Recombinant Mycoplasma pneumoniae Uncharacterized protein MPN_143 (MPN_143)

What is Recombinant Mycoplasma pneumoniae Uncharacterized protein MPN_143?

Recombinant Mycoplasma pneumoniae Uncharacterized protein MPN_143 is a protein expressed through molecular cloning techniques where the gene encoding MPN_143 is inserted into an expression vector and transfected into host cells (typically E. coli). Based on similar recombinant M. pneumoniae proteins, MPN_143 would likely be expressed with affinity tags (such as N-terminal His-tag and C-terminal Myc-tag) to facilitate purification from cell lysates through affinity chromatography, achieving purity levels typically greater than 85% as measured by SDS-PAGE .

What expression systems are most suitable for producing MPN_143?

For laboratory-scale production of MPN_143, E. coli expression systems are commonly employed due to their cost-effectiveness and high yield. The typical methodology involves:

• Cloning the MPN_143 gene into an expression vector containing appropriate promoters and tags

• Transforming the construct into an E. coli strain optimized for protein expression

• Inducing protein expression under controlled conditions

• Harvesting cells and lysing to release the recombinant protein

• Purifying via affinity chromatography using the engineered tags

For structural studies requiring eukaryotic post-translational modifications, alternative systems such as insect cells (baculovirus expression system) or mammalian cells might be preferable despite lower yields .

What is the known or predicted function of MPN_143 in Mycoplasma pneumoniae?

While specific information about MPN_143 function is limited, its characterization as an "uncharacterized protein" indicates its biological role remains to be fully elucidated. Based on research on M. pneumoniae pathogenesis, it may potentially be involved in:

• Host cell adhesion and colonization (similar to the P1 adhesin protein)

• Stimulation of pro-inflammatory cytokine production

• Modulation of host immune responses

• Potential role in the Th1/Th2 imbalance observed during M. pneumoniae infection

Computational predictions using sequence homology, structural modeling, and domain analysis would provide initial insights into potential functions.

What strategies can optimize the expression and purification of MPN_143 for structural studies?

Optimizing MPN_143 expression and purification requires systematic testing of multiple parameters:

For crystallography studies, screening multiple constructs with different N- and C-terminal boundaries may be necessary to identify stable domains amenable to crystallization .

How can researchers investigate the role of MPN_143 in M. pneumoniae pathogenesis?

A comprehensive investigation would employ multiple complementary approaches:

• Gene knockout/knockdown studies:

  • CRISPR-Cas9 or transposon mutagenesis to disrupt MPN_143

  • Assessment of mutant phenotypes in growth, adhesion, and virulence assays

• Protein-protein interaction studies:

  • Pull-down assays using tagged recombinant MPN_143

  • Yeast two-hybrid screening to identify binding partners

  • Surface plasmon resonance to quantify binding affinities

• Host response analysis:

  • Measure cytokine production (particularly IL-5 and IFN-γ) in response to purified MPN_143

  • Assess effects on Th1/Th2 balance in immune cell cultures

• Animal model studies:

  • Compare wild-type and MPN_143-deficient strains in appropriate infection models

  • Evaluate pathological changes and immune responses

• Transcriptomic and proteomic profiling:

  • Analyze differential gene/protein expression during infection stages

  • Determine when MPN_143 is expressed during the infection cycle

What methods are most effective for investigating protein-protein interactions involving MPN_143?

Multiple complementary techniques should be employed to robustly characterize MPN_143 interactions:

Integration of data from multiple methods increases confidence in identifying true interaction partners of MPN_143.

How does the immune response to MPN_143 compare to other M. pneumoniae proteins?

While specific data on immune responses to MPN_143 is not available, research on M. pneumoniae infections provides a framework for investigation:

• Humoral immunity: Measure antibody responses (IgM, IgG) to MPN_143 in patient sera compared to known immunogenic proteins

• Cellular immunity: Compare T-cell responses to MPN_143 versus other M. pneumoniae proteins

  • Analyze cytokine profiles, particularly focusing on IL-5 and IFN-γ which are elevated in severe M. pneumoniae pneumonia

  • Evaluate impact on CD3+ T-cell populations which show significant decreases in MPP patients

• Inflammatory potential: Compare proinflammatory cytokine induction by MPN_143 versus other proteins

  • Examine effects on inflammatory markers like C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and serum ferritin (SF) that are elevated in severe infection

• Potential diagnostic value: Assess whether antibodies against MPN_143 could serve as biomarkers for infection or disease severity

What analytical methods should be used to assess the quality of purified recombinant MPN_143?

A comprehensive quality assessment would include:

A protein purity of >85% measured by SDS-PAGE would be expected following affinity purification, similar to other M. pneumoniae recombinant proteins .

How can researchers design functional assays to characterize the biological activity of MPN_143?

