Recombinant Treponema pallidum Uncharacterized protein TP_0031 (TP_0031)

What is the basic structure and properties of TP_0031?

TP_0031 is an uncharacterized protein from Treponema pallidum with a full length of 92 amino acids . The complete amino acid sequence is: MDLGQRVVRVIPLAPLPVRVYNAGGLRVDFFPRFFGRSPQGVGVGFARLKLSASVGSNGFRLTRAVWIFWLCFLVSGLSRAFLVYFLSVIRI . This protein is cataloged in UniProt with the ID O83074, and while it remains functionally uncharacterized, its small size (92 amino acids) suggests it may serve as a regulatory protein or protein domain within the Treponema pallidum proteome .

For research purposes, working with recombinant versions of this protein typically involves the addition of tags to facilitate purification and detection. The commercial preparations available include His-tagged versions expressed in E. coli systems, which are supplied as lyophilized powders with greater than 90% purity as determined by SDS-PAGE analysis .

How does TP_0031 compare to other uncharacterized proteins in T. pallidum?

T. pallidum contains numerous uncharacterized proteins that represent knowledge gaps in understanding this pathogen's biology. While the search results don't provide specific comparative information between TP_0031 and other uncharacterized proteins, we can infer from general T. pallidum research that:

Unlike several other T. pallidum proteins that have shown evidence of recombination events between subspecies, there is no specific mention of TP_0031 in the list of 12 genes with detected recombination events described in comprehensive genome analysis studies . The evolutionary analysis of T. pallidum has identified recombination in genes like tp0136, tp0865, and others, but TP_0031 was not highlighted among these recombination-prone genes .

When designing comparative studies between TP_0031 and other T. pallidum proteins, researchers should consider evolutionary conservation, predicted structural motifs, and potential functional domains, even in the absence of characterized function.

What expression systems are recommended for TP_0031 production?

E. coli expression systems are the predominant choice for TP_0031 recombinant production, as evidenced by commercial preparations and general recombinant protein production practices . For optimal expression in E. coli, researchers should consider the following methodological approach:

• Vector selection: pET expression systems with T7 promoters provide high-level expression control, similar to the commercially available TP_0031 preparations .

• E. coli strain selection: BL21(DE3) derivatives, particularly those with enhanced mRNA stability like BL21 Star(DE3), may improve expression yields similar to what has been demonstrated for other recombinant proteins .

• Expression conditions: Statistical experimental design approaches, as demonstrated for other recombinant proteins, can significantly optimize TP_0031 expression by systematically evaluating multiple variables simultaneously .

How can statistical experimental design improve TP_0031 expression yields?

Statistical experimental design methodologies offer significant advantages over traditional univariate approaches when optimizing recombinant protein expression, including TP_0031. Rather than changing one variable at a time, multivariate analysis allows researchers to:

• Evaluate multiple variables simultaneously, accounting for their interactions

• Characterize experimental error systematically

• Compare normalized variable effects

• Gather high-quality information with fewer experiments

For TP_0031 expression optimization, researchers should implement a fractional factorial design similar to that used for other recombinant proteins from pathogenic organisms. A recommended approach includes:

• Variable selection: Focus on eight key variables that significantly impact expression:

  • Media components (yeast extract, tryptone, NaCl concentrations)

  • Carbon sources (glucose, glycerol concentrations)

  • Antibiotic concentration

  • Induction conditions (inducer concentration, absorbance at induction)

  • Expression temperature

• Experimental matrix: Implement a 2^8-4 fractional factorial design with central point replicates to maintain statistical orthogonality while reducing the total number of experiments .

• Response measurement: Assess protein production through multiple metrics:

  • Cell growth (measured by final OD600)

  • Specific protein activity (using appropriate functional assays)

  • Volumetric productivity (mg of protein per liter of culture)

This approach has demonstrated success in optimizing recombinant protein expression, achieving yields of 250 mg/L for other proteins, and similar methodologies should be applicable to TP_0031 expression .

What factors most significantly affect soluble expression of TP_0031?

Based on general principles of recombinant protein expression and the specific challenges of expressing T. pallidum proteins, the following factors likely have the greatest impact on soluble TP_0031 expression:

• Translation initiation efficiency: Research indicates that the accessibility of translation initiation sites modeled using mRNA base-unpairing across the Boltzmann's ensemble significantly impacts recombinant protein expression success . For TP_0031, optimizing the accessibility of the translation initiation region through synonymous codon substitutions in the first nine codons may substantially improve expression levels .

• Temperature control: Lower post-induction temperatures (25°C compared to 37°C) often increase soluble protein fraction by slowing protein synthesis and allowing proper folding .

