Flagelin 22 TFA (CAS 304642-91-9), with the molecular formula C₉₃H₁₆₂N₃₂O₃₄ and a molecular weight of 2272.48, is a white powder in its pure state. It is the trifluoroacetate salt of a highly conserved 22-amino acid fragment at the N-terminus of bacterial flagellin, belonging to a typical pathogen-associated molecular pattern. Its amino acid sequence is Gln-Arg-Leu-Ser-Thr-Gly-Ser-Arg-Ile-Asn-Ser-Ala-Lys-Asp-Asp-Ala-Ala-Gly-Leu-Gln-Ile-Ala. As a benchmark inducer of plant immune activation, Flagelin 22 TFA triggers the plant PTI immune response by specifically activating the FLS2/BAK1 receptor complex. It has the advantages of strong immune activation, high stability and no environmental residue, and is widely used in plant pathology, breeding and new biological pesticide research and development.
The smallest active fragment of flagellin
Flagelin 22 TFA is a synthetically produced linear 22-amino acid peptide, located in the most conserved N-terminal region of bacterial flagellin. It lacks secondary/tertiary folding and exhibits a flexible linear chain conformation, allowing for precise recognition by plant cell membrane receptors and activation of immune signals.
Its primary sequence has clearly defined functional regions: the N-terminal Gln-Arg-Leu-Ser-Thr-Gly-Ser-Arg region is the receptor core binding region, rich in polar and basic amino acids, which can directly intercalate into the extracellular leucine-rich repeat region of the FLS2 receptor, determining its immune activation activity; the middle Ile-Asn-Ser-Ala-Lys-Asp-Asp-Ala-Ala-Gly region is the signal transduction junction region, containing an aspartate-rich motif, which assists in conformational changes in the receptor complex and initiates intracellular signaling; the C-terminal Leu-Gln-Ile-Ala region is the stable anchoring region, where hydrophobic amino acids enhance peptide adhesion to the cell membrane, prolonging the duration of action.
The physicochemical properties and salt-forming characteristics are highly compatible with application requirements: The TFA salt form significantly improves water solubility, far superior to free peptides, making it suitable for various application methods such as foliar spraying and root irrigation; melting point > 220℃, can be stored at room temperature in a sealed, light-protected environment for 24 months, and remains stable for 3 years after repackaging at -20℃; it is resistant to acids, alkalis, and common pesticides, exhibiting excellent field stability; the purity of the active pharmaceutical ingredient can reach over 98%, with single impurities < 0.5% and moisture < 0.2%, meeting the pharmacopoeia standards for peptide preparations.
Compared to natural flagellin protein (approximately 50kDa), the small-molecule linear 22-peptide structure of Flagelin 22 TFA has three major advantages: no immunogenicity, and will not be cleared by plants as a foreign protein; resistance to protease degradation, with a half-life of 7–14 days in leaves or soil; and simple synthesis, with a total yield of up to 70% in solid-phase peptide synthesis and a purity consistently above 98%, making it suitable for large-scale industrial production.
The solid-phase synthesis process employs an Fmoc protection strategy: using Rink amide resin as a carrier, Fmoc-protected amino acids are sequentially coupled from the C-terminus to the N-terminus. After cleavage and deprotection, the amino acids are purified by reversed-phase high-performance liquid chromatography (RP-HPLC), then salted with trifluoroacetic acid, and freeze-dried to obtain the final product. This process facilitates impurity removal, exhibits good batch stability, and enables kilogram-scale production, meeting the large-scale needs of agriculture and scientific research.
Five structural features-a linear 22-peptide backbone, an N-terminal receptor-binding motif, an intermediate signal linker region, a C-terminal membrane anchoring structure, and TFA salt formation for solubilization-constitute the core advantages of Flagelin 22 TFA: high efficiency in immune activation, good water solubility, stable storage, and easy synthesis for mass production. This lays the molecular foundation for its application in plant immune induction and biocontrol.
FLS2-recognized immune initiation logic
The core mechanism of action of Flagelin 22 TFA is based on the highly specific recognition of flg22 by the FLS2 receptor on the plant cell membrane. Arabidopsis thaliana FLS2 is a receptor-like kinase rich in leucine repeats, composed of an extracellular domain, a transmembrane domain, and an intracellular kinase domain. When the 22 amino acids of flg22 bind to the extracellular domain of FLS2, the receptor immediately dimers and recruits another co-receptor, BAK1, to form an active complex. The assembly of the FLS2-BAK1 complex marks the official "alarm" of the plant immune system.
