Recombinant Coxiella burnetii UPF0301 protein CBU_2093 (CBU_2093)

What are the optimal expression systems for Recombinant Coxiella burnetii UPF0301 protein CBU_2093?

Several expression systems have been validated for CBU_2093 production, each with specific advantages. E. coli and yeast expression systems offer the highest yields and most rapid production timelines for basic structural studies . Insect cells with baculovirus vectors and mammalian cell systems provide the necessary posttranslational modifications that may be critical for maintaining proper protein folding and biological activity . For projects requiring high-throughput expression, E. coli remains the preferred system, while functional studies requiring native protein characteristics benefit from insect or mammalian expression systems.

When designing your expression strategy, consider the following comparison of expression systems:

What is the amino acid sequence and structural characteristics of CBU_2093?

The full-length CBU_2093 protein consists of 194 amino acids with the following sequence: MRLSGDPRIL SVIMVKTNIL SNHFLVAMPQ LNDFTFTKAV IYVSQHDAKG ALGIIINRPL ALTLGKVLEH LNIEIAQPQI ANHPVLMGGP IGQEHGFIVY EQESPQGAEI LLSASKDMLD DIAKNKGPDD FLITLGYAGW EAGQLENEIA RNDWLVVPFN RKILFETPLK SRWQKAAALI GVDINQLSGQ IGHA .

Structural analysis indicates potential functional domains involved in cellular processes, though the precise function remains under investigation. The protein's molecular weight is approximately 21.5 kDa before considering any post-translational modifications or expression tags. When designing experiments, be mindful that different expression tags may alter protein behavior and that the native structure may be sensitive to purification conditions.

How can CBU_2093 be incorporated into vaccine development research?

CBU_2093 has been identified as a potential subunit vaccine antigen candidate for Coxiella burnetii vaccines . When developing subunit vaccine formulations incorporating this protein, consider the following methodological approach:

• Expression optimization: Select expression systems that preserve immunogenic epitopes

• Adjuvant selection: CBU_2093 has been studied in conjunction with TLR (Toll-Like Receptor) agonists and various adjuvant formulations

• Conjugation strategies: Both non-specific and site-specific conjugation methods have been developed

Research indicates that CBU_2093 may function effectively as part of multi-antigen vaccine formulations. When designing experiments, consider evaluating CBU_2093 both independently and in combination with other C. burnetii antigens such as CBU1061a, CBU0499, and CBU0381 . The immunogenicity of these combinations may provide synergistic effects that are not observable with single-antigen formulations.

What are the critical considerations for designing TLR agonist-conjugated CBU_2093 experiments?

When designing experiments incorporating CBU_2093 with TLR agonists for enhanced immune responses, researchers should consider multiple conjugation strategies:

• Non-specific NHS-mediated conjugation: While simpler to implement, this approach results in heterogeneous products with varied conjugation sites

• Site-specific conjugation: Using techniques like tris-NTA mediated complexation to control the conjugation location and stoichiometry

• Multi-TLR agonist systems: Research suggests that tri-agonist systems targeting multiple TLRs simultaneously (such as TLR4_7_9 combinations) may provide more robust immune responses compared to single TLR activation

When evaluating the efficacy of these conjugated systems, implement comprehensive immune response profiling including:

• Cytokine expression profiling (both in vitro and in vivo)

• Antibody titer assessment

• T-cell response characterization

• Protection assessment in challenge models

How should researchers approach troubleshooting expression issues with CBU_2093?

When encountering expression difficulties with recombinant CBU_2093, implement a systematic troubleshooting approach:

• Expression system evaluation:

  • For E. coli expression: Test multiple strains (BL21(DE3), Rosetta, etc.) and optimize induction parameters (temperature, IPTG concentration, induction time)

  • For yeast systems: Evaluate Pichia pastoris vs. Saccharomyces cerevisiae and optimize methanol induction protocols

  • For insect/mammalian systems: Optimize viral titer and harvest timing

• Solubility enhancement strategies:

  • Fusion protein approaches (MBP, SUMO, GST tags)

  • Co-expression with chaperones

  • Adjustment of lysis buffer composition (detergents, salt concentration)

• Purification optimization:

  • Multi-step purification combining affinity, ion exchange, and size exclusion chromatography

  • Stability assessment at different pH and buffer conditions

  • Implementing protease inhibitors throughout the purification process

Particularly challenging cases may require resorting to denaturation and refolding strategies, though this approach often yields lower activity than native purification methods.

What purification strategies are most effective for obtaining high-purity CBU_2093?