• Induction parameters: The combination of inducer concentration and cell density at induction time can dramatically affect soluble protein yields. Statistical analysis of experimental data is essential to identify optimal combinations, as these parameters often display significant interaction effects .

• Media composition: A balanced medium that supports both cell growth and protein expression is crucial. For TP_0031, starting with a medium containing moderate levels of yeast extract (5 g/L), tryptone (5 g/L), NaCl (10 g/L), and a defined glucose concentration (1 g/L) provides a reasonable baseline for optimization .

A methodical approach to optimizing these variables through factorial design will likely achieve the highest soluble yields of TP_0031.

How can mRNA structure optimization improve TP_0031 expression?

mRNA secondary structure, particularly around the translation initiation site, is a critical determinant of recombinant protein expression efficiency. For TP_0031 expression optimization, researchers should consider:

• Translation initiation site accessibility: Research has demonstrated that the accessibility of translation initiation sites significantly outperforms alternative features in predicting expression success . Computational tools that model mRNA base-unpairing across the Boltzmann's ensemble can predict the likelihood of successful expression .

• Synonymous codon optimization: Using tools like TIsigner that implement simulated annealing algorithms to modify the first nine codons of the mRNA with synonymous substitutions can substantially improve expression levels without altering the amino acid sequence .

• Balance of expression and cellular burden: Stochastic simulation models indicate that higher mRNA accessibility leads to higher protein production but slower cell growth, reflecting a protein cost where cell growth is constrained during overexpression . Finding the optimal balance for TP_0031 expression will require systematic testing.

The methodological approach should include:

• Computational analysis of the original TP_0031 sequence using mRNA structure prediction algorithms

• Generation of synonymously modified variants with improved translation initiation site accessibility

• Experimental validation of expression levels for these variants

• Selection of the optimal sequence for large-scale expression

What approaches can determine the function of this uncharacterized protein?

Determining the function of TP_0031 requires a multi-faceted approach combining computational predictions, experimental characterization, and comparative analysis:

• Bioinformatic analysis:

  • Sequence homology searches against characterized proteins

  • Structural prediction using tools like AlphaFold or RoseTTAFold

  • Identification of conserved domains or motifs

  • Genomic context analysis to identify potential operons or functional associations

• Experimental characterization:

  • Protein-protein interaction studies (pull-down assays, yeast two-hybrid)

  • Subcellular localization using tagged versions of TP_0031

  • Gene knockout or knockdown studies in T. pallidum (if genetic manipulation systems are available)

  • Heterologous expression in model organisms to observe phenotypic effects

• Structural biology approaches:

  • X-ray crystallography or NMR spectroscopy of the purified recombinant protein

  • Hydrogen-deuterium exchange mass spectrometry to identify potential binding regions

• Comparative analysis with other T. pallidum proteins:

  • Expression pattern analysis during different stages of infection

  • Evolutionary conservation analysis across Treponema subspecies and related spirochetes

When planning these studies, researchers should consider that TP_0031's small size (92 amino acids) may indicate it functions as part of a larger complex or has a regulatory role rather than an enzymatic function .

How reliable are computational predictions for TP_0031 function?

Computational predictions for uncharacterized proteins like TP_0031 have both strengths and limitations that researchers should carefully consider:

Strengths:

Limitations:

• Small proteins like TP_0031 (92 amino acids) often have fewer distinctive features for algorithms to leverage

• T. pallidum has many unique proteins with limited homology to well-characterized proteins from model organisms

• Function prediction algorithms struggle with proteins that have evolved through recombination events, which are common in T. pallidum

For TP_0031 specifically, computational predictions should be treated as hypotheses requiring experimental validation. A recommended methodological approach includes:

• Apply multiple prediction algorithms and look for consensus predictions

• Evaluate prediction confidence scores carefully

• Design experiments that can directly test the highest-confidence predictions

• Consider the evolutionary context of T. pallidum when interpreting predictions

Given the evidence of extensive recombination in T. pallidum genomes, which has played a key role in the evolution and recent expansion of syphilis bacteria, predictions should consider the possibility that TP_0031 may have chimeric functional elements from different evolutionary sources .

What role might TP_0031 play in T. pallidum pathogenesis?

