Values of ellipticity were converted to molar ellipticity. Fluorescent probe The tetramethylrhodamine (TMR) labeled peptide with sequence Gly-Gly-GLy-Ser-Lys-(TMR)was purchased from Genscript. therefore enhancing RBD immunogenicity. Thus, multimeric RBD-BLS particles are promising candidates for a protein-based vaccinewith a stability, biophysical properties and immunogenicity similar to those of RBD produced in mammalian HEK-2913T cells12. Remarkably, the production of RBD in yeast cells is highly cost effective, and the fermentation bioprocess can be easily scaled up under Good Manufacturing Practice conditions. In pilot-scale experiments in a bioreactor we attained yields of 180?mg L?1 of?~?90% of pure RBD in (BLS) is a highly immunogenic and very stable decameric protein ideally suited to function as an immunogenic carrier. Five BLS subunits (17?kDa each) form a pentamer, and two pentamers a very stable decamer, which can be decorated with any protein whose antigenicity needs to be increased by coupling it to the N-terminus of BLS16C18. Moreover, BLS activates dendritic cells in vitro, increasing the levels of co-stimulatory molecules and the secretion of proinflammatory cytokines, and also recruits dendritic cells in vivo, in both cases in a TLR4-dependent manner19. In addition, BLS chimeras are extremely effective to rapidly activate specific CD8+?lymphocytes, and to induce significant cytotoxic activity20. While other carrier proteins have been non-covalently attached to target proteins through a pair of LY500307 interacting proteins (e.g. X-dockerin-cohesin-Y21 where X and Y are proteins expressed as a fusion protein with the dockerin and cohesin domains, respectively), such non-covalent interactions might be relatively weak particularly for immunization purposes, which normally require the use of strong adjuvants. The translational fusion of target proteins and peptides LY500307 with BLS has allowed researchers to overcome this problem22. However, given that BLS fusions are usually expressed at high yields in in their native state, using the Sortase A enzyme. Sortase A and its variants can efficiently catalyze a transpeptidation reaction that creates a covalent link between two native proteins or peptides23. This reaction requires the N-terminal protein to be covalently linked to contain a specific signal located in its C-terminal stretch: ProteinN-Leu-Pro-X-Thr-Gly (Fig.?1). This site should be preferably in an unstructured context, and the Gly residue is usually followed by a histidine tag to facilitate its purification. The C-terminal protein to be LY500307 covalently linked should contain three LY500307 Gly amino acid residues in its N-terminal region (Gly-Gly-Gly-ProteinC). After the Sortase A transpeptidation reaction, both subunits are joined to produce the covalently-linked product ProteinN-Leu-Pro-X-Thr-Gly-Gly-Gly-ProteinC (Fig.?1). Thus, the Sortase A-mediated transpeptidation reaction can couple preassembled oligomeric carriers, such as BLS or virus-like particles (VLPs), to an immunogen expressed in a different system24. An additional advantage of Sortase A-mediated coupling is that the scar of the transpeptidation reaction in the protein product is only seven residues, of which three are glycines. Open in a separate window Figure 1 The strategy: Sortase A-mediated transpeptidation reaction. (A) The reaction is exemplified by the coupling of a BLS subunit (red) and the SARS-Co-V2 RBD domain (green). The enzymatic intermediary form (RBD covalent bound to Sortase A) is shown, as Tetracosactide Acetate well as its subsequent hydrolysis to yield the by-product RBD-Leu-Pro-Glu-Thr. The latter cannot be reused in the coupling reaction. The reaction catalyzed by TEV protease to yield Gly-Gly-Gly-BLS, the substrate of Sortase A is also shown. (B).