Recombinant Tropheryma whipplei UPF0102 protein TW312 (TW312)

What are the optimal expression systems for producing Recombinant Tropheryma whipplei UPF0102 protein TW312?

When expressing Recombinant Tropheryma whipplei UPF0102 protein TW312, researchers should consider multiple expression systems, each with distinct advantages. E. coli and yeast expression systems typically provide the highest yields and shorter turnaround times for UPF0102 family proteins . For optimal expression in E. coli, consider the following methodology:

• Strain selection: BL21(DE3) or Rosetta strains are preferred for proteins that may contain rare codons

• Temperature optimization: Lower temperatures (16-25°C) often improve folding for complex proteins

• Induction parameters: IPTG concentration (0.1-1.0 mM) and induction timing (mid-log phase, OD600 = 0.6-0.8)

• Media supplementation: Addition of glucose (0.5-1%) may reduce basal expression and improve yield

For more complex post-translational modifications, insect cell-baculovirus or mammalian expression systems should be considered, though these typically result in lower yields compared to microbial systems.

What purification strategies are most effective for isolating TW312 with high purity and biological activity?

A multi-step purification strategy is essential for obtaining high-purity TW312 protein suitable for structural and functional studies:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using His-tags is recommended for primary isolation

• Intermediate purification: Ion exchange chromatography to separate based on charge properties

• Polishing: Size exclusion chromatography to remove aggregates and ensure monodispersity

For UPF0102 family proteins, optimal buffer conditions typically include:

• pH range: 7.0-8.0

• Salt concentration: 150-300 mM NaCl

• Addition of reducing agents: 1-5 mM DTT or 0.5-2 mM TCEP

• Protease inhibitors: PMSF (1 mM) or commercial cocktails during initial extraction

Activity preservation often requires careful optimization of buffer components, as UPF0102 proteins may require specific cofactors or conditions to maintain their native conformations and biological functions.

How can researchers verify the structural integrity of purified TW312 protein?

Multiple complementary analytical techniques should be employed to verify structural integrity:

• Circular Dichroism (CD) Spectroscopy:

  • Far-UV CD (190-260 nm): Determines secondary structure composition

  • Near-UV CD (250-350 nm): Assesses tertiary structure organization

• Thermal Shift Assays:

  • Differential Scanning Fluorimetry (DSF) to determine melting temperature (Tm)

  • Buffer optimization parameters that increase Tm typically enhance stability

• Size Exclusion Chromatography - Multi-Angle Light Scattering (SEC-MALS):

  • Determines absolute molecular weight and oligomeric state

  • Identifies potential aggregation issues

• Limited Proteolysis:

  • Trypsin digestion at varying enzyme:protein ratios (1:100 to 1:1000)

  • Analysis of digestion patterns by SDS-PAGE and mass spectrometry

  • Well-folded proteins exhibit characteristic resistance patterns

For UPF0102 family proteins, which may have diverse structural characteristics, establishing these baseline parameters is essential before proceeding to functional assays.

What functional assays are appropriate for characterizing the biological activity of TW312 protein?

Developing appropriate functional assays requires understanding the predicted molecular functions of UPF0102 family proteins. While specific assays for TW312 would need to be tailored to its exact function, consider these methodological approaches:

• Protein-Protein Interaction Studies:

  • Pull-down assays using tagged versions of TW312

  • Surface Plasmon Resonance (SPR) for kinetic binding parameters

  • Microscale Thermophoresis (MST) for quantitative binding in solution

• Enzymatic Activity Analysis:

  • If TW312 has predicted enzymatic activity, design substrate-based assays

  • Monitor reaction products by HPLC, mass spectrometry, or spectrophotometric methods

  • Determine kinetic parameters (Km, Vmax, kcat) under varying conditions

• Cell-Based Functional Assays:

  • Overexpression studies in relevant cell lines

  • Analysis of effects on cell cycle progression, as observed with related proteins like TW-37

  • Assessment of effects on signaling pathways that may be modulated by UPF0102 proteins

For quantitative measurements, design experiments with appropriate controls, statistical power calculations, and multiple independent replicates to ensure reproducibility.

How can researchers investigate potential protein-protein interactions involving TW312 in Tropheryma whipplei pathogenesis?