A robust purification strategy for CBU_2093 typically involves a multi-stage approach:

• Initial capture: Affinity chromatography utilizing the protein's expression tag (His-tag, Avi-tag, etc.)

  • For His-tagged proteins: IMAC purification with Ni-NTA or Co-NTA resins

  • For biotinylated proteins: Streptavidin affinity chromatography

• Intermediate purification: Ion exchange chromatography

  • Based on theoretical pI of the protein

  • Generally, anion exchange at pH 8.0 provides good separation from contaminants

• Polishing: Size exclusion chromatography

  • Removes aggregates and separates monomeric protein

  • Allows buffer exchange into final storage formulation

For maximum purity (>95%), combine these techniques sequentially, analyzing purity by SDS-PAGE and/or Western blot at each stage. When working with CBU_2093 expressed in E. coli, be particularly vigilant about endotoxin removal, which can be accomplished through specific endotoxin removal resins or Triton X-114 phase separation.

What analytical methods should be employed to verify CBU_2093 quality and activity?

Comprehensive quality assessment of purified CBU_2093 requires multiple analytical approaches:

• Purity assessment:

  • SDS-PAGE with densitometry analysis (target >85% purity)

  • SEC-HPLC for aggregation analysis

  • Mass spectrometry for identification and verification

• Structural integrity:

  • Circular dichroism for secondary structure verification

  • Thermal shift assays for stability assessment

  • Limited proteolysis to identify domain boundaries

• Functional assessment:

  • Binding assays with potential interaction partners

  • For in vivo biotinylated variants: Streptavidin binding assays to confirm biotinylation

• Biological activity:

  • Cell-based assays relevant to hypothesized function

  • For vaccine applications: In vitro dendritic cell activation assays

Following purification, protein stability should be monitored under various storage conditions. Lyophilized preparations generally offer extended stability compared to solution formulations . For critical applications, activity assessment should be performed immediately before use rather than relying solely on quality control performed at the time of purification.

How can CBU_2093 be effectively incorporated into TLR tri-agonist immunization strategies?

When designing TLR tri-agonist strategies incorporating CBU_2093, consider the following methodological approach:

• Conjugation optimization:

  • Direct protein conjugation to TLR agonist complexes

  • Admixture vs. conjugate formulations (conjugates typically provide superior localization of antigen and adjuvant to the same antigen-presenting cell)

• Formulation development:

  • Evaluation of TLR1/2_4_7, TLR2/6_4_7, TLR1/2_4_9, TLR2/6_4_9, and TLR4_7_9 combinations

  • Assessment of emulsion vs. aqueous formulations

• Immunization protocol design:

  • Prime-boost strategies with varying intervals

  • Route of administration optimization (subcutaneous vs. intramuscular vs. intranasal)

Research indicates that TLR tri-agonist formulations can dramatically enhance immune responses to subunit antigens through synergistic activation of multiple pattern recognition pathways . When designing experiments, implement comprehensive immune profiling including cytokine expression, antibody titers (including subclass distribution), and T-cell responses (both CD4+ and CD8+).

What are the key considerations in experimental design for evaluating CBU_2093 stability and degradation pathways?

When designing stability studies for CBU_2093, implement a comprehensive approach addressing multiple degradation pathways:

• Physical stability assessment:

  • Differential scanning calorimetry to determine melting temperature

  • Dynamic light scattering to monitor aggregation propensity

  • Freeze-thaw stability through multiple cycles

• Chemical stability evaluation:

  • Oxidation susceptibility (methionine and cysteine residues)

  • Deamidation risk (asparagine residues)

  • Proteolytic sensitivity

• Formulation optimization:

  • pH screening (typically pH 6.0-8.0)

  • Buffer composition comparison

  • Excipient screening (sugars, amino acids, surfactants)

For accelerated stability studies, employ stress testing at elevated temperatures (25°C, 37°C, 45°C) with regular sampling for analytical characterization. Stability in biological matrices (serum, cell culture media) should be separately evaluated through activity retention measurements after incubation .

How should researchers design challenge studies to evaluate CBU_2093-based vaccine candidates?

When designing challenge studies to evaluate vaccine candidates containing CBU_2093, consider this methodological framework:

• Animal model selection:

  • Mouse models for initial screening

  • Guinea pig models for more translatable results

  • Selection based on susceptibility to C. burnetii infection

• Vaccination protocol:

  • Prime-boost regimens with optimized intervals

  • Dosage determination through dose-escalation studies

  • Adjuvant comparison (alum vs. oil-in-water vs. TLR agonist formulations)

• Challenge protocol design:

  • Route of challenge (aerosol preferred for C. burnetii studies)

  • Challenge dose optimization

  • Timing between final immunization and challenge

• Protection evaluation:

  • Bacterial burden quantification in tissues

  • Histopathological assessment

  • Clinical scoring systems

  • Survival analysis where appropriate

When comparing to established vaccines like Q-Vax, implement identical challenge protocols to enable direct efficacy comparisons . Comprehensive immune profiling pre-challenge can help identify correlates of protection that may guide future vaccine optimization efforts.

