Treponema p17 16.4kDa

What is Treponema p17 16.4kDa and what is its structural composition?

Treponema p17 16.4kDa is a recombinant protein derived from Treponema pallidum, expressed in Escherichia coli expression systems. This protein is a non-glycosylated polypeptide chain with a molecular mass of 16.4kDa and typically features a His-tag fusion at the N-terminus to facilitate purification and detection in various applications . The protein is part of Treponema pallidum's limited protein repertoire, reflecting the organism's highly specialized parasitic lifestyle and reduced genome (1.14 million base pairs) . Understanding its structure is crucial as T. pallidum is a gram-negative spirochaete bacterium with distinct morphological features, including a helical structure enabling its characteristic corkscrew movement through viscous media such as mucus .

What are the optimal storage conditions for maintaining Treponema p17 16.4kDa stability?

To maintain the structural integrity and functional properties of Treponema p17 16.4kDa, researchers should store the protein below -18°C in its lyophilized form . While the protein demonstrates notable stability at room temperature for up to 4 weeks, long-term storage requires freezer conditions . It is critical to prevent freeze-thaw cycles, which can significantly decrease protein activity through denaturation and aggregation . For research requiring extended use periods, it is advisable to prepare aliquots of the reconstituted protein to minimize repeated freeze-thaw events. Proper storage conditions are essential for maintaining experimental reproducibility and ensuring that research outcomes accurately reflect the protein's native characteristics rather than artifacts of improper handling.

What is the recommended reconstitution protocol for Treponema p17 16.4kDa?

For optimal reconstitution of lyophilized Treponema p17 16.4kDa, researchers should use sterile 18M-cm H₂O at a concentration not less than 100μg/ml . This initial stock solution can then be further diluted to appropriate working concentrations in various aqueous buffers depending on the experimental requirements. The reconstitution process should be performed under sterile conditions to prevent microbial contamination which could interfere with experimental results or degrade the protein. After reconstitution, the solution should be handled gently to avoid protein denaturation through excessive physical forces. The manufacturer's specific recommendations indicate that the protein is initially lyophilized from a 1mg/ml solution in 20mM sodium carbonate at pH 10 , which provides insights into compatible buffer systems for subsequent experimental applications.

How can Treponema p17 16.4kDa be employed in immunoassay development?

When developing immunoassays using Treponema p17 16.4kDa, researchers should first consider the protein's high purity (>95% as determined by SDS-PAGE) , which makes it suitable for generating specific antibodies or as a standard in detection systems. For ELISA applications, the recombinant protein can serve as a coating antigen at concentrations typically ranging from 1-10 μg/ml in carbonate buffer (pH 9.6). The His-tag fusion facilitates oriented immobilization on nickel-coated plates, potentially improving assay sensitivity.

When developing a sandwich ELISA, researchers should:

• Optimize antibody pairs to ensure one recognizes the native epitope and another recognizes a different region

• Establish standard curves using purified Treponema p17 in the range of 0.1-1000 ng/ml

• Validate assay specificity by testing against closely related Treponema species proteins

• Implement stringent washing steps (typically 3-5 washes with PBS-Tween 0.05%)

The high stability of the protein at room temperature for up to 4 weeks allows for convenient assay development without constant freeze-thaw cycles during optimization phases.

What considerations should be made when using Treponema p17 16.4kDa for structural biology studies?

For X-ray crystallography applications:

• The protein should be further purified to achieve >99% homogeneity using techniques such as size exclusion chromatography

• Screening for crystallization conditions should begin with sparse matrix screens, paying particular attention to pH conditions around the protein's stable formulation (pH 10)

• Consider tag removal through specific proteases if the tag interferes with crystallization

For NMR studies, the protein can be isotopically labeled with ¹⁵N and ¹³C during expression in E. coli using minimal media with these isotopes as sole nitrogen and carbon sources. The protein's relatively small size (16.4kDa) is advantageous for solution NMR studies, potentially allowing for detailed structural analysis without requiring deuteration.

How can researchers optimize Western blot protocols for Treponema p17 16.4kDa detection?

