Recombinant Mycobacterium bovis UPF0233 membrane protein JTY_0011 (JTY_0011)

How is recombinant JTY_0011 protein typically expressed and purified?

Recombinant JTY_0011 protein is typically expressed in E. coli expression systems with an N-terminal His-tag to facilitate purification. The expression methodology involves:

• Cloning the JTY_0011 gene (crgA) into an appropriate expression vector

• Transforming the construct into a compatible E. coli strain

• Inducing protein expression under optimized conditions

• Harvesting cells and lysing to release the protein

• Purifying via affinity chromatography using the His-tag

The purified protein is often provided as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE. For storage, it is recommended to reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL and add 5-50% glycerol before aliquoting for long-term storage at -20°C/-80°C .

What are the optimal storage conditions for recombinant JTY_0011 protein?

The optimal storage conditions for recombinant JTY_0011 protein are:

It is important to avoid repeated freeze-thaw cycles as they can compromise protein integrity and activity. Centrifuging vials before opening ensures the contents are at the bottom of the tube. The addition of glycerol helps prevent protein denaturation during freezing .

How can researchers design experiments to investigate JTY_0011's role in membrane protein biogenesis?

To investigate JTY_0011's role in membrane protein biogenesis, researchers should consider a multifaceted experimental approach:

• Gene knockout/knockdown studies:

  • CRISPR-Cas9 or antisense RNA techniques to downregulate JTY_0011 expression

  • Phenotypic analysis focusing on membrane integrity and cell division

  • Complementation studies with wild-type and mutant JTY_0011 variants

• Protein-protein interaction studies:

  • Co-immunoprecipitation with potential interacting partners

  • Bacterial two-hybrid screening

  • Cross-linking studies followed by mass spectrometry

• Localization studies:

  • Fluorescent protein tagging to determine subcellular localization

  • Immunoelectron microscopy to precisely locate the protein within the membrane

• Functional reconstitution:

  • Incorporation of purified JTY_0011 into liposomes

  • Measuring membrane insertion/translocation activities

Since membrane protein biogenesis often involves the Oxa1 and SecY family proteins, experiments should be designed to test whether JTY_0011 interacts with these pathways. The relatively short transmembrane domains of JTY_0011 suggest it might utilize the Oxa1 family for insertion rather than the SecY channel, which typically handles membrane proteins with longer translocated segments .

What methodologies are recommended for analyzing potential contradictions in experimental data regarding JTY_0011 function?

When encountering contradictory data regarding JTY_0011 function, researchers should employ the following methodological approaches:

• Context analysis:

  • Carefully evaluate experimental conditions across studies (expression systems, buffer compositions, assay temperatures)

  • Consider strain-specific differences in M. bovis isolates

  • Assess whether His-tagging or other modifications might affect function in different experimental setups

• Cross-validation with multiple techniques:

  • Employ orthogonal methods to test the same hypothesis

  • Confirm protein-protein interactions using at least three independent methods

  • Validate functional assays with both in vitro and in vivo approaches

• Statistical rigor:

  • Apply appropriate statistical tests, controlling for multiple comparisons

  • Consider Bayesian approaches to reconcile contradictory evidence

  • Use meta-analytical techniques when sufficient studies are available

• Experimental design optimization:

  • Implement factorial designs to systematically explore interaction effects

  • Use time-series experiments to detect temporal dynamics that might explain contradictions

  • Consider the regression-discontinuity design for threshold-dependent behaviors

• Collaborative confirmation:

  • Establish inter-laboratory validation protocols

  • Share reagents and standardized protocols to reduce technical variability

When contradictions persist, they often signal previously unrecognized complexity in the system under study, potentially leading to novel discoveries about JTY_0011's context-dependent functions.

How might JTY_0011 interact with other mycobacterial membrane proteins in co-translational insertion mechanisms?