Investigating protein-protein interactions (PPIs) involving TW312 requires a multi-faceted approach:

• Computational Prediction Methods:

  • Sequence-based prediction tools (STRING, IntAct)

  • Structural homology modeling to identify potential interaction surfaces

  • Molecular docking simulations with predicted partners

• In Vitro Interaction Studies:

  • Co-immunoprecipitation (Co-IP) using antibodies against TW312

  • GST pull-down assays with cellular lysates

  • Crosslinking mass spectrometry (XL-MS) to identify interaction sites

• Cellular Validation:

  • Bimolecular Fluorescence Complementation (BiFC)

  • Förster Resonance Energy Transfer (FRET) microscopy

  • Proximity Ligation Assay (PLA) in infected cells

• Functional Validation:

  • Mutagenesis of predicted interaction interfaces

  • Competition assays with peptides derived from interaction sites

  • Effects of disrupting interactions on bacterial survival and pathogenesis

Similar approaches revealed that TW-37, another protein with therapeutic potential, functions by inhibiting Bcl-2 family proteins , suggesting the importance of protein interactions in determining functional outcomes.

What role might epigenetic modulators play in regulating expression systems for TW312 and how can these be experimentally manipulated?

Epigenetic regulation may significantly impact heterologous expression of TW312 and can be experimentally manipulated:

• Histone Deacetylase (HDAC) Modulation:

  • Treatment with HDAC inhibitors (e.g., Trichostatin A, SAHA) may enhance expression

  • These compounds can relieve transcriptional repression, as demonstrated with other proteins

  • Experimental design: Dose-response experiments (0.1-10 μM range) with time-course analysis

• DNA Methylation Analysis:

  • Assessment of CpG islands in promoter regions using bisulfite sequencing

  • Treatment with 5-azacytidine (1-5 μM) to inhibit DNA methylation

  • Quantification of expression changes via qRT-PCR and Western blotting

• Chromatin Immunoprecipitation (ChIP) Assays:

  • Identification of histone modifications associated with the gene encoding TW312

  • Analysis of transcription factor binding to regulatory regions

  • Integration with expression data to establish causative relationships

The strategic use of epigenetic modulators may significantly enhance expression levels, particularly in mammalian systems where chromatin structure plays a critical role in gene regulation.

What are the optimal conditions for studying TW312 protein stability and preventing aggregation during in vitro experiments?

Protein stability and prevention of aggregation require careful optimization:

Methodology for stability assessment:

• Time-course stability studies: Aliquot protein and store under various conditions, testing activity/structure at regular intervals (0, 1, 3, 7, 14, 30 days)

• Freeze-thaw stability: Subject protein to 0, 1, 3, 5 freeze-thaw cycles and assess structural integrity

• Chemical denaturation: Titrate denaturants (urea, guanidinium HCl) and monitor unfolding by tryptophan fluorescence

• Aggregation kinetics: Monitor by right-angle light scattering during thermal ramping experiments

For UPF0102 family proteins, which may have challenging stability profiles, establishing these parameters is crucial for generating reproducible experimental results.

How can researchers design effective cell-based assays to study the function of TW312 in models of Tropheryma whipplei infection?

Designing cell-based assays requires careful consideration of physiological relevance:

• Cell Line Selection:

  • Primary macrophages (most relevant for T. whipplei infection)

  • THP-1 monocytic cells (differentiated with PMA)

  • Intestinal epithelial cell lines (Caco-2, HT-29) for host-pathogen interactions

• Infection Protocol Optimization:

  • MOI (multiplicity of infection) titration (1:1 to 100:1)

  • Time-course experiments (4, 24, 48, 72 hours post-infection)

  • Quantification by qPCR, immunofluorescence, or flow cytometry

• Genetic Manipulation Approaches:

  • CRISPR/Cas9 knockout of host interacting partners

  • siRNA knockdown for transient depletion studies

  • Overexpression of wild-type vs. mutant TW312 to assess function

• Readout Selection:

  • Bacterial survival and replication rates

  • Host cell responses (cytokine production, cell death pathways)

  • Subcellular localization studies using confocal microscopy

Similar approaches have been used to study TW-37's effects on cell cycle regulation and apoptosis in cancer cells, revealing S-phase arrest mechanisms , which could provide methodological insights applicable to TW312 research.

What computational approaches can predict the structure-function relationship of TW312 when crystallographic data is unavailable?

