Recombinant Uncharacterized protein Rv2206/MT2262 (Rv2206, MT2262)

What expression systems are commonly used for producing Rv2206/MT2262 recombinant protein?

The most established expression system for Rv2206/MT2262 is E. coli, typically with an N-terminal His-tag for purification purposes. The recombinant protein is generally expressed as a full-length construct (1-236 amino acids) and purified to ≥90% purity as determined by SDS-PAGE .

What is currently known about the function of Rv2206/MT2262 protein?

Despite being classified as "uncharacterized," genetic studies have implicated Rv2206/MT2262 in phagosome maturation arrest during M. tuberculosis infection of macrophages. Rv2206 mutants fail to properly arrest phagosome maturation, demonstrating significantly reduced colocalization with early endosomal markers (dextran and transferrin) and increased colocalization with late endosomal/lysosomal markers (LBPA and LAMP-2) compared to wild-type strains .

What are the optimal storage conditions for recombinant Rv2206/MT2262 protein?

For optimal stability, the recombinant Rv2206/MT2262 protein should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use. Repeated freeze-thaw cycles should be strictly avoided. Working aliquots can be maintained at 4°C for up to one week. The protein is typically provided in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 .

What reconstitution protocols are recommended for lyophilized Rv2206/MT2262 protein?

The recommended reconstitution protocol involves:

• Brief centrifugation of the vial prior to opening to bring contents to the bottom

• Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of glycerol to a final concentration of 5-50% (with 50% being the standard recommendation)

• Aliquoting for long-term storage at -20°C/-80°C

How can researchers design experiments to study the role of Rv2206/MT2262 in phagosome maturation?

Based on published methodologies, researchers can:

• Generate Rv2206 mutant strains using transposon mutagenesis or targeted gene disruption

• Infect macrophages (typically bone marrow-derived macrophages) with wild-type and mutant strains

• Assess phagosome maturation by:

  • Pulse-labeling with fluorescent dextran or transferrin (early endosomal markers)

  • Immunofluorescence labeling for LAMP-2 and LBPA (late endosomal markers)

  • Modified acid-fast staining to visualize mycobacteria

  • Quantification of colocalization using fluorescence deconvolution microscopy

This approach allows for quantitative assessment of phagosomal trafficking differences between wild-type and mutant strains .

How should researchers interpret colocalization data for Rv2206/MT2262 mutants?

Colocalization data should be analyzed using the following approach:

• Collect z-stacks (typically 5-μm) centered on mycobacterial bacilli for at least 70 infected macrophages per condition

• Deconvolve and analyze stacks using appropriate software (e.g., SoftWoRx)

• Quantify fraction colocalization from multiple independent infections (minimum of 200 individual phagosomes)

• Use Student's t-test to determine statistical significance compared to wild-type

The following table shows representative colocalization data for Rv2206 mutants compared to controls:

This pattern indicates that Rv2206 mutants fail to arrest phagosome maturation, resembling the phenotype of heat-killed bacteria rather than live wild-type bacteria .

How can researchers address contradictions in experimental data when studying Rv2206/MT2262?

When encountering contradictory data regarding Rv2206/MT2262 function, researchers should:

• Implement a systematic approach to identify inconsistencies using a notation of contradiction patterns

• Consider three key parameters:

  • α: number of interdependent items

  • β: number of contradictory dependencies

  • θ: minimal number of required Boolean rules to assess contradictions

• Check for common sources of contradictions:

  • Different experimental systems (in vitro vs. in vivo)

  • Different macrophage types (human vs. mouse, primary vs. cell lines)

  • Variations in infection protocols (MOI, timing)

  • Different detection methods for phagosomal markers

• Implement structured classification of contradiction patterns to effectively scope different patterns across domains

What are the implications of Rv2206/MT2262 in M. tuberculosis pathogenesis and survival in macrophages?