Western blot optimization for Treponema p17 16.4kDa detection requires careful consideration of sample preparation, electrophoresis, and detection parameters. The protein's molecular weight of 16.4kDa dictates the use of appropriate percentage acrylamide gels (typically 12-15%) to achieve optimal resolution in this molecular weight range.

Recommended Western blot protocol optimizations include:

• Sample preparation:

  • Denature samples in loading buffer containing SDS and DTT or β-mercaptoethanol

  • Heat at 95°C for 5 minutes to ensure complete denaturation

  • Load 10-50 ng of purified protein for positive control lanes

• Electrophoresis conditions:

  • Use Tris-Glycine or Tris-Tricine buffer systems for improved resolution of lower molecular weight proteins

  • Run at 120V constant until the dye front reaches the bottom of the gel

• Transfer parameters:

  • For proteins in this molecular weight range, semi-dry transfer systems at 15V for 30 minutes often provide efficient transfer

  • Use PVDF membranes with 0.22 μm pore size for optimal binding of smaller proteins

• Detection strategy:

  • Primary antibody dilutions should be determined empirically, typically starting at 1:1000

  • The His-tag provides an alternative detection method using anti-His antibodies

  • Enhanced chemiluminescence systems offer sensitive detection with low background

This optimized protocol leverages the protein's high purity (>95%) to establish reliable positive controls for clinical or experimental sample analysis.

How can Treponema p17 16.4kDa be utilized in host-pathogen interaction studies?

Treponema p17 16.4kDa represents a valuable tool for investigating host-pathogen interactions involving Treponema pallidum. The protein can be employed to study specific immune responses and potential roles in pathogenesis. Researchers should consider that T. pallidum has evolved with one of the smallest bacterial genomes at 1.14 million base pairs, reflecting adaptation through genome reduction to its specialized parasitic lifestyle in mammalian tissue .

For host-immune response studies, protocols should include:

• Stimulation of immune cells:

  • Treat peripheral blood mononuclear cells (PBMCs) with purified Treponema p17 at concentrations ranging from 1-10 μg/ml

  • Monitor cytokine production (particularly IL-6, TNF-α, and IFN-γ) at 24, 48, and 72 hours post-stimulation

  • Compare responses between healthy donors and syphilis patients to identify differential activation patterns

• Cellular localization studies:

  • Conjugate purified Treponema p17 with fluorescent labels (ensuring the labeling doesn't interfere with protein function)

  • Observe interactions with host cells using confocal microscopy

  • Complement with subcellular fractionation and Western blotting to confirm localization data

• Receptor identification:

  • Employ co-immunoprecipitation assays using Treponema p17 as bait to identify potential host cell receptors

  • Validate interactions through surface plasmon resonance or bio-layer interferometry to determine binding kinetics

The non-glycosylated nature of the recombinant protein should be noted as a potential limitation when extrapolating to native interactions, as the natural protein may contain post-translational modifications not present in the E. coli-expressed version.

What strategies can be employed for epitope mapping of Treponema p17 16.4kDa?

Epitope mapping of Treponema p17 16.4kDa provides crucial insights into immunodominant regions that may serve as diagnostic targets or vaccine candidates. Given the high purity (>95%) of the commercially available recombinant protein , several approaches can be implemented for comprehensive epitope characterization.

Table 1: Comparative Analysis of Epitope Mapping Techniques for Treponema p17 16.4kDa

For peptide scanning approaches, researchers should:

• Generate overlapping peptides (typically 15-mers with 5 amino acid overlap) spanning the entire Treponema p17 sequence

• Test reactivity with polyclonal sera from infected individuals and monoclonal antibodies

• Identify immunodominant regions showing consistent reactivity across multiple samples

• Further define minimal epitopes through alanine scanning mutagenesis of reactive peptides

The stability of Treponema p17 at room temperature for up to 4 weeks facilitates extended experimental protocols without compromising protein integrity, which is particularly advantageous for time-intensive epitope mapping procedures.

How does the His-tag affect functional studies of Treponema p17 16.4kDa?

