High quality reagents for innate immunity research, vaccine development and drug discovery

		Recombinant S. Typhimurium FljB protein with central SARS-CoV2 RBD domain
		Background Flagellin is the principal structural protein of bacterial flagella, the helical appendages that enable bacterial motility [1]. Almost uniquely among proteins, flagellin contains regions that are sufficiently conserved across bacterial species to be recognised by two distinct pattern recognition receptors (PRRs) of the mammalian innate immune system. Flagellin may bind Toll-like receptor 5 (TLR5) on the surface of immune cells, triggering signalling pathways that result in the production of pro-inflammatory cytokines and other immune responses [2]. Alternatively, flagellin that enters the cytosol may be recognised by the NAIP / NLRC4 inflammasome, resulting in the processing of pro-IL1β to the active form of IL1β, and pyroptosis via cleavage of gasdermin D [3]. As these responses make flagellin a potent activator of dendritic cells and adaptive immunre responses more generally, it has received much interest as a vaccine adjuvant and carrier in both pre-clinical models and clinical trials [4]. The flagellin molecule can be thought of as comprising four major domains, with domains D0 and D1 being highly conserved, and containing the motifs responsible for recognition by TLR5 and NLRC4. The D3 domain, by contrast, is highly variable, and is the dominant epitope for anti-flagellin antibodies arising from natural infection as it is exposed on the surface of the flagellar filament. Fusion proteins in which antigens of interest either replace the D3 domain, or are attached at the C-terminus of flagellin, have been shown to be potent inducers of humoral and T-cell responses to the target antigen [4]. The spike (S) protein of the SARS-CoV-2 virus, which is responsible for the respiratory illness referred to as COVID-19, is essential for the virus’s ability to infect human cells [5]. Specifically, the receptor-binding domain (RBD) of the spike protein is responsible for directly interacting with the angiotensin-converting enzyme 2 (ACE2) receptor on target cells, enabling virus attachment and entry. Being prominently exposed on the surface of the viral particle, the RBD is also a dominant antigen of the humoral immune response to SARS-CoV-2 infection. For these reasons, it has become the most commonly targeted antigen for the generation of candidate and approved vaccines, and for use in studies of serological conversion [6.7]. Caithness Biotech recombinant Salmonella enterica serovar Typhimurium FljB flagellin with central C-terminal SARS-CoV2 RBD domain comprises amino acids Met 1 to Ala 190 of S. Typhimurium FljB protein, then Arg 319 to Phe 541 of the SARS-CoV2 spike protein (strain Wuhan-Hu-1, Pango lineage A, Nextstrain Clade 19A [5]) replacing the central D3 domain, then FljB Val 293 - Arg 506 of S. Typhimurium FljB protein. This product retains potent TLR5 stimulatory capacity, and is expressed in mammalian cells to maximise purity and minimise presence of contaminating bacterial stimulants of other TLRs, such as TLR2 and TLR4. Potential applications of flagellin fusion proteins include use in studies of innate immune signalling, and for vaccine development.
		Description
		Product	Recombinant S. Typhimurium FljB protein with central SARS-CoV2 RBD domain
		Size / volume	20 μg
		Expression system	HEK-293 cells
		Amino acids	FljB Met 1 to Ala 190, SARS-CoV2 Arg 319 to Phe 541, FljB Val 293 - Arg 506, accession numbers P52616 and NCBI reference sequence YP_009724390.1
		Tags	C-terminal 6x His tag
		Sequence graphic
		Intended use	For laboratory research only, not for clinical or diagnostic use.
		Specifications
		Format	Lyophilised from sterile PBS (pH 7.4) with trehalose as protectant and without additional carrier protein.
		Purity	>95% by SDS PAGE
		Molecular weight	Migrates at ~ 75 kDa (glycosylation present)
		LPS content	< 0.1 ng / μg (by HEK-293-TLR4 bioassay, relative to E. coli LPS standard)
		BLP content	< 0.1 ng / μg (by HEK-293-TLR2 bioassay, relative to Pam3CSK4 standard)
		Amino acid sequence	See Technical data sheet.
		Applications	ELISA / bioassay / SDS PAGE / binding studies / immunoassays
		Pricing
		Catalogue number	FLA-R1
		Price per unit	£100 per vial of 20 μg
		Bespoke flagellin fusion protein service We also offer cloning, expression and purification of bespoke flagellin fusion proteins for vaccine research and development. Your antigen of interest may be fused either to the C-terminus, or within a central domain replacing the D3 loop. Please click here for futher information regarding our bespoke flagellin services.
		Figure 1: SDS PAGE analysis 1 μg of recombinant protein was separated by reducing SDS PAGE and visualised by Coomassie Blue staining. Caithness Biotech recombinant FljB-RBD ΔD3 migrates at approximately 75 kDa due to glycosylation.		Figure 2: Validation of the capacity of recombinant FljB-RBD to stimulate TLR5-signalling HEK-293 cells were transfected with NF-κB reporter and TLR5, then treated with indicated concentrations of the reconstituted protein (FLA-R1) or native (non-recombinant) S. typmimurium flagellin (StF) overnight. NF-κB activation was measured by luminometry.
		Product flyer Please click here to download the product flyer.		Technical data sheet Please click here to download the product technical data sheet.
	References
	[1] Samatey FA, Imada K, Nagashima S, Vonderviszt F, Kumasaka T, Yamamoto M, Namba K. Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling. Nature 410:331-7 (2001) [2] Smith KD, Andersen-Nissen E, Hayashi F, Strobe K, Bergman MA, Barrett SLR, Cookson BT, Aderem A. Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat Immunol 4:1247-53 (2003) [3] Zhao Y, Yang J, Shi J, Gong Y-N, Lu Q, Xu H, Liu L, Shao F. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature 477:596-600 (2011) [4] Huleatt JW, Jacobs AR, Tang J, Desai P, Kopp EB, Huang Y, Song L, Nakaar V, Powell TJ. Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity. Vaccine 25:763-75 (2007) [5] Zhou P, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579:270-273 (2020) [6] Kleanthous H, Silverman JM, Makar KW, Yoon I-K, Jackson N, Vaughn DW. Scientific rationale for developing potent RBD-based vaccines targeting COVID-19. npj Vaccines 6:128 (2021) [7] Amanat F, et al. A serological assay to detect SARS-CoV-2 seroconversion in humans. Nature Medicine 26:1033-1036 (2020)

 

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Caithness Biotechnologies Ltd., 72 Boston Road, Leicester, UK, LE4 1HB. Telephone: +44 (0) 116 326 3802 | email: contact@caithnessbiotechnologies.com