Recombinant Borrelia burgdorferi Uncharacterized protein BBD24 (BB_D24)

What is the structure and sequence of Recombinant Borrelia burgdorferi Uncharacterized Protein BBD24?

Recombinant Borrelia burgdorferi Uncharacterized Protein BBD24 (BB_D24) is a 76 amino acid protein that can be produced with an N-terminal His tag in E. coli expression systems. The full amino acid sequence is:

MTAIIVYSCLTMCVIYFHLQLKTFFTKLIRFCKKCFDIFLLLIEMLKLIFYLLIINNKFYIFIIISIALITINTMI

The protein is relatively small at 76 amino acids, and while its tertiary structure has not been fully characterized, the primary sequence suggests potential membrane-associated properties based on its hydrophobic amino acid content. Researchers should note that the His-tag addition facilitates purification but may impact structural studies if not removed prior to analysis .

How should I store and reconstitute BBD24 for experimental use?

For optimal results when working with Recombinant BBD24 protein, follow these methodological guidelines:

• Upon receipt, briefly centrifuge the vial to bring contents to the bottom

• Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended) for long-term storage

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

• For long-term storage, keep aliquots at -20°C/-80°C

The protein is typically supplied in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0. Repeated freeze-thaw cycles can significantly reduce protein activity, so creating multiple single-use aliquots is strongly recommended for maintaining experimental consistency .

What are the recommended applications for BBD24 protein in basic research?

While BBD24 is currently listed primarily for SDS-PAGE applications , researchers can utilize this protein for multiple basic research purposes:

• Antibody production and validation

• Protein-protein interaction studies

• Structural analysis (following tag removal)

• Immunological research exploring host-pathogen interactions

• Comparative studies with other Borrelia proteins

When designing experiments, consider that BBD24 remains largely uncharacterized, making it valuable for discovery-based research. Standard validation techniques should confirm protein identity through western blotting with anti-His antibodies and mass spectrometry before proceeding to more complex applications. For interaction studies, techniques such as pull-down assays, co-immunoprecipitation, or yeast two-hybrid screens can help identify potential binding partners and begin elucidating this protein's function.

How can I design growth inhibition assays to test the potential antimicrobial activity of BBD24 against Borrelia burgdorferi?

To assess whether BBD24 exhibits antimicrobial activity against Borrelia burgdorferi, researchers can adapt protocols similar to those used for SCGB1D2 protein . A methodological approach would include:

• Culture B. burgdorferi B31A3-GFP strain in BSK-H media with 6% rabbit serum at 37°C

• Determine bacterial concentration using flow cytometry

• Prepare 96-well plates with approximately 150,000 spirochetes per well

• Test different concentrations of recombinant BBD24 protein (e.g., 2, 4, 8, and 16 μg/mL)

• Include appropriate controls:

  • Medium-only negative control

  • Bacteria-only control (no protein)

  • Known antimicrobial positive control

• Incubate and monitor bacterial growth using automated imaging systems (e.g., IncuCyte®)

• Analyze GFP-expressing bacteria count per image at multiple time points (24h, 72h, 140h)

What experimental approaches can determine if BBD24 has roles in host-pathogen interactions similar to other Borrelia proteins?

To investigate potential roles of BBD24 in host-pathogen interactions, consider these methodological approaches:

• Cell binding assays: Assess binding of labeled BBD24 to various human cell types (epithelial cells, fibroblasts, immune cells) using flow cytometry or microscopy

• Expression analysis during infection stages: Examine BBD24 expression levels in different phases of Borrelia infection using RT-qPCR or proteomics

• Comparative genomics approach:

  • Analyze BBD24 sequence conservation across Borrelia strains

  • Identify potential functional domains through bioinformatic analysis

  • Compare with characterized virulence factors

• Gene knockout/knockdown studies:

  • Generate BBD24-deficient Borrelia strains

  • Evaluate changes in bacterial fitness and virulence

  • Assess host immune responses to mutant strains

• Human tissue expression mapping:

  • Determine if BBD24 interacts with specific human tissues

  • Investigate expression patterns in skin, joint, and neurological tissues (common sites of Lyme disease manifestations)

When designing these experiments, incorporate appropriate controls and consider that different Borrelia proteins may have strain-specific functions in pathogenesis.

How can I assess the potential role of BBD24 in immune evasion mechanisms of Borrelia burgdorferi?