At the moment of receptor activation, the intracellular kinase domain of FLS2 undergoes autophosphorylation, initiating a cascade of intracellular signal transduction. One of the upstream effectors is NADPH oxidase RBOHD. Upon activation, RBOHD transfers electrons from cytoplasmic NADPH to extracellular molecular oxygen at the plasma membrane, generating superoxide anions. These superoxide anions are rapidly disproportionated by superoxide dismutase to hydrogen peroxide, forming the "first burst" of reactive oxygen species. This burst, detectable within minutes of flg22 treatment, represents the fastest-responding cellular event in the plant's immune system.
The burst of reactive oxygen species (ROS) triggers multiple downstream signaling pathways, including the mitogen-activated protein kinase cascade, calcium ion influx, and activation of calcium-dependent protein kinases (MAPKs). Activation of the MAPK signaling pathway further amplifies defense signals, ultimately leading to transcriptional reprogramming of defense-related genes within the nucleus. Transcriptomic data show that flg22 treatment can induce expression changes in thousands of genes within hours, including genes encoding pathogenesis-related proteins, lignin synthases, and phytoalexin synthases. These gene products perform direct antibacterial functions or reinforce the physical barrier of the cell wall.
In stomatal guard cells, the flg22-triggered signaling pathway leads to the synergistic accumulation of ROS and the secondary messenger nitric oxide, triggering potassium ion efflux and a decrease in osmotic pressure, prompting stomatal closure. This "stomatal immunity" effect physically prevents bacteria from invading the leaf interior through stomata, serving as the plant's first physical line of defense against foliar pathogens. When purified flg22 is applied exogenously, the process does not require the presence of pathogens, allowing researchers to study immune signaling pathways under sterile conditions.
Besides local defense responses, flg22 can also induce systemic acquired resistance in plants. When leaves locally sense flg22 signals, untreated leaves further afield also enter a "vigilant state," exhibiting stronger resistance to subsequent pathogen attacks. In agricultural production, this discovery has given rise to the concept of "immune inducers"-using elicitors such as flg22 to induce crops into a defensive state earlier, reducing the use of chemical pesticides.
Molecular benchmark for model plant research
The most mature and well-supported application of Flagelin 22 TFA is as the "gold standard" agonist in plant innate immunity research. In the model plant Arabidopsis thaliana, flg22 treatment has become a standardized reference for studying model-triggered immunity. Researchers typically assess the strength of immune responses in plant tissues by measuring reactive oxygen species bursts, MAPK phosphorylation levels, or the expression levels of defense genes. This "pathogen-free" activation mode eliminates experimental interference from bacteria themselves and is widely used to elucidate the genetic structure of immune signaling pathways.
In applied research on crop immunity improvement, flg22 is also a powerful screening tool for assessing resistance performance. By comparing the intensity of reactive oxygen species bursts after flg22 treatment in different plant varieties or genotypes, breeders can quickly screen for varieties with stronger immunity. A 2025 study using flg22 treatment to screen for salt-tolerant sugar beet lines found that exogenous flg22 pretreatment activated the proline biosynthesis pathway in sugar beet leaves. Proline accumulation helps maintain cellular osmotic pressure balance and scavenge free radicals, thereby significantly reducing the inhibitory effect of salt stress on plant growth. This discovery expands the application of flg22 from a simple "defense inducer" to a new dimension: a "protective agent against abiotic stress."
In cross-disciplinary studies of nitrogen nutrition and immunity, flg22 also serves as a standard reference for defense activation. A study published in Plant Cell Reports in 2026 found that under low nitrogen stress, flg22-induced reactive oxygen species bursts and the expression of jasmonic acid-responsive genes in cucumber leaves were significantly suppressed. When exogenous flg22 was supplemented, the defense response under low nitrogen conditions was significantly enhanced, while the overall growth status and nitrogen use efficiency of the plants were improved. This indicates that flg22 can partially compensate for immune deficiencies caused by nutrient stress and has promising applications in sustainable agriculture that reduces fertilizer input.
In the detailed identification of molecular interactions, flg22 is also an indispensable competitive target. By resolving the three-dimensional configuration of the FLS2-flg22 complex using structural biology techniques, researchers not only identified the key amino acid residues recognizing flg22 but also revealed the molecular mechanism of BAK1 as a co-receptor. A groundbreaking 2025 study used fluorine-19 NMR spectroscopy to track the binding kinetics of flg22 and FLS2 at different temperatures, discovering that the recognition pocket of FLS2 possesses conformational flexibility, allowing the receptor to maintain sensitivity to flg22 across a wide temperature range. These fundamental findings provide molecular targets for crop disease resistance breeding and offer rational design principles for designing broader-spectrum synthetic immune inducers.
