Recombinant Mycobacterium tuberculosis UPF0233 membrane protein MRA_0013 (MRA_0013)

What is MRA_0013 and what is its role in Mycobacterium tuberculosis?

MRA_0013 belongs to the UPF0233 family of membrane proteins found in Mycobacterium tuberculosis. The UPF (Uncharacterized Protein Family) designation indicates limited functional characterization to date. Based on comparative analysis with other membrane proteins in mycobacteria, MRA_0013 likely participates in cellular processes related to bacterial survival, membrane integrity, or pathogenesis.

The functional characterization of MRA_0013 would typically involve multiple complementary approaches:

• Comparative genomic analysis across mycobacterial species

• Transcriptomic profiling under various growth conditions

• Loss-of-function studies using CRISPR interference (CRISPRi)

• Protein-protein interaction studies to identify binding partners

• Localization studies to determine membrane distribution

Similar to research approaches for other membrane proteins, CRISPRi techniques can be particularly valuable for studying MRA_0013 function through targeted gene repression .

What expression systems are most suitable for recombinant MRA_0013 production?

Several expression systems can be considered for recombinant production of MRA_0013, each with distinct advantages:

For mycobacterial membrane proteins, the rBCG (recombinant Bacillus Calmette-Guérin) system has shown particular promise for maintaining native protein conformation. This approach has been successfully used for expressing other mycobacterial proteins and could be adapted for MRA_0013 expression .

The choice of expression system should be guided by downstream applications. For structural studies requiring high purity, E. coli systems may be preferable, while for functional studies, mycobacterial expression systems provide a more native-like environment that preserves proper folding and post-translational modifications .

How can I verify successful expression of recombinant MRA_0013?

Verification of successful MRA_0013 expression requires a multi-faceted approach:

• Transcriptional verification: Quantitative PCR (qPCR) can confirm expression at the mRNA level before proceeding to protein detection, similar to approaches used for verifying expression of other membrane proteins .

• Protein detection: Western blotting using antibodies against epitope tags (His, FLAG, etc.) incorporated into the recombinant construct. For untagged constructs, custom antibodies against MRA_0013 would be necessary.

• Mass spectrometry verification: SDS-PAGE followed by mass spectrometry to confirm protein identity and integrity. This approach can also identify any post-translational modifications.

• Membrane localization confirmation: Membrane fractionation followed by Western blotting to verify proper targeting to the membrane fraction. This is essential for confirming proper folding and insertion of membrane proteins.

• Functional assays: If molecular function becomes known, specific activity assays can verify not just expression but also proper folding and activity.

When designing expression constructs, careful consideration should be given to tag placement, as N-terminal or C-terminal tags may interfere with membrane insertion or protein function .

What purification strategies work best for recombinant MRA_0013?

Purification of membrane proteins like MRA_0013 presents specific challenges requiring specialized approaches:

• Membrane extraction: Selection of appropriate detergents is critical for solubilization while maintaining protein structure and function. Common detergents include:

  • n-Dodecyl β-D-maltoside (DDM)

  • n-Octyl β-D-glucopyranoside (OG)

  • Digitonin (for gentle extraction)

  • CHAPS (zwitterionic detergent)

• Affinity chromatography: His-tag purification is commonly employed as the first step, but buffer composition must be optimized to maintain protein stability. Inclusion of glycerol (10-20%) and appropriate detergent concentrations is essential.

• Size exclusion chromatography: This serves as a critical second purification step to obtain homogeneous protein preparations and remove aggregates.

• Alternative membrane mimetics: Transition from detergents to more stable environments:

  • Nanodiscs for a native-like lipid bilayer environment

  • Amphipols for enhanced stability

  • Lipid cubic phase for crystallization attempts

Detergent-resistant membrane fractions have been successfully used for purifying certain membrane proteins, and similar approaches might be adapted for mycobacterial membrane proteins like MRA_0013, with consideration for the different membrane composition of mycobacteria .

How do I assess the stability and quality of purified recombinant MRA_0013?

Multiple complementary techniques should be employed to comprehensively assess the stability and quality of purified MRA_0013:

• Thermal shift assays: These determine protein thermal stability under different buffer conditions, detergents, or stabilizing additives. The melting temperature (Tm) provides a quantitative measure of stability.

