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T3SS Inner Rod protein fused to Lethal Factor - NLRC4 agonist
Mouse NLRC4/NAIP Inflammasome Inducer
InvivoGen provides LFn-Rod, a model of NLRC4/NAIP inflammasome agonist [1-3]. The Inner Rod protein is a component of the type III secretion systems (T3SS) of intracellular bacteria described as an NLRC4/NAIP ligand [1-5]. It is fused to the amino-terminal domain of B. anthracis lethal factor (LFn). This fusion system, when co-administred with the anthrax toxin’s protective antigen (PA), allows intracellular delivery of the bacterial ligand . The combination of LFn-Rod with the anthrax protective antigen (PA) is named Rod-Tox .
InvivoGen's LFn-Rod recombinant protein features S. typhimurium T3SS Inner Rod protein [M1-S101] fused to the amino-terminal domain [A34-R296] of anthrax toxin’s lethal factor (LFn) protein in N-terminal. Its ability to induce the murine NLRC4 inflammasome has been validated using RAW-ASC and RAW-ASC KO-NLRC4 cell lines.
LFn-Needle is also available and is a potent inducer of human NLRC4/NAIP inflammasome.
The NLRC4 (aka Ipaf) inflammasome is an intracellular multi-protein complex that plays a central role in innate immunity [7, 8]. It is activated by a two-step process; a first signal (‘priming’) is provided by microbial molecules such as TLR ligands, while the second signal is provided by intracellular bacterial molecules such as Flagellin from the motility apparatus, or Inner Rod and Needle proteins from the bacterial type III or IV secretion systems (T3SS or T4SS). NLCR4 is an indirect sensor: it interacts with NAIPs (NLR family apoptosis inhibitory proteins) that directly bind to Flagellin, Needle, and Inner Rod. While a single NAIP operates upstream of NLRC4 in humans and recognizes each of these activators , multiple NAIPs have been described in mice with different affinities for each molecule [1-4]. The NLRC4 inflammasome appears to protect mucosal barriers, such as the lung, stomach, and intestine, from invading bacteria .
Features of LFn-Rod:
- Inner Rod (PrgJ) from S. typhimurium T3SS
- Potent inducer of the murine NLRC4 inflammasome in vitro
- Produced in Sf9 insect cells
- Each lot is functionally tested
1. Zhao Y. et al., 2011. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature. 477(7366):596-600.
2. Kofoed E.M. & Vance R.E., 2011. Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature. 477(7366):592-595.
3. Rauch I. et al., 2016. NAIP proteins are required for cytosolic detection of specific bacterial ligands in vivo. The Journal of Exp. Med. 213(5):657-665.
4. Zhao Y. et al., 2016. Genetic functions of the NAIP family of inflammasome receptors for bacterial ligands in mice. J Exp Med. 213(5):647-656.
5. Worrall L.J. et al., 2011. Structural overview of the bacterial injectisome. Curr Opin Microbiology. 14(1):3-8.
6. Ballard J.D. et al., 1996. Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo. PNAS. 93(22):12531-12534.
7. Platnich J.M. & Muruve D.A., 2019. NOD-like receptors and inflammasomes: A review of their canonical and non-canonical signaling pathways. Arch Biochem Biophys. 670:4-14.
8. Bauer R. & Rauch I., 2020. The NAIP/NLRC4 inflammasome in infection and pathology. Mol Aspects Med. 76:100863.
9. Yang J. et al., 2013. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation. PNAS. 110(35):14408-14413.
Secretion of mature IL-1β by RAW-ASC KO-NLRC4 cells and their parental RAW-ASC cells upon inflammasome activation.
~2x105 RAW-ASC (WT) and RAW-ASC KO‑NLRC4 (KO) cells were incubated for 3h at 37°C with Pam3CSK4 (100 ng/ml) (priming) and then incubated (activation) with inflammasome inducers: Nigericin (5 μM), transfected Poly (dA:dT) (1 μg/ml), Rod-Tox (2 μg/ml). After 24h, the secretion of mature IL-1β was assessed in the culture supernatant using an ELISA assay.
Note: Rod-Tox is a combination of LFn-Rod (2 μg/ml) with the anthrax protective antigen (PA) (1 μg/ml). PA allows LFn-Rod translocation into the cytosol.
Secretion of mature IL-1β and pyroptosis upon RAW-derived cell activation using LFn-Rod.
~2x105 RAW-ASC (WT) parental and RAW-ASC KO‑NLRC4 (KO) cells were incubated for 3h at 37°C with Pam3CSK4 (100 ng/ml) (priming) and then incubated (activation) with LFn-Rod (0.64 ng -10 μg/ml). After 24h activation, the secretion of mature IL-1β was assessed in the culture supernatant using an ELISA assay (A), and cell death was assessed using the lactate dehydrogenase (LDH) assay (B).
- Protein construction: T3SS Inner Rod (PrgJ) protein [M1-S101] fused to the amino-terminal domain [A34-R296] of anthrax toxin’s lethal factor (LFn) protein in N-terminal.
- Accession sequence: WP_00002043 (Inner Rod; PrgJ sequence)
- Species: Salmonella typhimurium
- Source: Sf9 insect cells
- Tag: N-terminal poly-histidine (6 x His)
- Total protein size: 384 a.a. (secreted form)
- Molecular weight: ~ 48 kDa (SDS-PAGE gel)
- Purification: Ni2+ affinity chromatography
- Purity: >90% (SDS-PAGE)
- The protein has been validated by functional assays using RAW-ASC and RAW-ASC KO-NLRC4 cell lines.
- The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cellular assays.
Note: B. anthracis protective antigen (PA) is not provided
- 50 μg of lyophilized LFn-Rod protein
- 1.5 ml of endotoxin-free water
The product is shipped at room temperature.
Lyophilized protein should be stored at -20 ̊C up to 6 months.
Resuspended protein is stable up to 6 months when stored at -20°C
Avoid repeated freeze-thaw cycles.Back to the top