As one of the key virulence factors of S. aureus, Hla plays a crucial role in the pathogenesis of many experimental infections, particularly those involving epithelial tissues such as skin abscesses and pneumonia. Upon secretion, Hla exists as a soluble monomer. It oligomerizes on the surface of host cells by binding to its high-affinity metalloprotease receptor, a disintegrin, and metalloprotease 10 (ADAM10). Finally, it forms a 1-3-nm pore that spans the lipid bilayer of the target cell membrane. It is widely recognized that Hla has diverse effects on a variety of host cells. As a secreted pore-forming toxin, Hla targets a range of cell types, including lymphocytes, macrophages, alveolar epithelial cells, pulmonary endothelium, and erythrocytes. The recombinant protein HlaH35L is genetically detoxified that lacks the ability to form pores. Its lytic activity may be neutralized by antibodies against it.
Fig.1 Structure of Hla. (Berube & Wardenburg, 2013)
CD BioGlyco provides a recombinant expression plasmid HlaH35L with one glycoside which is based on the detoxified version of S. aureus Hla. The mature HlaH35L protein's coding sequence is fused downstream and in frame with the signal sequence of E. coli DsbA. A hexahistidine tag is added at the C-terminus. We introduce a glycosylation site at amino acid position 130 of the fused sequence through rational design. We synthesize the DNA encoding the mature protein, including the signal sequence-cleaved form of HlaH35L, the glycosylation site, and the hexahistidine tag. In addition, we subclone it into the NheI and SalI sites of plasmid p150, yielding plasmid p570 for subsequent experiments.
We utilize a state-of-the-art glycoengineering technique for developing a complex vaccine against S. aureus infections. In our advanced approach, the coexpression of genes that encode for S. aureus Capsular Polysaccharides (CPS) biosynthesis, the protein carrier S. aureus Hla, and PglB (an oligosaccharyl transferase from Campylobacter) are coexpressed in Escherichia coli. Our approach takes advantage of E. coli's N-linked glycosylation capabilities, in which O-antigens are transferred to specific protein carrier sites by using the oligosaccharyltransferase PglB. To achieve the multicomponent vaccine, we purify the bioconjugate vaccine by multiple purification steps including immobilized metal affinity, anionic exchange, hydroxyapatite, and size-exclusion chromatography.
Fig.2 The production process of bioconjugate vaccine. (CD BioGlyco)
At CD BioGlyco, our research team specializes in Carbohydrate-based Vaccine Development through our cutting-edge Glyco™ Vaccine Development Platform. Our unique platform allows for the precise design and manipulation of carbohydrate-based vaccines for a range of infectious diseases. CD BioGlyco delivers innovative solutions for vaccine development to clients all over the world. If our services attract you, please contact us so that we provide you with more detailed information.
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