• Dynamic light scattering (DLS): This non-destructive technique assesses the homogeneity of protein preparations and can detect aggregation. Monodisperse samples typically indicate well-folded protein.

• Circular dichroism (CD) spectroscopy: This provides information about secondary structure content, confirming proper folding. For membrane proteins, CD can verify the alpha-helical content expected in transmembrane domains.

• Limited proteolysis: Controlled digestion with proteases can identify stable, well-folded domains that resist proteolysis. The digestion pattern can be analyzed by SDS-PAGE or mass spectrometry.

• Analytical ultracentrifugation: This determines the oligomeric state and homogeneity of the protein preparation.

For membrane proteins, environment-sensitive probes can assess proper integration into membrane mimetics, similar to approaches used for other membrane proteins .

What approaches can be used to study MRA_0013 localization in the mycobacterial membrane?

Several sophisticated techniques can be applied to study MRA_0013 localization and dynamics:

• Fluorescent protein fusions: Creating fusions with fluorescent proteins (GFP, mCherry) allows visualization in live bacteria. Care must be taken to ensure the fusion doesn't disrupt protein function or localization.

• Super-resolution microscopy: Techniques that overcome the diffraction limit provide detailed localization information:

  • Stimulated emission depletion (STED) microscopy

  • Photoactivated localization microscopy (PALM)

  • Single-molecule localization microscopy (SMLM)

• Immunogold electron microscopy: This provides nanometer-scale resolution for precise localization within the complex mycobacterial cell envelope.

• Single-particle tracking: This reveals protein dynamics within the membrane, including diffusion rates and potential confinement in specific domains.

• Environment-sensitive probes: These can detect the protein's association with specific membrane environments, similar to techniques used to study plant plasma membrane proteins .

Advanced imaging studies can determine whether MRA_0013 is distributed uniformly or segregated into functional domains within the membrane, similar to the nanodomain organization observed in plant membrane proteins .

How can I develop a CRISPRi system to study MRA_0013 function in Mycobacterium tuberculosis?

Developing a CRISPRi system for studying MRA_0013 function requires a systematic approach:

• Generation of a stable M. tuberculosis strain expressing dCas9-KRAB: Create a strain expressing the catalytically inactive Cas9 fused to a transcriptional repressor domain, similar to the approach described for human cell lines .

• sgRNA design for MRA_0013: Design multiple sgRNAs targeting the promoter region or transcription start site (TSS). Based on documented CRISPRi approaches, designing sgRNAs that target the proximity of the TSS is critical for effective gene repression .

• Validation of knockdown efficiency: Perform qPCR analysis to assess the efficiency of different sgRNAs. As demonstrated in other CRISPRi systems, significant variation in knockdown efficiency can occur between different sgRNAs targeting the same gene .

• Off-target analysis: Conduct in silico analysis using tools like Cas-OFFinder to identify potential off-targets. This is essential for confirming the specificity of the CRISPRi approach .

• Phenotypic analysis: Perform assays to assess the impact of MRA_0013 knockdown on bacterial growth, survival under stress conditions, and virulence in infection models.

The development of inducible CRISPRi systems allows for temporal control of gene knockdown, which is particularly valuable for studying essential genes or for time-course experiments .

What techniques are most suitable for studying MRA_0013 interactions with other proteins?

Several complementary techniques can be employed to comprehensively characterize protein-protein interactions involving MRA_0013:

• Co-immunoprecipitation (Co-IP): This identifies interacting protein partners in a near-native context. For membrane proteins like MRA_0013, specialized detergents that preserve protein-protein interactions should be used.

• Crosslinking mass spectrometry: This identifies interaction interfaces and is particularly valuable for membrane proteins. Chemical crosslinkers with different spacer lengths can capture both direct and proximal interactions.

• Bacterial two-hybrid systems: These are useful for screening potential interacting partners in a bacterial context, though may be challenging for full-length membrane proteins.

• Surface plasmon resonance (SPR): This provides quantitative binding information including association and dissociation rates. For membrane proteins, specialized sensor chips containing lipid bilayers can be utilized.

