Recombinant Uncharacterized protein Rv2658c/MT2734.1 (Rv2658c, MT2734.1)

What is Rv2658c/MT2734.1 and why is it significant in tuberculosis research?

Rv2658c/MT2734.1 is an uncharacterized protein from Mycobacterium tuberculosis, the causative agent of tuberculosis. This protein is of interest because:

• It belongs to the proteome of M. tuberculosis, a pathogen responsible for a significant global health burden

• Uncharacterized proteins may represent novel drug targets or vaccine candidates

• Understanding its function could provide insights into M. tuberculosis pathogenesis and survival mechanisms

M. tuberculosis is an obligate aerobic organism with an optimum growth temperature of 37°C, and it cannot grow below 30°C. The bacterium can invade multiple organs, with pulmonary tuberculosis being the most common manifestation. Understanding proteins like Rv2658c may reveal mechanisms that contribute to the bacterium's survival during infection .

How is Rv2658c annotated in different M. tuberculosis reference genomes?

The protein is annotated as:

• Rv2658c in the H37Rv reference strain

• MT2734.1 in the CDC1551 strain of M. tuberculosis

This dual nomenclature reflects different annotation systems used for these reference genomes. The "c" in Rv2658c indicates that the gene is encoded on the complementary strand of DNA .

What experimental approaches are most effective for initial characterization of Rv2658c?

For initial characterization of an uncharacterized protein like Rv2658c, implement a multi-faceted approach:

These approaches should be conducted in parallel to develop a comprehensive understanding of Rv2658c function .

How can researchers address analytical contradictions when studying Rv2658c?

When encountering contradictory results:

• Apply the Same Analysis Approach (SAA) methodology:

  • Type 1: Apply the same analysis method to variables of the experimental design

  • Type 2: Apply the same analysis to empirical control data

  • Type 3: Apply the same analysis to simulated null data

• Identify potential sources of error:

  • Mismatch between experimental design and analysis methods

  • Counterbalancing issues in crossover designs

  • Linear decoding of nonlinear effects

• Implement positive and negative control analyses:

  • Positive controls test if design variables that should influence outcomes yield significant results

  • Negative controls test if variables that should not influence outcomes do not yield significant results

• Document all conditions systematically:

  • Maintain detailed records of experimental conditions

  • Report both positive and negative findings

  • Distinguish clearly between data and interpretation

What expression systems are optimal for recombinant production of Rv2658c?

Several expression systems can be considered for Rv2658c production:

According to available information, E. coli is commonly used for initial attempts at expressing mycobacterial proteins, with sources including E. coli, yeast, baculovirus, or mammalian cell systems .

What purification challenges are specific to Rv2658c and how can they be addressed?

While specific challenges for Rv2658c are not detailed in the available literature, mycobacterial proteins often present the following purification challenges:

• Solubility issues:

  • Use solubility-enhancing tags (MBP, SUMO, Trx)

  • Optimize buffer conditions (pH, salt concentration, additives)

  • Consider mild detergents for membrane-associated proteins

• Stability concerns:

  • Include stabilizing agents (glycerol, arginine, trehalose)

  • Maintain reducing conditions if cysteine residues are present

  • Consider on-column refolding protocols

• Purity verification:

  • Employ SDS-PAGE and western blotting for identity confirmation

  • Use mass spectrometry for molecular weight verification

  • Apply dynamic light scattering to assess aggregation state

For recombinant Rv2658c specifically, sources indicate it can be produced from E. coli or alternative expression systems, suggesting that standard affinity purification approaches may be applicable .

How can transcriptomic data inform our understanding of Rv2658c function?

Transcriptomic analysis can provide valuable insights into Rv2658c function:

• Expression correlation analysis:

  • Identify genes co-expressed with Rv2658c

  • Map these to known functional pathways

  • Infer potential involvement in specific cellular processes

• Condition-dependent expression:

  • Analyze expression during:

    • In vitro stress conditions (starvation, hypoxia, acid stress)

    • Macrophage infection

    • Animal model infection stages

  • Compare to expression patterns of genes with known functions

• Regulatory network analysis:

  • Identify potential transcription factors regulating Rv2658c

  • Map the gene to known regulons in M. tuberculosis

Drawing from studies of other M. tuberculosis genes, researchers should be cautious about transcript orientation and potential small RNAs. For example, the study of Rv2660c revealed that the upregulated transcript during starvation was actually a small RNA encoded on the opposite strand, not the protein-coding gene as initially thought .

What approaches can determine if Rv2658c plays a role in M. tuberculosis drug resistance?

To investigate potential involvement in drug resistance:

The increasing prevalence of multi-resistant strains of M. tuberculosis globally highlights the importance of understanding all potential resistance mechanisms, including uncharacterized proteins that might contribute to resistance .

