative amino acid sources (eg; Tryptone and Casamino acids) as appropriate substitutes for the commercial RTS mix to reduce costs by 100-fold. In future, our developed method can be used as a platform to successfully synthesise of glycoproteins in a cost-effective way using Pichia pastoris cell-free protein synthesis system.