• Biolayer interferometry (BLI): This provides real-time binding data with minimal protein consumption, making it suitable for difficult-to-express membrane proteins.

• Protein-lipid interactions: Lipidomic analysis could reveal interactions with specific lipids, which may be essential for proper function. This approach has been used successfully for other membrane proteins that co-purify with specific lipids like sterols and phosphoinositides .

The combination of multiple interaction detection methods provides the most comprehensive and reliable characterization of protein-protein interactions.

How can I analyze the role of MRA_0013 in Mycobacterium tuberculosis pathogenesis?

Analyzing the role of MRA_0013 in pathogenesis requires a multi-faceted approach spanning in vitro and in vivo models:

• In vitro infection models:

  • Macrophage infection assays comparing wild-type versus MRA_0013 knockdown strains

  • Analysis of bacterial survival, growth, and host cell responses

  • Evaluation of cytokine responses and inflammasome activation

• Animal models:

  • Infection studies in mice using wild-type versus modified strains

  • Measurement of bacterial burden in lungs and other organs

  • Histopathological assessment of tissue damage

  • Survival studies to assess virulence

• Immune response analysis:

  • Characterization of T cell responses using flow cytometry

  • Analysis of memory T cell populations (TCM, TEM, TRM)

  • Cytokine profiling to assess Th1/Th17 polarization

Based on approaches used in M. tuberculosis vaccine research, analyzing T cell responses could include flow cytometry characterization of:

• Polyfunctional T cells producing multiple cytokines

• Central memory T cells (TCM)

• Effector memory T cells (TEM)

• Resident memory T cells (TRM)

• Host-pathogen interaction studies:

  • Identification of host factors that interact with MRA_0013

  • Impact on phagosomal maturation and survival

These approaches provide comprehensive assessment of how MRA_0013 contributes to pathogenesis and modulates host immune responses .

What structural analysis techniques are appropriate for determining the structure of MRA_0013?

Several complementary techniques can be applied to determine the structure of membrane proteins like MRA_0013:

A combined approach using multiple structural techniques typically provides the most comprehensive structural understanding of membrane proteins .

What are the common challenges in working with recombinant MRA_0013 and how can they be addressed?

Working with membrane proteins like MRA_0013 presents several challenges that require specific strategies:

• Low expression yields:

  • Solution: Optimize codon usage for mycobacterial expression

  • Test different promoters and induction conditions

  • Use specialized strains designed for membrane protein expression

  • Consider fusion partners such as MBP or SUMO that enhance solubility

• Protein aggregation:

  • Solution: Screen multiple detergents and concentrations

  • Use milder extraction conditions to prevent denaturation

  • Consider nanodiscs or amphipols for stabilization

  • Include stabilizing additives like glycerol or specific lipids

• Loss of function during purification:

  • Solution: Validate function at each purification step

  • Use detergent-free extraction methods when possible

  • Reconstitute into liposomes to restore native environment

  • Minimize exposure to harsh conditions

• Difficulty in crystallization:

  • Solution: Use lipidic cubic phase crystallization

  • Try antibody fragment co-crystallization

  • Consider fusion with crystallization chaperones

  • Use surface entropy reduction mutations

• Limited stability of purified protein:

  • Solution: Optimize buffer conditions (pH, salt, additives)

  • Include specific lipids that might stabilize the protein

  • Store at optimal temperature with appropriate protease inhibitors

Approaches similar to those used for expressing other challenging membrane proteins can be adapted for MRA_0013 .

How can I design experiments to determine if MRA_0013 is essential for Mycobacterium tuberculosis survival?

Determining the essentiality of MRA_0013 requires carefully designed genetic approaches:

• Conditional knockdown systems:

  • CRISPRi with inducible promoters controlling dCas9 or sgRNA expression

  • Tetracycline-responsive repression systems

  • Degradation tag systems for protein-level depletion

• Transposon mutagenesis:

  • Transposon insertion sequencing (TnSeq)

  • Analysis of insertion patterns relative to gene essentiality models

  • Conditional essentiality testing under different growth conditions

• Targeted gene replacement:

  • Attempt to create a clean deletion with complementation

  • Merodiploid approach with second copy before deletion attempt

  • If deletion is only possible with complementation, this suggests essentiality

• Growth kinetics analysis:

  • Monitor growth rates after protein depletion

  • Assess survival under different stress conditions

  • Evaluate recovery potential after temporary depletion

Based on approaches used in other studies, a CRISPRi system would allow for titratable repression of MRA_0013 to determine the threshold levels required for survival and to characterize phenotypes resulting from partial depletion .