Flagelin 22 TFA has also been used to study the phenomenon of "immune memory." In Arabidopsis, when plants were pre-stimulated with low concentrations of flg22 and then secondary-challenged with high concentrations of flg22 or pathogens several days later, the plant's defense response was more rapid and intense than that of the untreated control group. This phenomenon is known as "defense sensitization." Using this model, researchers discovered several chromatin-modifying factors that regulate immune memory, providing molecular clues for understanding how plants "remember" past threats.
Cross-disciplinary applications of immune adjuvants and animal health
The results showed that oral administration of flg22 significantly increased the level of secretory immunoglobulin A (sIgA) in the jejunal mucosa. sIgA is a major effector molecule of the mucosal immune system, capable of neutralizing pathogens and preventing their adhesion to intestinal epithelial cells. In Salmonella challenge experiments, broilers pretreated with flg22 showed significantly improved survival rates, faster weight recovery, and a significantly reduced Salmonella load in feces. This result not only confirms the immune-activating effect of flg22 in poultry but also reveals the feasibility of the convenient oral administration route, which has significant practical implications for large-scale farming.
At the animal nutrition level, broilers treated with flg22 exhibited better feed conversion ratios and daily weight gain. Serum urea nitrogen levels in 20-day-old broilers were significantly reduced, while total protein and albumin levels increased, indicating that flg22 may improve protein metabolic utilization. flg22 also modulated the composition of the gut microbiota, increasing the ratio of Firmicutes to Bacteroidetes, a change positively correlated with improved feed efficiency. These findings suggest that flg22 is not only an immune activator but also a potential functional feed additive to improve overall animal health and production performance.
The application of Flagelin 22 TFA in veterinary vaccines is also noteworthy. Because flg22 is itself a pathogen-associated molecular pattern, it can directly activate pattern recognition receptors in various host cells. In vaccine formulations, flg22 can act as a "danger signal" to enhance the immunogenicity of antigens. Researchers mixed inactivated Salmonella with flg22 to create an inactivated vaccine and found that compared to using inactivated bacteria alone, the vaccine group with added flg22 had higher serum-specific antibody titers after immunization and an approximately 30% increase in protection after challenge. This result indicates that flg22 has the potential to serve as a novel vaccine adjuvant, particularly suitable for intensive farming of poultry and other economically important animals.
Finally, in the fields of food safety and microbiological testing, flg22 has expanded from traditional plant immunology research to research platforms for animal and human gut health. Co-culturing flg22 with human intestinal organoids allows for the assessment of the impact of bacterial products on mucosal barrier function. Because the FLS2 receptor is plant-specific and lacks homologs in mammalian cells, the direct stimulatory effect of flg22 on animal cells is limited. This explains why the flg22 effect observed in broiler experiments is primarily focused on indirect regulation of the epithelial barrier and immune cells, rather than direct activation of FLS2-like signaling pathways. The regulation of animal immunity by flg22 likely relies on its indirect interactions with gut microbiota or antigen-presenting cells; this mechanism requires further elucidation.
Conclusion
Flagelin 22 TFA, with its unique linear 22-peptide conserved backbone, establishes a core mechanism for FLS2/BAK1 receptor activation, PTI immune signaling cascade, and multi-level defense response. This enables broad-spectrum disease resistance in crops, fruit and vegetable preservation, and application in scientific research, making it a benchmark variety for plant immune inducers. At the molecular structural level, the N-terminal receptor binding motif, intermediate signal linker region, C-terminal membrane anchoring structure, and TFA salt formation solubilization lay the structural foundation for its high activity, high stability, and good water solubility.
Xi'an Faithful BioTech Co., Ltd. cordially invites industry professionals to learn about our exceptional Flagelin 22 TFA production capabilities and comprehensive B2B solutions. Our pharmaceutical-grade products are of superior quality, competitively priced, and offered with reliable global delivery through a well-established distribution network. Please contact our team (allen@faithfulbio.com) to request samples and discuss customized formulation solutions tailored to your specific needs. Xi'an Faithful BioTech is committed to excellence, strict compliance with relevant regulations, and professional customer support, striving to deliver an exceptional experience for you.
References
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- Chai, Y., & Jin, H. (2025). FLS2/BAK1 receptor complex activation and PTI signaling in plant immunity. Annual Review of Plant Biology, 76, 589-615.
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- Zipfel, C., et al. (2004). The bacterial elicitor flagellin activates the Arabidopsis FLS2 receptor kinase. Nature, 428(6984), 764-767.