Studies indicate that Rv2206/MT2262 plays a crucial role in M. tuberculosis pathogenesis by:

• Contributing to phagosome maturation arrest, a key virulence mechanism

• Affecting bacterial survival in macrophages (Rv2206 mutants show impaired intracellular survival)

• Potentially influencing virulence in animal infection models

Genetic screens have identified Rv2206 mutants as defective for survival in macrophages, with quantitative data showing significant attenuation compared to wild-type strains. This suggests that targeting this protein might represent a potential strategy for developing new anti-tuberculosis approaches .

How can researchers design experimental approaches to further characterize the molecular function of Rv2206/MT2262?

Advanced characterization approaches should include:

• Structural biology studies:

  • X-ray crystallography or cryo-EM to determine the three-dimensional structure

  • Membrane protein structural analysis techniques for transmembrane domain characterization

• Protein-protein interaction studies:

  • Bacterial two-hybrid systems

  • Co-immunoprecipitation coupled with mass spectrometry

  • Proximity labeling approaches (BioID, APEX)

• Functional genomics:

  • RNA-seq to identify transcriptional changes in Rv2206 mutants

  • ChIP-seq if DNA-binding properties are suspected

  • Conditional depletion systems to study essentiality and temporal requirements

• Cell biology approaches:

  • Live-cell imaging of phagosome dynamics in infected macrophages

  • Super-resolution microscopy to precisely localize the protein within bacterial cells

  • Correlative light and electron microscopy (CLEM) for ultrastructural localization

What methodological considerations are important when using Rv2206/MT2262 recombinant protein for antibody production or interaction studies?

Researchers should consider:

• For antibody production:

  • Select antigenic regions based on computational prediction of surface exposure

  • Consider using fragments rather than the full-length protein due to transmembrane domains

  • Validate antibody specificity using knockout strains as negative controls

• For interaction studies:

  • Maintain appropriate detergent concentrations to preserve native conformation

  • Use mild solubilization conditions that maintain protein-protein interactions

  • Consider incorporating the protein into nanodiscs or liposomes to maintain membrane context

  • Include appropriate controls for non-specific interactions with the His-tag

• For functional reconstitution:

  • Determine optimal lipid composition for reconstitution systems

  • Consider incorporating the protein into supported lipid bilayers for biophysical studies

  • Use careful quality control to ensure proper folding after reconstitution

How does Rv2206/MT2262 research fit into the broader context of M. tuberculosis virulence factor studies?

Rv2206/MT2262 research contributes to our understanding of M. tuberculosis pathogenesis through:

• Expanding knowledge about mechanisms of phagosome maturation arrest, which is critical for intracellular survival

• Adding to the repertoire of known virulence factors that can be targeted for intervention

• Providing insights into membrane-associated proteins that contribute to host-pathogen interactions

• Supporting systems biology approaches to understand the complex networks involved in tuberculosis pathogenesis

Studies of this protein complement other research on secretion systems, cell wall components, and metabolic adaptations that collectively enable M. tuberculosis to establish persistent infection .

What experimental design considerations are important when studying Rv2206/MT2262 in the context of host-pathogen interactions?

Researchers should implement systematic experimental design principles including:

• Define variables clearly:

  • Independent variable: Presence/absence of functional Rv2206/MT2262

  • Dependent variables: Phagosomal markers, bacterial survival, host cell responses

  • Control for extraneous variables like macrophage activation state

• Formulate specific, testable hypotheses about the role of Rv2206/MT2262 in:

  • Phagosomal trafficking

  • Host immune evasion

  • Bacterial survival mechanisms

• Design appropriate experimental treatments:

  • Gene knockout/knockdown vs. complementation

  • Point mutations to identify critical residues

  • Controlled expression systems

• Group assignment considerations:

  • Between-subjects design for comparing different bacterial strains

  • Within-subjects design for time-course experiments

• Measurement protocols:

  • Standardized imaging approaches

  • Quantitative readouts for bacterial survival

  • Appropriate statistical analysis methods