What computational methods can predict the structure of Rv2658c in the absence of experimental data?

In the absence of experimental structures, several computational approaches can predict Rv2658c structure:

• Homology modeling:

  • Identify templates through sensitive sequence searches (HHpred, HMMER)

  • Generate models using programs like MODELLER or SWISS-MODEL

  • Validate using energy minimization and Ramachandran plot analysis

• Ab initio and deep learning methods:

  • AlphaFold2 for high-confidence predictions

  • RoseTTAFold as an alternative approach

  • I-TASSER for threading-based modeling

• Integrative modeling:

  • Combine predictions with sparse experimental data

  • Use coevolutionary information to predict contacts

  • Apply molecular dynamics simulations for refinement

• Functional site prediction:

  • Identify conserved residues through multiple sequence alignment

  • Predict binding pockets using CASTp or COACH

  • Map conservation onto the structural model using ConSurf

These approaches can provide a starting point for structure-function hypotheses that can be tested experimentally.

What experimental structures would provide the most informative comparisons for Rv2658c?

When analyzing predicted or experimental structures of Rv2658c, comparing to these structural classes would be most informative:

• Structures of other M. tuberculosis proteins with similar predicted secondary structure profiles

• Proteins involved in mycobacterial cell wall synthesis or modification

• Structures of stress response proteins from related organisms

• Proteins involved in dormancy or persistence mechanisms

• Structures of proteins with similar domain architectures, even from distant organisms

How might Rv2658c contribute to M. tuberculosis survival during infection?

Several potential roles for Rv2658c in M. tuberculosis pathogenesis can be hypothesized:

• Stress response:

  • Adaptation to nutrient limitation during infection

  • Response to oxidative or nitrosative stress in macrophages

  • Adaptation to hypoxic conditions in granulomas

• Cell wall modulation:

  • Contribution to cell envelope integrity

  • Modification of cell surface to evade immune recognition

  • Alteration of permeability to antibiotics or host factors

• Metabolic adaptation:

  • Role in alternative metabolic pathways during persistence

  • Contribution to utilization of host-derived nutrients

  • Involvement in energy conservation during dormancy

• Regulatory functions:

  • Potential role as a transcriptional or post-transcriptional regulator

  • Signal transduction during host-pathogen interaction

M. tuberculosis is known to adapt to various stress conditions during infection, including nutrient starvation, which has been shown to induce expression of certain genes. Studies of similar uncharacterized proteins have revealed important roles in survival and persistence .

What is known about Rv2658c immunogenicity and potential as a vaccine component?

While specific information about Rv2658c immunogenicity is limited in the provided sources, assessment as a vaccine candidate would involve:

• Antigenicity evaluation:

  • In silico epitope prediction

  • T-cell stimulation assays with synthetic peptides

  • B-cell epitope mapping

• Conservation analysis:

  • Sequence conservation across clinical isolates

  • Presence in BCG and other vaccine strains

  • Absence in environmental mycobacteria

• Safety assessment:

  • Homology to human proteins

  • Potential molecular mimicry concerns

  • Cross-reactivity with commensal microbiota

• Immunological properties:

  • Ability to induce Th1/Th17 responses

  • Memory T-cell generation

  • Protective efficacy in animal models

Recombinant proteins from M. tuberculosis, including uncharacterized proteins like Rv2658c, are being investigated for vaccine development, though their use is currently limited to research purposes as noted in source materials .

How can CRISPR-based approaches advance our understanding of Rv2658c function?

CRISPR technologies offer powerful tools for studying Rv2658c:

• CRISPRi (interference) applications:

  • Tunable knockdown of Rv2658c expression

  • Temporal control of repression to study essentiality at different growth phases

  • Multiplexed targeting to study genetic interactions

• CRISPR-Cas9 genome editing:

  • Clean deletion or modification of Rv2658c

  • Introduction of point mutations to test specific residues

  • Insertion of reporter tags at the endogenous locus

• CRISPRa (activation) approaches:

  • Overexpression of Rv2658c from its native locus

  • Study of dose-dependent phenotypes

  • Combination with stress conditions to identify synthetic phenotypes

• CRISPR-based screening:

  • Library-scale assessment of conditions where Rv2658c becomes essential

  • Identification of genetic interactions through double-knockdown screens

These approaches overcome many limitations of traditional genetic manipulation in slow-growing mycobacteria.

What protein-protein interaction methods are most suitable for studying Rv2658c in its native context?

To study Rv2658c interactions in a near-native context:

For a protein like Rv2658c with unknown function, employing multiple complementary methods is recommended to build confidence in the identified interactions.