While the specific role of TP_0031 in T. pallidum pathogenesis remains uncharacterized, we can propose potential functions based on general patterns in bacterial pathogenesis and T. pallidum biology:

• Potential involvement in host-pathogen interactions:

  • Small proteins often mediate specific interactions with host factors

  • The relatively high conservation of proteins compared to polysaccharides in T. pallidum suggests proteins may be key antigenic determinants

• Possible regulatory functions:

  • Small proteins frequently serve as regulators of virulence gene expression

  • T. pallidum adapts to different host environments during infection, requiring sophisticated regulatory networks

• Contribution to immune evasion:

  • T. pallidum is known for its ability to evade host immune responses

  • Small, surface-exposed proteins can contribute to antigenic variation

• Evolutionary considerations:

  • Recombination between T. pallidum subspecies has played a key role in the evolution and recent expansion of syphilis bacteria

  • While TP_0031 was not specifically identified among the 12 genes with detected recombination events, its evolutionary history may still influence its current function

To investigate TP_0031's role in pathogenesis, researchers should consider:

• Expression analysis during different stages of infection

• Interaction studies with host proteins and tissues

• Immunological studies to determine if TP_0031 is recognized by the host immune system

• Comparative analysis across T. pallidum subspecies (pallidum, pertenue, and endemicum)

How does TP_0031 fit into evolutionary patterns of T. pallidum subspecies?

TP_0031 should be evaluated within the context of T. pallidum's complex evolutionary history, characterized by recombination and selection pressures across subspecies:

T. pallidum encompasses several subspecies responsible for different diseases: subsp. pallidum (TPA) causes syphilis, subsp. pertenue (TPE) causes yaws, and subsp. endemicum (TEN) causes bejel . Comprehensive genomic analysis has identified significant intersubspecies recombination, primarily from TPE/TEN to TPA, involving 12 genes and 21 recombination events .

While TP_0031 was not specifically identified among the 12 genes showing strong evidence of recombination, researchers should still investigate:

• Sequence conservation of TP_0031 across subspecies to determine its evolutionary stability

• Potential involvement in subspecies-specific functions by comparing expression patterns

• Whether TP_0031 shows evidence of selection pressure (positive or purifying selection)

• Possible functional relationships with the identified recombination-prone genes, which include tp0136, tp0164, tp0462, tp0483, tp0548, tp0558, tp0699, tp0856, tp0865, and others

The methodological approach should include:

• Comparative sequence analysis across multiple strains of each subspecies

• Calculation of dN/dS ratios to assess selection pressure

• Phylogenetic analysis to determine if TP_0031 follows species evolution or shows evidence of horizontal transfer

• Functional studies comparing TP_0031 from different subspecies

What purification strategies yield the highest purity and activity for TP_0031?

Purification of recombinant TP_0031 requires a strategic approach designed for this specific protein:

• Initial capture:

  • For His-tagged TP_0031, immobilized metal affinity chromatography (IMAC) provides an efficient first purification step

  • Optimal conditions include buffers containing 20-50 mM imidazole to reduce non-specific binding while maintaining target protein affinity

• Intermediate purification:

  • Ion exchange chromatography based on TP_0031's theoretical isoelectric point

  • Hydrophobic interaction chromatography as an orthogonal separation technique

• Polishing:

  • Size exclusion chromatography to achieve final purity >95% and remove potential aggregates

  • Yields can be assessed using typical protein quantification methods (Bradford/BCA assays)

• Activity preservation:

  • Commercial preparations of TP_0031 are stabilized using Tris/PBS-based buffer with 6% trehalose at pH 8.0

  • For long-term storage, addition of 5-50% glycerol and storage at -20°C/-80°C is recommended

  • Aliquoting is necessary to avoid repeated freeze-thaw cycles that can compromise activity

• Quality control:

  • SDS-PAGE analysis for purity assessment (target >90%)

  • Mass spectrometry for identity confirmation

  • Functionality assays (when developed for this uncharacterized protein)

Commercial preparations achieve >90% purity , but research applications may require higher purity levels depending on the specific experimental requirements.

How can contradictory expression data for TP_0031 be reconciled?

When researchers encounter contradictory expression data for TP_0031, a systematic troubleshooting approach should be implemented:

• Methodology standardization:

  • Implement statistical experimental design to systematically evaluate variables

  • Apply a 2^8-4 fractional factorial design with center points to efficiently identify significant variables affecting expression

  • Standardize key measurements (cell growth, protein quantity, activity) across experiments

• Data analysis:

  • Apply multivariate statistical methods to identify interactions between variables that may explain contradictory results

  • Evaluate whether differences in mRNA translation initiation site accessibility could explain expression variability

  • Consider how synonymous codon usage affects expression levels across different systems

• Specific variables to reconcile:

  • Growth medium composition (particularly yeast extract, tryptone, and carbon source concentrations)

  • Induction parameters (cell density at induction, inducer concentration, temperature)

  • Host strain variations (different E. coli strains may show dramatically different expression patterns)

• Protein property considerations:

  • Assess whether solubility vs. inclusion body formation differs between methods

  • Evaluate if protein activity measurements are standardized appropriately

  • Consider if the recombinant tag affects expression or solubility

For example, when facing contradictory results, researchers might discover that induction at higher cell densities (OD600 = 0.8) with lower inducer concentrations (0.1 mM IPTG) at reduced temperatures (25°C) consistently yields more active protein, as demonstrated for other recombinant proteins .

