Recombinant Bordetella bronchiseptica Adenylosuccinate synthetase (purA)
Description
Enzymatic Function and Biological Role
Adenylosuccinate synthetase (PurA) catalyzes the conversion of inosine monophosphate (IMP) and aspartate to adenylosuccinate in the de novo purine biosynthesis pathway. This ATP-dependent reaction is essential for nucleotide metabolism, supporting bacterial growth and survival. In Bordetella bronchiseptica, purine metabolism is linked to virulence and host adaptation, as purine secretion modulates nitrogen utilization and host immune responses.
2.1. Expression Systems
Recombinant PurA from Bordetella species (e.g., B. parapertussis and B. avium) is typically expressed in E. coli, yeast, or mammalian cells. For example:
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Source: E. coli-expressed B. parapertussis PurA (1–435 aa) shows high purity and stability.
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Yield: Production scales range from milligrams to grams, depending on the host system.
3.1. Vaccine Development
Recombinant Bordetella proteins, including outer membrane porins (PPP) and lipoproteins (PL), have been tested as subunit vaccine candidates due to their immunogenicity. Although PurA itself has not been directly evaluated for vaccine use, its role in essential metabolic pathways makes it a potential target for antimicrobial strategies.
3.2. Metabolic Studies
Genome-scale metabolic models of B. pertussis highlight purine secretion as a key nitrogen-release mechanism. Similar pathways in B. bronchiseptica likely involve PurA, suggesting its utility in studying bacterial nutrient utilization and persistence.
Comparative Analysis of Bordetella PurA Proteins
| Property | B. parapertussis PurA | B. avium PurA | B. bronchiseptica (Inferred) |
|---|---|---|---|
| Amino Acid Range | 1–435 | 1–431 | ~1–430 (estimated) |
| Molecular Weight | ~49 kDa | ~48 kDa | ~48–50 kDa |
| Expression Host | E. coli | E. coli | E. coli (hypothesized) |
| Key Function | IMP → adenylosuccinate | IMP → adenylosuccinate | IMP → adenylosuccinate |
Challenges and Future Directions
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Knowledge Gaps: Direct structural and functional data on B. bronchiseptica PurA remain scarce. Studies on homologs (e.g., WbmF in B. bronchiseptica) provide indirect insights but warrant validation.
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Biotechnological Potential: Engineering PurA-deficient strains could elucidate its role in virulence, while recombinant PurA might serve as a diagnostic antigen or enzyme inhibitor target.
References in Context
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Structural Homology: SDR family enzymes in Bordetella share NAD-binding domains critical for redox reactions.
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Metabolic Flexibility: B. bronchiseptica's ability to use diverse carbon sources (e.g., citrate, lactate) relies on functional TCA cycle enzymes, indirectly supporting PurA’s role in nucleotide cofactor synthesis.
Product Specs
Note: We will prioritize shipping the format we have in stock. However, if you have specific requirements for the format, please indicate them when placing your order, and we will fulfill your request.
Note: All of our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please inform us in advance, as additional fees will apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: bbr:BB3165
STRING: 257310.BB3165
Q&A
FAQs for Researchers on Recombinant Bordetella bronchiseptica Proteins
The following FAQs address key methodological and analytical considerations for studying recombinant Bordetella bronchiseptica proteins, based on experimental designs and data from peer-reviewed studies. While the provided sources focus on proteins such as outer membrane porin (PPP), lipoprotein (PL), and fusion proteins (rF1P2), the methodologies and analytical frameworks can be extrapolated to other recombinant antigens like adenylosuccinate synthetase (PurA).
What experimental steps are critical for cloning and expressing recombinant B. bronchiseptica proteins?
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Cloning: Target genes (e.g., pl, ppp) are amplified via PCR, ligated into expression vectors (e.g., pET-28a), and transformed into E. coli BL21(DE3) cells. Sequence homology (>97% with reference genes) must be confirmed .
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Expression: Recombinant proteins are induced with IPTG and expressed as inclusion bodies. Optimization includes varying induction times and temperatures .
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Purification: Proteins are solubilized in 8 M urea and purified via nickel-affinity chromatography under denaturing conditions. Purity is validated using SDS-PAGE (e.g., 12% resolving gel) and Western blot with convalescent sera .
How are antibody titers quantified in murine vaccination studies?
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ELISA Protocol:
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Coat polystyrene plates with purified recombinant protein (1 µg/mL).
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Incubate with serial dilutions of mouse sera (e.g., 1:100).
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Detect bound IgG using HRP-conjugated secondary antibodies (1:5,000 dilution) and TMB substrate.
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Measure absorbance at 450 nm; titers are defined as the highest serum dilution yielding an OD450 ≥ 2.1× background .
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Statistical thresholds: A significant increase in antibodies (e.g., P < 0.005 vs. PBS controls) is required to confirm immunogenicity .
How can contradictions in protective efficacy between recombinant proteins be resolved?
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Comparative analysis: Use survival rates post-challenge (e.g., 62.5% for PPP vs. 12.5% for controls) and IgG subtype ratios (IgG1:IgG2a) to differentiate Th2-dominant (humoral) vs. Th1 (cellular) responses. For example, PL induces IgG1:IgG2a = 4.2 (Th2-skewed), while PPP shows a balanced ratio (1.38) .
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Mechanistic studies:
What models best evaluate mucosal vs. systemic immune responses to subunit vaccines?
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Murine intranasal challenge:
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Inflammation metrics:
Table 1. Protective Efficacy of Recombinant B. bronchiseptica Proteins
Table 2. Key Metrics for Subunit Vaccine Candidates
Methodological Recommendations
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Challenge models: Use intraperitoneal injection (1.74×10⁷ CFU, LD₅₀ = 2.42×10⁶ CFU) for lethal dose studies .
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Adjuvants: Freund’s complete adjuvant enhances immunogenicity but may skew Th1/Th2 balance; consider alternatives like alum for human-translatable studies .
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Contradiction resolution: Cross-validate ELISA data with Western blot to confirm antigen specificity and avoid false positives from denatured epitopes .
