Recombinant Treponema pallidum Uncharacterized protein TP_0055 (TP_0055)

What is TP_0055 and what is known about its structural characteristics?

TP_0055 is an uncharacterized protein from Treponema pallidum, the bacterium responsible for syphilis infection. Based on available data, TP_0055 is a relatively small protein consisting of 78 amino acids with the sequence: MNQIRLFAQSALVSVMGMGMVFAFLLLLICVVRCVGALVSSFGWDRGPDEGVGAAVPAGGALAAAIAVAVHEKARSTS . The protein appears to contain hydrophobic regions suggesting possible membrane association, though its precise functional role remains under investigation. The recombinant form commonly used in research includes an N-terminal His-tag to facilitate purification and detection .

What expression systems are recommended for producing recombinant TP_0055?

E. coli is the predominant expression system used for recombinant TP_0055 production . The methodology involves:

• Cloning the TP_0055 gene sequence into an appropriate expression vector

• Transforming the construct into a compatible E. coli strain

• Inducing protein expression under optimized conditions

• Harvesting and lysing cells

• Purifying the His-tagged protein using affinity chromatography

When designing your expression system, consider codon optimization for E. coli if expression yields are suboptimal. Alternative expression systems such as yeast or insect cells might be considered for specific research applications requiring eukaryotic post-translational modifications, though these are rarely necessary for basic structural or immunological studies of T. pallidum proteins.

How should recombinant TP_0055 be stored to maintain stability and functionality?

Optimal storage conditions for recombinant TP_0055 are as follows:

When handling the protein:

• Centrifuge vials briefly before opening to collect contents

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol (recommended final concentration: 50%) before aliquoting for freezer storage

• Avoid repeated freeze-thaw cycles as they significantly reduce protein integrity

What purification methods yield the highest purity of recombinant TP_0055?

For His-tagged recombinant TP_0055, immobilized metal affinity chromatography (IMAC) is the primary purification method. The typical workflow includes:

• Cell lysis under native or denaturing conditions

• Binding to Ni-NTA or similar metal-chelating resin

• Washing to remove non-specifically bound proteins

• Elution with imidazole gradient or pH shift

• Buffer exchange to remove imidazole

To achieve >90% purity as noted in commercial preparations , secondary purification steps may be necessary:

• Size exclusion chromatography to separate based on molecular weight

• Ion exchange chromatography for charge-based separation

• Endotoxin removal for applications sensitive to bacterial lipopolysaccharides

Confirm purification success using SDS-PAGE and/or Western blotting with anti-His antibodies.

How can researchers design experiments to evaluate TP_0055's potential role in syphilis pathogenesis?

Designing robust experiments to investigate TP_0055's role in pathogenesis requires a systematic approach:

• Formulate specific hypotheses about TP_0055's function based on:

  • Sequence analysis and structural predictions

  • Comparison with homologous proteins in other organisms

  • Temporal expression patterns during infection

• Design experimental treatments that manipulate your independent variables :

  • In vitro binding assays with host components

  • Protein localization studies

  • Expression level analysis under different conditions

• Use appropriate controls:

  • Other recombinant T. pallidum proteins with known functions

  • Mutated versions of TP_0055

  • Host cells or tissues from different sources

A potential experimental design might include:

Remember to control extraneous variables through randomization and blinding where appropriate .

How does TP_0055 compare to better-characterized T. pallidum antigens for diagnostic potential?

While specific data on TP_0055's diagnostic utility is limited in the provided search results, we can establish a framework for comparison based on known high-performance T. pallidum antigens:

Several recombinant T. pallidum proteins have demonstrated excellent diagnostic potential:

• Tp0453: 100% sensitivity and 100% specificity in syphilis diagnosis

• Tp92 (Tp0326): 98% sensitivity and 97% specificity

• Gpd (Tp0257): 91% sensitivity and 93% specificity

To evaluate TP_0055's diagnostic potential, researchers should:

• Conduct parallel ELISA assays using:

  • Sera from confirmed syphilis patients at different disease stages (n≥43)

  • Sera from patients with other spirochetal diseases like Lyme disease (n≥8) and relapsing fever (n≥8)

  • Sera from uninfected controls (n≥15)

• Calculate sensitivity and specificity values:

  • Sensitivity = true positives/(true positives + false negatives)

  • Specificity = true negatives/(true negatives + false positives)

• Compare reactivity patterns with conventional tests:

  • Venereal Disease Research Laboratory (VDRL) test

  • Microhemagglutination assay for T. pallidum (MHA-TP)

An advantage of recombinant T. pallidum antigens over crude antigen preparations is the standardized production process and reduced biosafety concerns .

What methods can be used to characterize protein-protein interactions involving TP_0055?