JTY_0011's interactions with other mycobacterial membrane proteins during co-translational insertion likely follow these mechanistic principles:

• Ribosome-membrane association:

  • JTY_0011 may function as part of a ribosome receptor complex that facilitates membrane-proximal protein synthesis

  • This would allow newly synthesized transmembrane domains to rapidly associate with the membrane, reducing aggregation risk

• Sequential insertion pathway:

  • Given its small size (93 amino acids) and transmembrane nature, JTY_0011 may participate in the insertion of multi-TMD proteins

  • The process would involve successive insertion of TMD pairs as they emerge from the ribosome

  • This mechanism reduces the exposure of hydrophobic domains to the cytosol

• Potential interacting partners:

  Protein Family	Likely Interaction	Functional Significance
  Oxa1 family	Direct interaction	Insertion of TMDs with short translocated segments
  SRP components	Indirect association	Targeting of nascent proteins to the membrane
  SecY complex	Possible crosstalk	Handoff of substrates between insertion pathways

• Regulatory role:

  • As a cell division protein (CrgA), JTY_0011 might coordinate membrane protein insertion with cell division events

  • It may regulate the spatial distribution of newly inserted membrane proteins relative to the division site

Experimental approaches to investigate these interactions should include in vitro reconstitution of the insertion machinery, real-time fluorescence microscopy to track protein movement, and cryo-electron microscopy to visualize the structural complexes formed during co-translational insertion.

What are the best experimental designs to evaluate JTY_0011's potential role in mycobacterial pathogenicity?

To evaluate JTY_0011's potential role in mycobacterial pathogenicity, researchers should implement the following experimental designs:

• Comparative genomics and transcriptomics:

  • Compare JTY_0011 sequence and expression levels between pathogenic and non-pathogenic mycobacterial strains

  • Analyze JTY_0011 expression changes during infection using RNA-seq data

• In vitro infection models:

  • Construct JTY_0011 knockout, knockdown, and overexpression strains

  • Assess phenotypes in macrophage infection assays (attachment, invasion, intracellular replication)

  • Measure cytokine responses in infected host cells

  • Employ the pretest-posttest control group design for robust statistical analysis

• Animal model studies:

  • Use the Solomon four-group design to control for testing effects :

    Group	Pretest	Treatment	Posttest
    R1	O	X	O
    R2	O	-	O
    R3	-	X	O
    R4	-	-	O

    Where R = randomization, O = observation, X = infection with wild-type or modified strain

  • Compare bacterial burden, histopathology, and survival rates between animals infected with wild-type versus JTY_0011-modified strains

• Immune response evaluation:

  • Assess if recombinant BCG expressing modified JTY_0011 variants alters immunogenic properties

  • Measure T-cell responses, antibody production, and protective efficacy

  • Implement time-series experimental designs to track the evolution of immune responses

• Structural biology approaches:

  • Determine if JTY_0011 structural features facilitate host-pathogen interactions

  • Investigate potential binding to host receptors or immune components

How should researchers optimize expression systems for functional studies of JTY_0011?

Optimizing expression systems for functional studies of JTY_0011 requires careful consideration of several parameters:

• Expression host selection:

  • E. coli-based systems offer simplicity but may not provide the proper membrane environment

  • Mycobacterial expression hosts (M. smegmatis) provide more native-like conditions

  • Comparative expression in both systems can reveal host-specific effects

• Vector design considerations:

  Feature	Recommendation	Rationale
  Promoter	Inducible (e.g., T7, tetracycline-responsive)	Allows tight control over expression timing and level
  Fusion tags	C-terminal His-tag or split tags	N-terminal tags may interfere with membrane insertion
  Solubility enhancers	Fusion with MBP or SUMO	May improve solubility of difficult-to-express constructs
  Cleavage sites	TEV or PreScission protease sites	Allows tag removal with minimal residual amino acids

• Expression optimization protocol:

  • Test multiple growth temperatures (18°C, 25°C, 30°C, 37°C)

  • Vary induction timing (early, mid, late log phase)

  • Optimize inducer concentration through titration experiments

  • Consider specialized media formulations for membrane protein expression

• Solubilization and purification strategies:

  • Screen multiple detergents (DDM, LDAO, Triton X-100) for efficient extraction

  • Compare gentle solubilization (native conditions) versus denaturing/refolding approaches

  • Implement multi-step purification (affinity, ion exchange, size exclusion)

  • Validate proper folding through circular dichroism or limited proteolysis

• Functional validation:

  • Develop activity assays specific to hypothesized JTY_0011 functions

  • Include positive and negative controls in all functional assessments

  • Confirm membrane localization through fractionation experiments

By systematically optimizing these parameters, researchers can produce JTY_0011 protein suitable for downstream functional characterization while minimizing artifacts associated with heterologous expression.