In the absence of crystallographic data, several computational approaches can predict structure-function relationships:

• Homology Modeling:

  • Identify structural templates using HHpred or SWISS-MODEL

  • Generate multiple models using different algorithms (MODELLER, Rosetta)

  • Validate models using PROCHECK, ERRAT, and Verify3D

• Molecular Dynamics Simulations:

  • Perform all-atom simulations in explicit solvent (100-500 ns)

  • Analyze conformational flexibility and stability

  • Identify potential binding pockets and functional regions

• Evolutionary Analysis:

  • Multiple sequence alignment of UPF0102 family proteins

  • Identification of conserved residues that may indicate functional importance

  • Analysis of co-evolving residues to predict interaction surfaces

• Integrative Modeling:

  • Combine low-resolution experimental data (SAXS, cryo-EM)

  • Incorporate cross-linking mass spectrometry constraints

  • Refine models iteratively with experimental validation

The refined models can guide the design of site-directed mutagenesis experiments to test functional hypotheses, similar to approaches used to characterize TW-37's interaction with Bcl-2 family proteins .

How can researchers develop specific inhibitors or activators targeting TW312 for therapeutic applications?

Development of specific modulators for TW312 requires a systematic approach:

• Target Validation:

  • Confirm essential role in pathogenesis through knockout/knockdown studies

  • Identify specific functional domains amenable to therapeutic intervention

  • Assess conservation across bacterial strains to predict spectrum of activity

• High-Throughput Screening:

  • Develop biochemical assays suitable for screening (enzymatic, binding)

  • Design cell-based phenotypic assays with appropriate readouts

  • Screen diverse chemical libraries (10,000-100,000 compounds)

• Structure-Activity Relationship (SAR) Analysis:

  • Synthesize analogs of initial hits

  • Perform systematic modification of chemical scaffolds

  • Correlate structural features with biological activity

• Mode of Action Studies:

  • Confirm direct binding using biophysical methods (SPR, ITC)

  • Map binding sites using hydrogen-deuterium exchange mass spectrometry

  • Generate co-crystal structures when possible

This approached has been successfully applied to develop TW-37, a small-molecule inhibitor of Bcl-2 family proteins that demonstrates efficacy in cancer models by targeting specific protein-protein interactions .

How might TW312 be involved in modulating host cell signaling pathways during Tropheryma whipplei infection?

Investigation of TW312's role in host cell signaling requires systematic analysis:

• Transcriptomic Analysis:

  • RNA-seq of infected cells with wild-type vs. TW312-deficient bacteria

  • Time-course analysis to capture dynamic signaling events

  • Pathway enrichment analysis to identify affected signaling networks

• Phosphoproteomics:

  • LC-MS/MS analysis of phosphorylated proteins in infected cells

  • Quantitative comparison of signaling pathway activation

  • Validation of key nodes using phospho-specific antibodies

• Targeted Pathway Analysis:

  • Investigation of specific pathways (e.g., NF-κB, MAPK, Notch)

  • Reporter assays for transcriptional activity

  • Pharmacological inhibition to validate pathway involvement

Research on related proteins like TW-37 has revealed effects on the Notch signaling pathway, specifically downregulating Notch-1 (ICN) and Jagged-1 expression , suggesting potential mechanisms by which bacterial proteins might modulate host signaling.

What experimental approaches can determine if TW312 exhibits cell cycle regulatory functions similar to other characterized proteins?

To investigate potential cell cycle regulatory functions:

• Synchronization Experiments:

  • Synchronize cells using double thymidine block or serum starvation

  • Introduce recombinant TW312 or activate endogenous expression

  • Monitor cell cycle progression by flow cytometry with propidium iodide staining

• Analysis of Cell Cycle Regulators:

  • Western blot analysis of cyclins, CDKs, and cell cycle inhibitors

  • qRT-PCR to assess transcriptional regulation

  • Immunofluorescence to determine subcellular localization

• BrdU Incorporation Assays:

  • Pulse-chase experiments to track S-phase progression

  • Co-staining with cell cycle markers to identify specific blocks

  • Quantitative image analysis for single-cell resolution data

• Targeted Gene Expression Analysis:

  • Focus on S-phase regulatory genes (E2F-1, Survivin, cdc25A)

  • Chromatin immunoprecipitation to assess promoter binding

  • Luciferase reporter assays to quantify transcriptional effects

These approaches parallel those used to demonstrate that TW-37 induces S-phase arrest in pancreatic cancer cells, accompanied by altered expression of cell cycle regulatory factors , providing a methodological framework for TW312 studies.