The N-terminal His-tag present on recombinant Treponema p17 16.4kDa warrants careful consideration in functional studies, as it may influence protein activity, structure, or interactions. Researchers should implement appropriate controls to distinguish between native protein functions and potential tag-related effects.

When designing functional studies:

• Consider tag removal options:

  • Incorporate a protease cleavage site between the tag and protein during construct design

  • Use commercial tag removal kits with specific proteases (such as TEV or Factor Xa)

  • Verify complete tag removal via Western blot with anti-His antibodies

• Include control proteins:

  • Test both tagged and untagged versions of the protein when possible

  • Use an irrelevant protein with the same tag as a negative control

  • Consider creating constructs with the tag positioned at the C-terminus for comparison

• Validate findings with native protein:

  • When feasible, compare results with native protein isolated from Treponema pallidum

  • Alternatively, use computational modeling to predict potential tag interference with functional domains

• Assay selection considerations:

  • For binding assays, the His-tag offers advantages for oriented immobilization on nickel surfaces

  • For structural studies, the tag may introduce flexibility affecting crystallization

  • In cell-based assays, the tag could potentially influence cellular uptake or trafficking

The high purity (>95%) of the recombinant protein as determined by SDS-PAGE suggests minimal contamination with E. coli proteins, reducing concerns about host cell protein interference in functional assays.

What are the common challenges in maintaining Treponema p17 16.4kDa stability after reconstitution?

Despite the high stability of lyophilized Treponema p17 16.4kDa at room temperature for up to 4 weeks , researchers frequently encounter stability issues after reconstitution. These challenges can significantly impact experimental reproducibility and data quality if not properly addressed.

Common stability issues and their solutions include:

• Protein aggregation:

  • Observable as precipitation or increased turbidity in solution

  • Prevention: Add carrier proteins such as BSA (0.1%) to dilute solutions

  • Solution: Centrifuge at 10,000 × g for 10 minutes to remove aggregates before use

  • Verification: Assess monodispersity via dynamic light scattering before experiments

• Proteolytic degradation:

  • Manifests as multiple bands on SDS-PAGE or loss of activity

  • Prevention: Add protease inhibitor cocktails to working solutions

  • Solution: Prepare fresh working dilutions from frozen stock for critical experiments

  • Verification: Regularly check protein integrity via SDS-PAGE

• Adsorption to storage containers:

  • Results in decreased effective concentration and experimental variability

  • Prevention: Use low-protein binding tubes and add 0.01-0.05% Tween-20 to buffers

  • Solution: Pre-coat storage containers with BSA before adding dilute protein solutions

  • Verification: Quantify protein concentration before and after storage using Bradford or BCA assays

• Oxidation of critical residues:

  • May cause functional alterations without visible aggregation

  • Prevention: Include reducing agents (0.1-1 mM DTT or β-mercaptoethanol) in buffers

  • Solution: Store under nitrogen or argon atmosphere for very sensitive applications

  • Verification: Assess functional activity using established assays before critical experiments

The recommendation to reconstitute Treponema p17 in sterile water at concentrations not less than 100μg/ml helps minimize some of these issues by maintaining a sufficiently high protein concentration to enhance stability.

How can researchers validate the antigenic integrity of Treponema p17 16.4kDa preparations?

Validating the antigenic integrity of Treponema p17 16.4kDa preparations is crucial for immunological studies, diagnostic development, and vaccine research. Several complementary approaches can be implemented to ensure that the recombinant protein maintains native-like antigenic properties.

Recommended validation methods include:

• Antibody recognition assays:

  • Test reactivity with monoclonal antibodies targeting known epitopes

  • Compare recognition patterns between recombinant and native protein (if available)

  • Implement both Western blot (for linear epitopes) and ELISA (for conformational epitopes)

  • Expected outcome: Similar recognition patterns indicate preserved antigenic structure

• Serological validation:

  • Test reactivity with serum samples from confirmed Treponema pallidum infections

  • Include negative controls from healthy individuals

  • Calculate sensitivity and specificity metrics

  • Expected outcome: High discrimination between positive and negative samples

• Circular dichroism (CD) spectroscopy:

  • Monitor secondary structure elements (α-helices, β-sheets)

  • Compare spectra before and after storage or experimental handling

  • Quantify changes in structural elements using spectral deconvolution software

  • Expected outcome: Minimal changes in spectral characteristics indicate structural stability

• Thermal shift assays:

  • Measure protein thermal stability using differential scanning fluorimetry

  • Compare melting temperatures across different batches or storage conditions

  • Identify buffer compositions that enhance stability

  • Expected outcome: Consistent melting temperatures indicate comparable folding stability

The high purity (>95%) of commercially available Treponema p17 as determined by SDS-PAGE provides a solid foundation for these validation studies by minimizing the influence of contaminants on antigenic assessment.

How can Treponema p17 16.4kDa be utilized in developing improved syphilis diagnostic assays?

Treponema p17 16.4kDa offers significant potential for enhancing syphilis diagnostic assays due to its high purity (>95%) and defined composition. Current diagnostic challenges include distinguishing active from past infections and reducing cross-reactivity with other spirochetes. Researchers can leverage this recombinant protein to address these limitations through several methodological approaches.

For diagnostic assay development, researchers should consider:

• Multiplex antigen panels:

  • Combine Treponema p17 with other recombinant T. pallidum antigens (TpN47, TpN15, TpN44.5)

  • Develop antigen ratios that optimize sensitivity and specificity

  • Validate with well-characterized clinical sample panels spanning different disease stages

  • Implement machine learning algorithms to identify signature patterns associated with disease progression

• Lateral flow assay development:

  • Immobilize Treponema p17 on nitrocellulose membranes at 0.5-1.0 mg/ml

  • Optimize gold nanoparticle conjugation conditions for detection antibodies

  • Determine limit of detection using standardized T. pallidum antibody preparations

  • Evaluate stability under various environmental conditions to assess field applicability

• Chemiluminescent immunoassay optimization:

  • Conjugate Treponema p17 to magnetic particles for automated platforms

  • Establish signal calibration curves using international reference standards

  • Implement signal amplification strategies for improved sensitivity

  • Validate assay precision with coefficient of variation <10% across the analytical range

The non-glycosylated nature of E. coli-expressed Treponema p17 should be considered when evaluating diagnostic performance, as glycosylation differences from the native protein might affect antibody recognition in some patient samples.

What are the key considerations when using Treponema p17 16.4kDa in vaccine development research?

Treponema p17 16.4kDa represents a potential vaccine candidate component for treponemal infections due to its defined composition and high purity. Researchers exploring its application in vaccine development should consider several methodological aspects to maximize efficacy and safety.

Critical considerations include:

• Adjuvant selection and formulation:

  • Test multiple adjuvant systems (alum, MF59, AS01, CpG) with Treponema p17

  • Evaluate protein stability in each adjuvant formulation over time

  • Measure particle size and zeta potential of formulations

  • Assess protein structural integrity after adjuvant incorporation using circular dichroism

• Immunization protocols:

  • Implement prime-boost strategies with varying dosages (typically 10-50 μg protein per dose)

  • Compare different administration routes (subcutaneous, intramuscular, intradermal)

  • Establish optimal intervals between immunizations (3-4 weeks typically)

  • Monitor antibody titer development and persistence over time

• Immune response characterization:

  • Analyze antibody isotype profiles (IgG1, IgG2, IgG3, IgG4) following immunization

  • Evaluate T-cell responses through cytokine profiling and proliferation assays

  • Assess neutralizing capacity of induced antibodies using in vitro assays

  • Determine epitope-specific responses through epitope mapping techniques

• Challenge studies (in appropriate animal models):

  • Establish protective correlates by comparing immunological parameters with protection metrics

  • Evaluate sterilizing immunity versus reduction in bacterial load

  • Monitor for potential enhancement effects or adverse reactions

  • Assess long-term protection through extended challenge timepoints

The recombinant protein's expression in E. coli provides consistency advantages for vaccine production but requires thorough testing for endotoxin contamination, which could confound immunological results or pose safety concerns in vaccine formulations.