How can I assess the impact of MRA_0013 on Mycobacterium tuberculosis response to oxidative stress?

Based on research showing that some membrane proteins affect oxidative stress responses, similar approaches could be applied to study MRA_0013's role:

• ROS measurement assays:

  • Dihydroethidium (DHE) or similar dyes to measure total ROS levels

  • Specific probes for different ROS species (H2O2, superoxide)

  • Comparison between wild-type and MRA_0013 knockdown strains

• Oxidative stress challenge experiments:

  • Expose strains to H2O2, cumene hydroperoxide, or other oxidants

  • Assess survival, growth rates, and recovery kinetics

  • Determine minimum inhibitory concentrations of oxidants

• Gene expression analysis:

  • RNA-seq or qPCR to analyze expression of oxidative stress response genes

  • Compare transcriptional responses between wild-type and MRA_0013-modified strains

  • Identify differentially regulated pathways

• Protein oxidation assessment:

  • OxyBlot or mass spectrometry to detect protein carbonylation

  • Redox proteomics to identify oxidation-sensitive proteins

  • Thiol oxidation state analysis

Similar to studies of TMEM97 knockdown effects on oxidative stress, comparison of ROS levels between control and MRA_0013-depleted bacteria under basal and stress conditions could reveal protective or sensitizing effects .

Through these comprehensive approaches, the specific contribution of MRA_0013 to oxidative stress resistance can be elucidated.

How should I interpret conflicting results when studying MRA_0013 function?

When confronted with conflicting results in MRA_0013 research, a systematic troubleshooting approach is essential:

• Consider context differences:

  • Different M. tuberculosis strain backgrounds may show genetic interactions

  • Growth media composition can significantly alter phenotypes

  • In vitro versus in vivo settings may yield different results

  • Environmental conditions (oxygen tension, pH, etc.) may affect outcomes

• Methodological validation:

  • Use multiple independent techniques to assess the same parameter

  • Include appropriate positive and negative controls for each method

  • Evaluate the sensitivity and specificity of each assay

  • Consider whether detection limits or dynamic ranges differ between methods

• Genetic complementation:

  • Ensure phenotypes can be rescued by wild-type gene expression

  • Use site-directed mutants to pinpoint critical residues

  • Create point mutations rather than wholesale deletions when possible

• Sequence verification:

  • Confirm the sequence of the MRA_0013 gene in your working strains

  • Check for suppressor mutations that might arise during manipulation

  • Verify constructs used for complementation or protein expression

• Collaborative validation:

  • Have key experiments replicated in independent laboratories

  • Use different methodological approaches to address the same question

Drawing from approaches used in membrane protein research, contradictory findings often reflect the complexity of membrane protein biology and may require multiple complementary techniques to resolve .

What statistical approaches are most appropriate for analyzing experimental data on MRA_0013?

Selection of appropriate statistical methods is critical for robust data analysis:

• For comparing two experimental groups:

  • Student's t-test for normally distributed data

  • Mann-Whitney U test for non-parametric data

  • Paired tests when appropriate (e.g., before/after treatments)

• For multiple group comparisons:

  • One-way ANOVA followed by post-hoc tests (Tukey, Bonferroni)

  • Two-way ANOVA for experiments with two factors

  • Kruskal-Wallis test for non-parametric data with multiple groups

• For time-course experiments:

  • Repeated measures ANOVA for normally distributed data

  • Mixed-effects modeling for complex experimental designs

  • Area under the curve (AUC) analysis for growth curves

• For survival studies:

  • Kaplan-Meier analysis with log-rank test

  • Cox proportional hazards models for multivariate analysis

• For correlation analyses:

  • Pearson correlation for linear relationships with normal distribution

  • Spearman correlation for non-parametric or non-linear relationships

• For complex datasets:

  • Principal component analysis for dimensionality reduction

  • Cluster analysis to identify patterns

  • Machine learning approaches for complex data integration

Similar statistical approaches to those used in studies examining the protection conferred by vaccine candidates could be appropriate, such as those used in the rBCG-LTAK63 vaccine research .

