Rhizavidin engineered for eukaryotic expression

Several technologies leverage avidin/biotin interactions for complexation, including the MAPS vaccine technology which utilizes rhizavidin, a biotin-binding protein derived from the proteobacterium Rhizobium etli, to complex antigens with biotinylated polysaccharides. Rhizavidin possesses five potential N-linked glycosylation sites which are not glycosylated in the native bacterium or when rhizavidin is recombinantly expressed in Escherichia coli. However, when expressed in eukaryotic cell systems, these sites undergo variable and non-physiologically relevant glycosylation that complicates purification. To overcome these challenges, we engineered rhizavidin with substitutions that abolish unnatural N-linked glycosylation while maintaining rhizavidin's biotin-binding functionality. As a proof of concept, this construct was genetically fused to the receptor binding domain of the spike protein from the SARS-CoV-2 virus, expressed in mammalian cells, and was successfully incorporated into MAPS technology-based vaccines. This newly engineered rhizavidin enhances the versatility of the MAPS technology, enabling the targeting of viruses and tumor-associated antigens that often require mammalian post-translational modifications.