To investigate whether BBD24 contributes to immune evasion by Borrelia burgdorferi, implement these methodological approaches:

• Complement resistance assays:

  • Incubate BBD24 with human serum components

  • Measure binding to complement factors (C3b, C4b, Factor H)

  • Assess effect on complement-mediated killing of sensitized bacteria

• Phagocytosis inhibition assays:

  • Pre-treat macrophages or neutrophils with recombinant BBD24

  • Add fluorescently-labeled Borrelia and measure phagocytic uptake

  • Compare with control protein treatments

• Antibody evasion studies:

  • Test if BBD24 binds to immunoglobulins non-specifically

  • Evaluate if BBD24 can mask surface antigens from antibody recognition

• Cytokine modulation:

  • Measure cytokine production by immune cells when exposed to BBD24

  • Assess changes in inflammatory response gene expression

• In vivo infection models:

  • Compare infection progression between wild-type and BBD24-deficient strains

  • Evaluate bacterial persistence and dissemination patterns

  • Measure host immune response parameters

When interpreting results, compare findings with known immune evasion mechanisms of other Borrelia proteins, as this may provide context for understanding BBD24's potential role in pathogenesis.

What are the optimal expression and purification methods for producing high-quality recombinant BBD24 protein?

For researchers seeking to express and purify BBD24 for their studies, these methodological details should be considered:

• Expression system optimization:

  • E. coli is the established system for BBD24 expression

  • Consider strain selection: BL21(DE3), Rosetta, or SHuffle for potentially challenging proteins

  • Optimize induction conditions: IPTG concentration (typically 0.1-1.0 mM), temperature (16°C, 25°C, 37°C), and duration (4-24 hours)

• Purification strategy:

  • Utilize IMAC (Immobilized Metal Affinity Chromatography) for His-tagged BBD24

  • Implement a two-step purification: IMAC followed by size exclusion chromatography

  • Consider buffer optimization to enhance protein stability:

  Buffer Component	Range to Test	Purpose
  NaCl	100-500 mM	Ionic strength
  Glycerol	5-20%	Stability enhancer
  pH	7.0-8.5	Optimize charge state
  Reducing agent	1-5 mM DTT or TCEP	Prevent oxidation
  Additives	Arginine, trehalose	Prevent aggregation

• Quality control assessments:

  • SDS-PAGE for purity (target >90%)

  • Western blot for identity confirmation

  • Dynamic light scattering for aggregation analysis

  • Circular dichroism for secondary structure verification

  • Mass spectrometry for sequence confirmation

• Tag removal considerations:

  • If tag-free protein is required, incorporate a protease cleavage site

  • Common proteases: TEV, PreScission, or thrombin

  • Perform reverse IMAC to remove cleaved tag and uncleaved protein

Optimizing these parameters will help ensure reproducible production of high-quality BBD24 protein suitable for downstream applications.

How can I design experiments to elucidate the structural characteristics of BBD24?

To characterize the structural properties of the uncharacterized BBD24 protein, implement these methodological approaches:

Given BBD24's small size (76 amino acids) , structural characterization should be feasible with these approaches, potentially revealing important insights about its functional domains.

How does BBD24 compare functionally with other characterized Borrelia burgdorferi proteins in research applications?

While BBD24 remains largely uncharacterized, comparative analysis with better-studied Borrelia proteins can provide research context:

*Note that SCGB1D2 is a human protein that inhibits Borrelia growth , not a Borrelia protein, but is included for methodological comparison.

When designing comparative experiments:

• Consider evolutionary conservation of BBD24 across Borrelia strains

• Assess expression patterns during different infection phases

• Evaluate functional redundancy with other uncharacterized proteins

• Test for complementary or antagonistic activities with known virulence factors

Growth inhibition assays as described for SCGB1D2 can be adapted to test whether BBD24 impacts growth of other bacterial species, potentially revealing antimicrobial properties or specificity for particular pathogens.

What approaches can determine if genetic variants of BBD24 exist and how might they affect protein function?

To investigate genetic variation in BBD24 and its functional consequences, implement these methodological approaches:

• Genetic screening strategy:

  • Sequence BBD24 gene from diverse Borrelia burgdorferi isolates

  • Compare clinical isolates from different geographical regions

  • Analyze isolates from different disease manifestations (skin, joint, neurological)

  • Identify single nucleotide polymorphisms and insertion/deletion variants

• Functional impact assessment:

  • Express and purify variant BBD24 proteins

  • Compare protein stability and solubility profiles

  • Assess structural differences using techniques outlined in Section 3.2

  • Evaluate functional differences in:

    • Growth characteristics

    • Host cell interactions

    • Immune response modulation

    • Antimicrobial susceptibility

• In silico prediction:

  • Use computational tools to predict impact of variants:

    • SIFT, PolyPhen for amino acid substitution effects

    • Molecular dynamics simulations for structural changes

    • Binding site prediction tools for interaction impacts

• Comparative approaches with known variants:

  • Study methodology from research on SCGB1D2 variant (P53L)

  • Adapt growth inhibition assays to compare variant functions

  • Consider dose-dependent effects as seen with SCGB1D2 variants

The methodology used to study the SCGB1D2 P53L variant demonstrates that genetic variants can significantly impact protein function, with the variant requiring approximately twice the concentration to achieve similar inhibition of Borrelia growth compared to the reference protein . Similar quantitative approaches could reveal functional differences in BBD24 variants.