title: Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli.
abstract: 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.
authors: University Hospital Freiburg, Internal Medicine 2/Molecular Biology, Hugstetter Str. 55, D-79106 Freiburg, Germany ; University of Freiburg, Biological Faculty, Schänzlestr. 1, D-79104 Freiburg, Germany.
authors: Philipp
authors: P
authors: Kolb
authors: University Hospital Freiburg, Internal Medicine 2/Molecular Biology, Hugstetter Str. 55, D-79106 Freiburg, Germany.
authors: Jolanta
authors: J
authors: Vorreiter
authors: University Hospital Heidelberg, Institute of Immunology, Im Neuenheimer Feld 305, D-69120 Heidelberg, Germany.
authors: Jüri
authors: J
authors: Habicht
authors: University of Freiburg, Institute of Physiology, Hermann-Herder-Str. 7, D-79104 Freiburg, Germany.
authors: Detlef
authors: D
authors: Bentrop
authors: University Hospital Heidelberg, Institute of Immunology, Im Neuenheimer Feld 305, D-69120 Heidelberg, Germany.
authors: Reinhard
authors: R
authors: Wallich
authors: University Hospital Freiburg, Internal Medicine 2/Molecular Biology, Hugstetter Str. 55, D-79106 Freiburg, Germany.
authors: Michael
authors: M
authors: Nassal
keywords: DsbA, thiol:disulfide interchange protein DsbA; GB1, immunoglobulin binding domain B1 of streptococcal protein G; GST, glutathione-S-transferase; HSQC, heteronuclear single quantum coherence; Lyme borreliosis; MBP, maltose-binding protein; OspC, outer surface protein C; Protein NMR; Saliva-assisted transmission; Salp15, salivary protein of 15 kDa; Solubility-enhanced fusion proteins; Tick saliva; Tick saliva antibodies; Trx, thioredoxin; eGFP, enhanced green fluorescent protein
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