Characterizing protein-protein interactions involving TP_0055 requires multiple complementary approaches:

• In vitro interaction assays:

  • Pull-down assays using His-tagged TP_0055 as bait

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • ELISA-based interaction assays

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Structural analysis techniques:

  • X-ray crystallography of TP_0055 with binding partners

  • NMR spectroscopy for dynamic interaction studies

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

  • Cryo-electron microscopy for larger complexes

• Computational approaches:

  • Molecular docking simulations

  • Sequence-based interaction prediction

  • Structural homology modeling

• Validation in cellular contexts:

  • Co-immunoprecipitation from T. pallidum or recombinant expression systems

  • Proximity labeling techniques (BioID, APEX)

  • Fluorescence resonance energy transfer (FRET)

When reporting interaction data, include quantitative measurements:

How can researchers address inconsistent experimental results when working with TP_0055?

When facing inconsistent results with TP_0055 experiments, employ a systematic troubleshooting approach:

• Evaluate protein quality:

  • Confirm purity by SDS-PAGE (>90% recommended)

  • Verify intact protein by mass spectrometry

  • Assess proper folding using circular dichroism

  • Check for batch-to-batch variations

• Optimize experimental conditions:

  • Test multiple buffer compositions

  • Evaluate pH dependence (typically pH 7.4-8.0 for T. pallidum proteins)

  • Determine temperature sensitivity

  • Assess the impact of additives (reducing agents, stabilizers)

• Apply statistical rigor:

  • Increase technical and biological replicates

  • Use appropriate statistical tests

  • Implement randomization and blinding when possible

  • Calculate effect sizes and confidence intervals

• Consider biological complexity:

  • Test for post-translational modifications

  • Evaluate oligomerization states

  • Investigate cofactor requirements

  • Assess native vs. recombinant protein differences

When reporting inconsistent results, present them transparently with a detailed discussion of possible sources of variation and their biological significance.

What considerations are important when designing immunoassays using recombinant TP_0055?

When designing immunoassays with recombinant TP_0055, consider these critical factors:

• Assay format selection:

  • Direct ELISA: Simple but may have higher background

  • Sandwich ELISA: Better specificity but requires two non-competing antibodies

  • Competitive ELISA: Useful for small proteins like TP_0055

• Optimization parameters:

  • Coating concentration (typically 1-10 μg/mL)

  • Blocking agent (BSA, casein, or commercial blockers)

  • Sample dilution series

  • Incubation times and temperatures

  • Detection system (colorimetric, fluorescent, chemiluminescent)

• Validation requirements:

  • Establish standard curves with purified TP_0055

  • Determine limit of detection and quantification

  • Assess cross-reactivity with related proteins

  • Evaluate matrix effects from biological samples

• Controls and normalization:

  • Include positive controls (sera from syphilis patients)

  • Include negative controls (sera from uninfected individuals)

  • Use reference standards for quantitative assays

  • Consider multiplexing with other T. pallidum antigens

For syphilis diagnostic applications, compare performance to established antigens like Tp0453, Tp92, and Gpd, which have shown excellent sensitivity and specificity in previous studies .

What is the optimal experimental design for evaluating TP_0055 functionality?

To evaluate TP_0055 functionality, consider these experimental design principles:

• Define clear variables :

  • Independent variables: TP_0055 concentration, experimental conditions

  • Dependent variables: Binding activity, immune response, structural changes

  • Control variables: Buffer composition, temperature, incubation time

• Formulate testable hypotheses :

  • Null hypothesis: TP_0055 does not interact with specific host components

  • Alternative hypothesis: TP_0055 binds to specific host receptors/proteins

• Design controls:

  • Positive controls: Known T. pallidum antigens with established functions

  • Negative controls: Irrelevant proteins of similar size/structure

  • Technical controls: Buffer-only samples, heat-denatured protein

• Determine appropriate sample size:

  • Conduct power analysis based on expected effect size

  • Plan for sufficient biological and technical replicates

  • Consider resource constraints and ethical considerations

• Select appropriate statistical approaches:

  • Parametric vs. non-parametric tests based on data distribution

  • Correction for multiple comparisons when testing several conditions

  • Analysis of variance for multi-factorial designs

Example experimental design for investigating TP_0055 host cell interactions:

How should researchers approach contradictory findings in TP_0055 research literature?