What analytical techniques are most appropriate for studying JTY_0011 interactions with membrane lipids?

Studying JTY_0011 interactions with membrane lipids requires specialized analytical techniques that can probe protein-lipid interactions while maintaining a native-like membrane environment:

• Liposome-based binding assays:

  • Prepare liposomes with defined lipid compositions mimicking mycobacterial membranes

  • Measure JTY_0011 binding using:

    • Co-flotation assays with density gradient centrifugation

    • Surface plasmon resonance with immobilized liposomes

    • Microscale thermophoresis for binding kinetics

• Biophysical characterization:

  • Differential scanning calorimetry to detect lipid phase transitions altered by protein binding

  • Fluorescence anisotropy with labeled lipids to measure membrane fluidity changes

  • Solid-state NMR to determine specific lipid-protein contacts

• Advanced microscopy techniques:

  Technique	Application	Advantage
  Atomic force microscopy	Topographical imaging of protein in membranes	Direct visualization of protein organization
  Cryo-electron microscopy	Structural analysis in membrane environment	Near-native conditions with minimal artifacts
  FRET microscopy	Measure protein-lipid proximity	Dynamic measurements in real-time

• Lipid specificity profiling:

  • Lipid overlay assays to screen for specific lipid binding partners

  • Lipidomics analysis of co-purifying lipids with JTY_0011

  • Competition binding assays to determine relative affinities

• Molecular dynamics simulations:

  • In silico modeling of JTY_0011-lipid interactions

  • Prediction of lipid binding sites and energetics

  • Generation of testable hypotheses for experimental validation

• Crosslinking approaches:

  • Photoactivatable lipid analogs to capture transient interactions

  • Mass spectrometry identification of crosslinked residues

  • Mapping of lipid interaction surfaces

By combining multiple analytical approaches, researchers can build a comprehensive understanding of how JTY_0011 interacts with membrane lipids, potentially revealing important functional insights about its role in mycobacterial membrane biology.

How can researchers design experiments to resolve contradictory findings about JTY_0011 topology?

Resolving contradictory findings about JTY_0011 topology requires a systematic approach utilizing complementary experimental techniques:

• Computational prediction refinement:

  • Apply multiple topology prediction algorithms (TMHMM, TOPCONS, Phobius)

  • Create a consensus model based on the outputs of different predictors

  • Identify regions of high confidence versus ambiguous segments

• Cysteine scanning mutagenesis:

  • Generate a comprehensive library of single-cysteine JTY_0011 mutants

  • Test accessibility using membrane-impermeable thiol-reactive reagents

  • Compare accessibility in intact cells versus membrane preparations

  • Data interpretation should follow this decision matrix:

  Reactivity in Intact Cells	Reactivity in Permeabilized Cells	Interpretation
  +	+	Extracellular/periplasmic location
  -	+	Cytoplasmic location
  -	-	Buried within protein or membrane

• Fusion protein reporter assays:

  • Create systematic N- and C-terminal fusions with dual reporters:

    • GFP (folds properly only in cytoplasm)

    • PhoA (active only in periplasm)

  • Quantitative measurement of both reporter activities

  • Iterative truncation to map internal topology transitions

• Protease protection assays:

  • Treatment of membrane preparations with proteases

  • Western blot analysis with domain-specific antibodies

  • Mass spectrometry identification of protected fragments

• Epitope insertion with immuno-detection:

  • Insert epitope tags (FLAG, HA, myc) at predicted loops

  • Differential antibody accessibility tests with and without permeabilization

  • Localization by immunoelectron microscopy for nanometer resolution

• Cross-validation approach:

  • Implement a Solomon four-group design to control for experimental artifacts

  • For each topology mapping technique, include appropriate positive and negative controls

  • Use Bayesian analysis to integrate evidence from multiple experimental approaches

• Structural biology techniques:

  • X-ray crystallography of purified protein (if achievable)

  • Cryo-EM analysis in membrane mimetics

  • Solid-state NMR for topology determination in native-like environments

By implementing this multi-technique approach with rigorous controls, researchers can resolve contradictory findings and establish a consensus model of JTY_0011 topology.

What role might JTY_0011 play in BCG vaccine development and immunological research?