How might TP_0031 contribute to vaccine development against T. pallidum?

Evaluating TP_0031 as a potential vaccine candidate requires considering both its characteristics and the broader challenges of T. pallidum vaccine development:

• Potential advantages of TP_0031 in vaccine development:

  • Protein-based vaccines offer advantages over polysaccharide-based approaches due to the higher level of amino acid sequence conservation compared to polysaccharide structure

  • Conserved proteins present in multiple T. pallidum serotypes, like pneumolysin (Ply), have shown effective protection against infections

  • If TP_0031 shows similar conservation across serotypes, it could potentially serve as a component in a multi-antigen vaccine

• Methodological approach for evaluation:

  • Assess conservation of TP_0031 sequence across T. pallidum isolates

  • Evaluate immunogenicity in animal models using purified recombinant protein

  • Determine if antibodies against TP_0031 can neutralize T. pallidum in vitro

  • Investigate whether TP_0031 is accessible to antibodies in intact T. pallidum

• Evolutionary considerations:

  • The evolutionary analysis of T. pallidum identified recombination patterns that have implications for vaccine candidate selection

  • Genes showing evidence of positive selection due to recombination may be under immune pressure and could be promising vaccine targets

  • Though TP_0031 was not specifically identified among recombination-prone genes, its conservation pattern should be evaluated in this context

• Integration with existing vaccine development efforts:

  • Current approaches focus on conserved proteins present in multiple serotypes

  • A successful vaccine may require multiple antigens targeting different aspects of T. pallidum pathogenesis

What techniques can resolve contradictory findings about TP_0031 structure?

Resolving structural contradictions for proteins like TP_0031 requires integrating multiple structural biology approaches:

• Complementary structural determination methods:

  • X-ray crystallography provides high-resolution static structures but requires protein crystallization

  • NMR spectroscopy offers solution-state structural information and dynamics but has size limitations

  • Cryo-electron microscopy can resolve structures without crystallization but may have resolution limitations for small proteins

  • Small-angle X-ray scattering (SAXS) provides low-resolution envelope information in solution

• Computational approaches:

  • Modern AI-based structure prediction (AlphaFold2, RoseTTAFold) can provide accurate models

  • Molecular dynamics simulations can explore conformational flexibility

  • Integrative modeling combines experimental data with computational predictions

• Methodological approach to resolve contradictions:

  • Systematically compare experimental conditions that led to different structural observations

  • Consider whether tag positions or purification methods affected structure

  • Evaluate whether the protein exists in multiple conformational states

  • Assess if the protein structure depends on binding partners or environmental conditions

• Specific considerations for TP_0031:

  • At 92 amino acids, TP_0031 is ideal for NMR structural determination

  • Different expression and purification protocols may yield proteins with different folding properties

  • Post-translational modifications present in native T. pallidum but absent in recombinant systems may affect structure

By integrating multiple structural biology approaches and carefully controlling experimental variables, researchers can reconcile seemingly contradictory structural data and develop a comprehensive understanding of TP_0031's structure-function relationship.

How can systems biology approaches integrate TP_0031 into T. pallidum pathogenesis models?

Integrating TP_0031 into systems biology models of T. pallidum pathogenesis requires a comprehensive approach that spans multiple biological scales:

• Network integration approaches:

  • Protein-protein interaction mapping to identify TP_0031 binding partners

  • Gene co-expression analysis across different infection stages

  • Metabolic network analysis to place TP_0031 in biochemical pathways

  • Regulatory network reconstruction to identify potential regulatory relationships

• Multi-omics data integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Use statistical and machine learning approaches to identify correlations between TP_0031 expression and other cellular processes

  • Develop predictive models of TP_0031 function based on integrated datasets

• Host-pathogen interaction modeling:

  • Map potential interactions between TP_0031 and host factors

  • Model immune responses to T. pallidum infection including potential responses to TP_0031

  • Simulate infection dynamics with and without functional TP_0031

• Evolutionary systems biology:

  • Integrate knowledge of recombination patterns in T. pallidum evolution

  • Compare systems biology models across T. pallidum subspecies

  • Identify how evolutionary processes like lateral gene transfer shape current pathogenesis mechanisms

• Methodological approach:

  • Generate condition-specific experimental data for TP_0031

  • Develop computational models that integrate diverse data types

  • Iteratively refine models based on experimental validation

  • Apply sensitivity analysis to identify key parameters in the system

This integrative approach can place TP_0031 within the broader context of T. pallidum biology and identify its contributions to pathogenesis, even without complete functional characterization.