Ticks transmit numerous pathogens, including borreliae, which cause
Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive
cysteine-rich secretory
glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodes ricinus. All tick-borne microbes benefit from the immunosuppression at the
tick bite site; in addition, borreliae exploit the binding of Salp15 to their
outer surface protein C (
OspC) for enhanced transmission. Hence, Salp15
proteins are attractive targets for anti-tick
vaccines that also target borreliae. However, recombinant Salp
proteins are not accessible in sufficient quantity for either
vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing
protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled
epitope mapping of two
monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with
OspC. Free Salp15 and Iric-1 from
protease-cleavable fusions, despite limited solubility, allowed the recording of (1)H-(15)N 2D NMR spectra, suggesting partial folding of the wild-type
proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in (13)C/(15)N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15
proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant
tick proteins.