How can computational approaches advance our understanding of MRA_0013?

Computational methods offer powerful tools for MRA_0013 research across multiple dimensions:

• Structural prediction and analysis:

  • Homology modeling based on related membrane proteins

  • Ab initio modeling for unique structural elements

  • Molecular dynamics simulations to study dynamics in a lipid bilayer

  • Identification of potential binding pockets or functional sites

• Functional prediction:

  • Identification of conserved domains and motifs

  • Prediction of transmembrane topology

  • Identification of potential post-translational modification sites

  • Metabolic pathway integration analysis

• Systems biology approaches:

  • Network analysis to place MRA_0013 in biological context

  • Integration with transcriptomic, proteomic, and metabolomic data

  • Flux balance analysis to predict metabolic impacts

  • Gene regulatory network analysis

• Evolutionary analysis:

  • Comparative genomics across mycobacterial species

  • Selection pressure analysis to identify functionally important regions

  • Identification of co-evolving proteins that may be functional partners

  • Horizontal gene transfer assessment

• Drug discovery applications:

  • Virtual screening for potential inhibitors

  • Binding site analysis and druggability assessment

  • Pharmacophore modeling for rational drug design

  • Prediction of resistance-conferring mutations

Similar to computational approaches used for off-target prediction in CRISPRi studies , bioinformatic methods can provide valuable insights while guiding experimental design for MRA_0013 research.

How might understanding MRA_0013 function contribute to tuberculosis vaccine development?

Research on MRA_0013 could impact TB vaccine development through several mechanisms:

• As a potential antigen candidate:

  • If MRA_0013 is surface-exposed, it could serve as an antigen target

  • Recombinant protein vaccines incorporating MRA_0013 epitopes

  • DNA vaccines encoding immunogenic regions of MRA_0013

• As a genetic adjuvant component:

  • Similar to the LTAK63 adjuvant approach described in TB vaccine research

  • Modification of MRA_0013 expression in attenuated vaccine strains

  • Co-expression with immunostimulatory molecules

• As a virulence modulator:

  • If involved in pathogenesis, attenuation through MRA_0013 modification

  • Creation of balanced attenuation for safety while maintaining immunogenicity

  • Development of auxotrophic strains dependent on exogenous factors

• For improved immunological memory:

  • If MRA_0013 affects immune response patterns, its modification could enhance memory T cell generation

  • Similar to how LTAK63 expression improved the generation of central memory T cells (TCM) and effector memory T cells (TEM)

The research on rBCG-LTAK63 vaccine demonstrates how recombinant BCG strains can enhance immune responses and improve protection against M. tuberculosis challenge, suggesting similar approaches could be applied using MRA_0013 modifications .

What role might MRA_0013 play in Mycobacterium tuberculosis drug resistance?

Potential involvement of MRA_0013 in drug resistance could occur through several mechanisms:

• Membrane permeability alterations:

  • If MRA_0013 affects membrane structure or composition

  • Could influence drug penetration into the cell

  • Might alter membrane fluidity or organization

• Stress response modulation:

  • If involved in stress responses similar to roles described for other membrane proteins

  • Could contribute to bacterial survival during drug exposure

  • Might activate adaptive responses to antibiotic pressure

• Biofilm formation:

  • If involved in surface properties or cell-cell interactions

  • Could contribute to biofilm-associated resistance

  • Might affect cell envelope remodeling during biofilm formation

• Signaling pathway involvement:

  • If part of stress sensing or signaling pathways

  • Could trigger compensatory mechanisms during drug exposure

  • Might coordinate multiple resistance mechanisms

Research approaches similar to those used for studying membrane protein roles in stress responses could be adapted to investigate MRA_0013's potential role in drug resistance mechanisms .