Peptide linker increased the stability of pneumococcal fusion protein vaccine candidate.

Autor:	Zane L; Laboratory of Vaccine Development, Butantan Institute, Sao Paulo, Brazil.; Interunits Graduate Program in Biotechnology, University of Sao Paulo, Sao Paulo, Brazil., Kraschowetz S; Laboratory of Vaccine Development, Butantan Institute, Sao Paulo, Brazil.; Interunits Graduate Program in Biotechnology, University of Sao Paulo, Sao Paulo, Brazil., Trentini MM; Laboratory of Vaccine Development, Butantan Institute, Sao Paulo, Brazil., Alves VDS; Laboratory of Vaccine Development, Butantan Institute, Sao Paulo, Brazil.; Interunits Graduate Program in Biotechnology, University of Sao Paulo, Sao Paulo, Brazil., Araujo SC; Laboratory of Vaccine Development, Butantan Institute, Sao Paulo, Brazil.; Interunits Graduate Program in Biotechnology, University of Sao Paulo, Sao Paulo, Brazil., Goulart C; Laboratory of Vaccine Development, Butantan Institute, Sao Paulo, Brazil.; Interunits Graduate Program in Biotechnology, University of Sao Paulo, Sao Paulo, Brazil., Leite LCC; Laboratory of Vaccine Development, Butantan Institute, Sao Paulo, Brazil., Gonçalves VM; Laboratory of Vaccine Development, Butantan Institute, Sao Paulo, Brazil.
Jazyk:	angličtina
Zdroj:	Frontiers in bioengineering and biotechnology [Front Bioeng Biotechnol] 2023 Jan 26; Vol. 11, pp. 1108300. Date of Electronic Publication: 2023 Jan 26 (Print Publication: 2023).
DOI:	10.3389/fbioe.2023.1108300
Abstrakt:	Streptococcus pneumoniae is a bacterial pathogen exclusive to humans, responsible for respiratory and systemic diseases. Pneumococcal protein vaccines have been proposed as serotype-independent alternatives to currently used conjugated polysaccharide vaccines, which have presented limitations regarding their coverage. Previously in our group, pneumococcal surface protein A (PspA) and detoxified pneumolysin (PdT) were genetically fused and the hybrid protein protected mice against pneumococcal challenge, offered higher cross-protection against different strains and showed greater opsonophagocytosis rate than co-administered proteins. As juxtaposed fusion was unstable to upscale production of the protein, flexible (PspA-FL-PdT) and rigid (PspA-RL-PdT) molecular linkers were inserted between the antigens to increase stability. This work aimed to produce recombinant fusion proteins, evaluate their stability after linker insertion, both in silico and experimentally, and enable the production of two antigens in a single process. The two constructs with linkers were cloned into Escherichia coli and hybrid proteins were purified using chromatography; purity was evaluated by SDS-PAGE and stability by Western blot and high performance size exclusion chromatography. PspA-FL-PdT showed higher stability at -20°C and 4°C, without additional preservatives. In silico analyses also showed differences regarding stability of the fusion proteins, with molecule without linker presenting disallowed amino acid positions in Ramachandran plot and PspA-FL-PdT showing the best scores, in agreement with experimental results. Mice were immunized with three doses and different amounts of each protein. Both fusion proteins protected all groups of mice against intranasal lethal challenge. The results show the importance of hybrid protein structure on the stability of the products, which is essential for a successful bioprocess development. Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2023 Zane, Kraschowetz, Trentini, Alves, Araujo, Goulart, Leite and Gonçalves.)
Databáze:	MEDLINE
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