When encountering contradictory findings about TP_0055 in the research literature, apply these systematic approaches:

• Conduct a thorough literature analysis:

  • Create a detailed comparison table of contradictory studies

  • Identify methodological differences (expression systems, purification methods, assay conditions)

  • Assess sample sizes and statistical power of each study

  • Evaluate the quality of controls and validation approaches

• Examine potential sources of variation:

  • Different recombinant constructs (tag position, fusion partners)

  • Varying buffer compositions and assay conditions

  • Distinct biological systems (cell lines, animal models)

  • Sample handling and storage differences

• Design reconciliation experiments:

  • Replicate key experiments using standardized protocols

  • Test multiple conditions spanning those reported in contradictory studies

  • Include appropriate positive and negative controls

  • Collaborate with authors of contradictory papers when possible

• Apply meta-analysis techniques:

  • Combine data across studies where methodologically appropriate

  • Weight findings by study quality and sample size

  • Calculate effect sizes rather than relying solely on p-values

  • Identify moderator variables that might explain discrepancies

• Report findings transparently:

  • Acknowledge limitations of your reconciliation attempts

  • Present all data, including those that don't support your hypothesis

  • Discuss remaining uncertainties and propose next steps

  • Consider pre-registering reconciliation studies to avoid bias

What are the best approaches for structural characterization of TP_0055?

Structural characterization of TP_0055 requires a multi-technique approach:

The 78-amino acid length of TP_0055 makes it particularly suitable for NMR structural determination, which can provide both structural and dynamic information.

How can researchers optimize expression and purification of TP_0055 for structural studies?

Optimizing expression and purification of TP_0055 for structural studies requires attention to several critical factors:

• Expression construct design:

  • Consider tag position (N- vs. C-terminal) based on structural predictions

  • Evaluate different fusion partners (MBP, SUMO, GST) for solubility enhancement

  • Include precision protease sites for tag removal

  • Optimize codon usage for the expression host

• Expression conditions:

  • Test multiple E. coli strains (BL21(DE3), Rosetta, SHuffle)

  • Optimize induction parameters (temperature, inducer concentration, time)

  • Consider auto-induction media for gradual protein expression

  • Evaluate cell lysis methods (sonication, high-pressure homogenization, detergents)

• Purification strategy:

  • Implement multi-step purification to achieve >95% purity

  • Consider on-column refolding for inclusion body purification

  • Optimize buffer compositions for stability (pH, salt, additives)

  • Remove the His-tag if it might interfere with structure or function

• Quality control measures:

  • Size-exclusion chromatography to assess oligomeric state

  • Dynamic light scattering for homogeneity analysis

  • Thermal shift assays to optimize buffer conditions

  • Activity assays to confirm functional integrity

• Specific considerations for structural techniques:

  • For crystallography: concentrate to 5-20 mg/mL, screen multiple crystallization conditions

  • For NMR: express in minimal media with 15N and 13C labels, concentrate to 0.5-1 mM

  • For cryo-EM: ensure sample homogeneity, optimize grid preparation

Expected yield benchmarks for recombinant TP_0055 production:

How can TP_0055 contribute to understanding T. pallidum pathogenesis?

While TP_0055's specific role remains uncharacterized, its investigation can contribute to understanding T. pallidum pathogenesis through:

• Comparative genomic analysis:

  • Determine conservation across T. pallidum subspecies

  • Identify homologs in other spirochetes

  • Assess evolutionary pressure through synonymous/non-synonymous mutation ratios

• Expression pattern analysis:

  • Determine temporal expression during different stages of infection

  • Investigate regulation in response to environmental stimuli

  • Examine expression in different tissue environments

• Host-pathogen interaction studies:

  • Test binding to extracellular matrix components

  • Evaluate interactions with immune cells

  • Assess impact on host cell signaling pathways

• Immunological profiling:

  • Characterize antibody responses to TP_0055 during natural infection

  • Determine T cell epitopes within the protein

  • Evaluate protective potential in animal models

• Structural insights:

  • Identify structural motifs associated with virulence

  • Map epitopes recognized by protective antibodies

  • Discover potential binding pockets for small molecule interventions

The inclusion of TP_0055 in comprehensive studies alongside better-characterized T. pallidum proteins can provide context for its role relative to known virulence factors.

What is the potential of TP_0055 as a biomarker for different stages of syphilis?

Evaluating TP_0055's potential as a stage-specific biomarker requires systematic investigation:

• Stage-specific antibody response analysis:

  • Test sera from patients with primary, secondary, latent, and tertiary syphilis

  • Compare antibody kinetics with established markers

  • Determine if TP_0055 antibodies correlate with disease progression

• Predictive value assessment:

  • Calculate positive and negative predictive values for each disease stage

  • Compare performance to current diagnostic standards

  • Evaluate in combination with other T. pallidum antigens

• Treatment response monitoring:

  • Measure antibody titer changes following antibiotic therapy

  • Determine if TP_0055 antibodies decline more rapidly than other markers

  • Assess correlation with clinical cure

• Special population studies:

  • Evaluate performance in HIV co-infected individuals

  • Test in congenital syphilis cases

  • Assess in neurosyphilis diagnosis

To determine if TP_0055 offers advantages over current diagnostic antigens, compare its performance to well-characterized recombinant proteins like Tp0453, Tp92, and Gpd, which have demonstrated excellent sensitivity and specificity in previous studies .