Soluble cysteine‐rich tick saliva proteins Salp15 and Iric‐1 fromE. coli. Issue 1 (24th December 2014)

• Record Type: Journal Article
• Authors: Kolb, Philipp; Vorreiter, Jolanta; Habicht, Jüri; Bentrop, Detlef; Wallich, Reinhard; Nassal, Michael
• Is Part Of: FEBS open bio. Volume 5:Issue 1(2015)
• Page Start: 42
• Page End: 55
• Publication Date: 2014-12-24
• Subjects: DsbA -- thiol:disulfide interchange protein DsbA -- GB1 -- immunoglobulin binding domain B1 of streptococcal protein G -- eGFP -- enhanced green fluorescent protein -- GST -- glutathione-S-transferase -- HSQC -- heteronuclear single quantum coherence -- MBP -- maltose-binding protein -- OspC -- outer surface protein C -- Salp15 -- salivary protein of 15 kDa -- Trx -- thioredoxin -- Lyme borreliosis -- Protein NMR -- Saliva-assisted transmission -- Solubility-enhanced fusion proteins -- Tick saliva -- Tick saliva antibodies; Molecular biology -- Periodicals; Cytology -- Periodicals; Life sciences -- Periodicals; Biological Science Disciplines -- Periodicals; Molecular Biology -- Periodicals; Cell Biology -- Periodicals; Cytology; Life sciences; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.fob.2014.12.002
• Languages: English
• ISSNs: 2211-5463

Abstract

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. Abstract : Tick saliva proteins Salp15 and Iric‐1 promote tick feeding and pathogen transmission. We established the first bacterial expression system for soluble Salp15 and Iric‐1. Using this system we mapped monoclonal antibody epitopes on Salp15 and Iric‐1. We defined the interaction sites with Borrelia outer surface protein C (OspC). We elucidated first secondary structure features in Iric‐1 by NMR.