JTY_0011 may have significant potential in BCG vaccine development and immunological research through several mechanisms:

• As an antigen carrier:

  • JTY_0011, as a membrane protein, could serve as a scaffold for presenting foreign antigens

  • Its membrane localization may enhance immune recognition and cross-presentation

  • Recombinant BCG (rBCG) expressing JTY_0011 fusion proteins could elicit robust immune responses against inserted epitopes

• Immunomodulatory functions:

  • JTY_0011 may influence the interaction between BCG and host immune cells

  • Modification of JTY_0011 expression could potentially enhance:

    • Antigen presentation to CD4+ and CD8+ T cells

    • Cross-priming capabilities

    • Major histocompatibility complex class I presentation

• Vaccine vector engineering:

  Modification Strategy	Potential Advantage	Experimental Approach
  JTY_0011 overexpression	Enhanced immunogenicity	Compare immune responses to wild-type vs. overexpressing strains
  Fusion with immunodominant epitopes	Targeted immune response	Measure epitope-specific T cell activation
  Structure-guided mutations	Optimized antigen presentation	Rational design based on membrane topology

• Safety and efficacy considerations:

  • JTY_0011 modifications should be evaluated for:

    • Impact on BCG growth and persistence

    • Safety profile in various models

    • Ability to induce long-lasting immune responses

  • Implementation of heterologous prime-boost strategies (e.g., rBCG-JTY_0011 prime followed by viral vector boost)

• Research applications:

  • JTY_0011 variants could serve as tools to:

    • Study mechanisms of cross-presentation

    • Investigate membrane protein trafficking in antigen-presenting cells

    • Develop improved adjuvant strategies for subunit vaccines

The experimental design for studying JTY_0011's role in vaccination should follow true experimental designs with proper controls and randomization to establish valid causal relationships between JTY_0011 modifications and immunological outcomes .

How can researchers utilize advanced structural biology techniques to characterize JTY_0011?

Characterizing the structure of JTY_0011 requires integrating multiple advanced structural biology techniques that address the challenges inherent to membrane proteins:

By integrating data from multiple techniques, researchers can overcome the challenges associated with membrane protein structural determination and build a comprehensive structural model of JTY_0011 that informs understanding of its function in mycobacterial biology.

What methodologies can researchers employ to study JTY_0011's role in mycobacterial cell division?

To elucidate JTY_0011's role in mycobacterial cell division, researchers should employ a comprehensive set of methodologies spanning genetic, biochemical, and imaging approaches:

• Genetic manipulation strategies:

  • Conditional knockdown systems (tetracycline-regulatable, CRISPRi)

  • CRISPR/Cas9-mediated knockout with complementation

  • Site-directed mutagenesis of key residues

  • Fluorescent protein tagging for localization

• Cell division phenotype characterization:

  • Time-lapse microscopy with microfluidic devices

  • Electron microscopy to visualize septum formation

  • Flow cytometry to analyze DNA content and cell size

  • Quantitative image analysis using specialized software

• Protein-protein interaction mapping:

  Method	Advantage	Application
  Bacterial two-hybrid	In vivo screening	Identify novel interaction partners
  Co-immunoprecipitation	Captures native complexes	Verify interactions in mycobacteria
  BioID proximity labeling	Identifies transient interactions	Map JTY_0011's interaction network
  FRET microscopy	Real-time dynamics	Visualize interactions during division

• Spatiotemporal dynamics:

  • Fluorescence recovery after photobleaching (FRAP) to measure protein mobility

  • Single-molecule tracking to determine diffusion properties

  • Correlative light and electron microscopy to integrate functional and structural data

  • Super-resolution microscopy (STORM, PALM) to visualize nanoscale organization

• Biochemical activity characterization:

  • In vitro reconstitution of division complexes

  • ATPase/GTPase activity assays of associated division proteins

  • Lipid binding and membrane remodeling assays

  • Structural studies of JTY_0011 in complex with division partners

• Systems biology approaches:

  • Transcriptomics of JTY_0011 mutants to identify compensatory mechanisms

  • Proteomics to detect changes in the divisome composition

  • Metabolomics to identify effects on cell wall synthesis

  • Network analysis to position JTY_0011 in the division pathway

• Experimental design considerations:

  • Implement time-series experiments to capture the dynamic process of cell division

  • Use multiple time-series design with control strains for comparative analysis

  • Apply regression-discontinuity analysis for threshold-dependent phenotypes

By integrating these methodologies, researchers can build a comprehensive understanding of JTY_0011's role in mycobacterial cell division, potentially revealing new targets for antimycobacterial therapy development.