What biotinylation approaches are most effective for CBU_2093 and what are their research applications?

In vivo biotinylation of CBU_2093 provides significant advantages for various applications. The methodology employs AviTag-BirA technology, where E. coli biotin ligase (BirA) catalyzes the covalent attachment of biotin to the specific lysine residue within the 15-amino acid AviTag peptide . This approach yields homogeneously biotinylated protein with predictable stoichiometry.

The biotinylated CBU_2093 enables numerous applications:

• Protein interaction studies:

  • Surface plasmon resonance with streptavidin surfaces

  • Pull-down assays with streptavidin beads

  • Protein microarray development

• Microscopy applications:

  • Single-molecule fluorescence studies

  • Super-resolution microscopy using streptavidin-fluorophore conjugates

  • Tracking protein localization in cellular contexts

• Vaccine technology applications:

  • Oriented coupling to streptavidin-coated nanoparticles

  • Site-specific conjugation to adjuvant systems

  • Multimerization to enhance immunogenicity

Alternative biotinylation approaches include chemical biotinylation using NHS-biotin reagents, though these typically result in heterogeneous products with multiple biotinylation sites that may impact protein function.

How can researchers effectively combine multiple C. burnetii antigens including CBU_2093 in subunit vaccine formulations?

When developing multi-antigen C. burnetii subunit vaccines incorporating CBU_2093, implement the following methodological approaches:

• Antigen selection and prioritization:

  • Combination of membrane-associated and cytosolic antigens

  • Consideration of epitope diversity and MHC binding predictions

  • Evaluation of CBU_2093 in combination with other identified immunogenic proteins (CBU1061a, CBU0499, CBU0471-r, CBU0381, etc.)

• Formulation strategies:

  • Protein mixtures with defined ratios

  • Co-expression of multiple antigens as fusion proteins

  • Protein nanoparticles displaying multiple antigens

• Adjuvant optimization:

  • TLR agonist combinations tailored to the antigen set

  • Emulsion formulations for enhanced delivery

  • Nanoparticle encapsulation for co-delivery

Research indicates that multi-antigen formulations often provide superior protection compared to single-antigen approaches . When designing experiments, implement factorial design approaches to efficiently evaluate various antigen combinations rather than testing each formulation independently.

How should researchers interpret conflicting data regarding CBU_2093 immunogenicity in different models?

When encountering conflicting immunogenicity data for CBU_2093 across different experimental models, implement this systematic evaluation approach:

• Model-specific factors assessment:

  • Species differences in immune recognition

  • Adjuvant interactions varying between models

  • Route of administration effects

• Antigen preparation variables:

  • Expression system differences affecting post-translational modifications

  • Conformational integrity verification

  • Endotoxin contamination evaluation

• Immune response profiling completeness:

  • Antibody response measurements (multiple isotypes)

  • T-cell response characterization (Th1/Th2/Th17 balance)

  • Innate immune activation assessment

• Statistical analysis robustness:

  • Power analysis verification

  • Appropriate statistical tests

  • Multiple testing correction application

When reconciling conflicting data, conduct comparative experiments using standardized protocols across models. Differences in protein preparation quality and adjuvant selection frequently account for apparent contradictions in immunogenicity data.

What analytical approaches can resolve challenges in detecting post-translational modifications of CBU_2093?

Detection and characterization of post-translational modifications in CBU_2093 requires multiple complementary analytical approaches:

• Mass spectrometry strategies:

  • Bottom-up proteomics for site identification

  • Top-down proteomics for intact mass analysis

  • Multiple fragmentation methods (CID, ETD, HCD)

• Enrichment strategies for specific modifications:

  • Phosphorylation: TiO2 or IMAC enrichment

  • Glycosylation: Lectin affinity or hydrazide chemistry

  • Ubiquitination: Ubiquitin remnant antibodies

• Site-directed mutagenesis:

  • Mutation of predicted modification sites

  • Functional impact assessment

  • Comparison with wild-type protein

When analyzing CBU_2093 expressed in different systems, be particularly attentive to system-specific modifications. E. coli-expressed protein will lack most eukaryotic modifications, while insect and mammalian cell expression will introduce various glycoforms that may affect protein function and immunogenicity.