How should researchers address potential data inconsistencies in JTY_0011 functional studies?

Addressing data inconsistencies in JTY_0011 functional studies requires a systematic approach to identify sources of variation and implement appropriate analytical solutions:

• Source identification:

  • Technical variability (expression systems, purification methods, assay conditions)

  • Biological variability (strain differences, growth phase, media composition)

  • Methodological differences (detection limits, equipment sensitivity)

  • Data analysis approaches (normalization methods, statistical tests)

• Structured analytical framework:

  • Implement a hierarchical experimental design to partition variance components

  • Distinguish between within-experiment and between-experiment variability

  • Apply mixed-effects statistical models to account for nested data structures

  • Use meta-analytical techniques to integrate findings across studies

• Validation and cross-verification strategies:

  Strategy	Implementation	Outcome
  Independent replication	Repeat key experiments in different laboratories	Confirms reproducibility
  Orthogonal methods	Test hypotheses using different techniques	Verifies technical robustness
  Positive/negative controls	Include established controls in all experiments	Validates assay performance
  Dose-response relationships	Test across concentration ranges	Establishes biological relevance

• Statistical approaches for reconciling contradictions:

  • Bayesian analysis to incorporate prior knowledge and update with new evidence

  • Sensitivity analysis to assess the impact of varying assumptions

  • Power analysis to ensure adequate sample sizes

  • Non-parametric methods when distributional assumptions are violated

• Systematic reporting:

  • Document all experimental conditions in detail

  • Report negative and inconclusive results alongside positive findings

  • Provide raw data and analysis scripts for transparency

  • Apply structured formats for sharing methods and materials

• Collaborative resolution:

  • Establish consortia to standardize protocols across laboratories

  • Implement round-robin testing of key hypotheses

  • Develop common reagents and reference materials

By implementing this comprehensive approach, researchers can distinguish genuine biological complexities of JTY_0011 function from technical artifacts, leading to more robust and reproducible findings in the field.

What statistical methods are most appropriate for analyzing complex datasets from JTY_0011 experiments?

Analyzing complex datasets from JTY_0011 experiments requires sophisticated statistical approaches tailored to the specific experimental designs and data types:

• For time-series experiments:

  • Repeated measures ANOVA for balanced designs

  • Linear mixed-effects models for handling missing data

  • Time-series analysis techniques including:

    • Autoregressive integrated moving average (ARIMA) models

    • Generalized additive models (GAMs) for non-linear trends

    • Change-point detection for identifying critical transitions

• For high-dimensional data:

  • Principal component analysis (PCA) for dimensionality reduction

  • Partial least squares (PLS) regression for linking multiple data types

  • Clustering methods (hierarchical, k-means, DBSCAN) for pattern identification

  • Random forest and other machine learning approaches for predictive modeling

• For interaction network analysis:

  Method	Application	Advantage
  Graph-theoretic metrics	Network topology characterization	Identifies key hub proteins
  Community detection	Functional module identification	Reveals coordinated processes
  Bayesian networks	Causal relationship inference	Models directional influences
  Differential network analysis	Condition-specific changes	Highlights context-dependent interactions

• For multi-level experimental designs:

  • Nested ANOVA for hierarchical sampling designs

  • Mixed-effects models incorporating both fixed and random effects

  • Generalized estimating equations (GEE) for correlated data

  • Multi-level Bayesian models for complex hierarchical structures

• For integrating heterogeneous data types:

  • Meta-analysis techniques for combining effect sizes across studies

  • Bayesian data integration frameworks

  • Multi-block statistical methods (DIABLO, MOFA)

  • Network-based data integration approaches

• Specialized approaches for specific data types:

  • Zero-inflated models for count data with excess zeros

  • Survival analysis for time-to-event data

  • Beta regression for proportion data

  • Circular statistics for periodic/angular measurements

• Multiple testing correction:

  • False discovery rate (FDR) control using Benjamini-Hochberg procedure

  • Family-wise error rate (FWER) control using Bonferroni or Holm method

  • Permutation-based significance testing for complex dependencies

Implementation should include appropriate validation steps such as cross-validation, bootstrapping, or independent test sets to ensure the robustness of findings and prevent overfitting to noise in the data.