Without known function, a systematic approach to functional characterization is required:

• Bioinformatic analysis: Predict potential functions based on:

  • Sequence homology to characterized proteins

  • Domain identification

  • Structural modeling

  • Cellular localization predictions

• Adhesion assays:

  • Fluorescently labeled MPN_143 binding to respiratory epithelial cells

  • Competition with known M. pneumoniae adhesins

  • Inhibition studies using generated antibodies

• Immunomodulation assays:

  • Measure cytokine production in response to purified MPN_143

  • Focus on IL-5 and IFN-γ levels, which are predictive markers for severe M. pneumoniae pneumonia

  • Assess impact on Th1/Th2 balance in appropriate immune cell models

• Enzyme activity screening:

  • Test for common enzymatic activities (protease, nuclease, etc.)

  • Substrate specificity determination if activity detected

• Cell signaling assays:

  • Monitor activation of key signaling pathways in host cells

  • Phosphorylation status of pathway components

Appropriate controls should include heat-denatured protein, tag-only constructs, and dose-response curves to confirm specific biological activities.

What considerations are important for developing antibodies against MPN_143?

Development of research-grade antibodies against MPN_143 requires:

• Antigen design options:

  • Full-length recombinant protein

  • Synthetic peptides from predicted surface-exposed regions

  • Combination approaches for broader epitope coverage

• Production strategies:

  • Polyclonal antibodies: Multiple epitope recognition but batch variation

  • Monoclonal antibodies: Consistent specificity but limited epitope coverage

  • Recombinant antibodies: Reproducible production without animals

• Validation requirements:

  • Western blot against recombinant protein and M. pneumoniae lysates

  • Immunoprecipitation capability testing

  • Immunofluorescence microscopy to confirm native protein recognition

  • Functional assays to identify neutralizing potential

• Application considerations:

  • Purification requirements (protein A/G, affinity purification)

  • Storage stability and format (whole IgG, Fab fragments)

  • Cross-reactivity testing with related Mycoplasma species

Research indicates that monoclonal antibodies against M. pneumoniae surface proteins can inhibit bacterial adhesion to respiratory epithelial cells, suggesting potential research applications for anti-MPN_143 antibodies .

How can MPN_143 research be integrated into broader studies of M. pneumoniae pathogenesis and immune evasion?

Integration of MPN_143 studies with broader M. pneumoniae research should address:

• Temporal expression patterns:

  • When during infection is MPN_143 expressed?

  • Does expression correlate with specific disease stages?

• Contribution to key pathogenic mechanisms:

  • Role in cytokine induction, particularly IL-5 and IFN-γ which are elevated in severe M. pneumoniae pneumonia

  • Impact on CD3+ T-cell populations, which show significant decreases in MPP patients

  • Potential involvement in the Th1/Th2 imbalance characteristic of infection

• Relation to clinical outcomes:

  • Correlation between MPN_143 antibody levels and disease severity

  • Potential as a biomarker for prognosis or treatment response

• Therapeutic targeting potential:

  • Vaccine antigen candidacy assessment

  • Evaluation as target for neutralizing antibodies or small molecule inhibitors

• Evolutionary considerations:

  • Conservation across M. pneumoniae strains

  • Presence of homologs in related pathogens

This integrated approach positions MPN_143 research within the context of understanding M. pneumoniae's complex pathogenesis mechanisms, particularly the balance between direct pathogen effects and host immune response contributions to disease .

What are the most effective storage conditions for maintaining MPN_143 stability?

Without specific data on MPN_143 stability, general protein storage principles should be applied:

Stability testing with regular analysis by SDS-PAGE and activity assays would determine optimal conditions specific to MPN_143.

What experimental controls are essential when studying MPN_143 function?

Rigorous experimental design requires appropriate controls:

• Protein-specific controls:

  • Tag-only protein (expressed from empty vector with same tags)

  • Heat-denatured MPN_143 (functionality lost but same composition)

  • Irrelevant protein of similar size and purification method

  • Dose-response testing to confirm specific effects

• Assay-specific controls:

  • Positive controls: Known M. pneumoniae proteins with established functions

  • Negative controls: Buffer-only treatments

  • Inhibitor controls where applicable

• Host cell controls:

  • Untreated cells establishing baseline responses

  • Cells treated with known stimulants (e.g., LPS) or inhibitors

  • Cell viability monitoring to distinguish specific effects from cytotoxicity

• Validation controls:

  • Multiple cell types/lines to confirm biological relevance

  • Independent methods confirming key findings

  • Blocking experiments using antibodies against MPN_143

These controls help distinguish specific MPN_143 effects from artifacts related to contaminants, tags, or experimental conditions.

How should researchers interpret contradictory results between different functional assays for MPN_143?

When facing contradictory results:

• Methodological considerations:

  • Examine differences in experimental conditions (pH, temperature, buffer components)

  • Assess protein quality and batch variation

  • Consider cell type differences and their physiological relevance

  • Evaluate sensitivity and specificity of each assay

• Biological explanations:

  • MPN_143 may have multiple functions with different activation requirements

  • Context-dependent effects based on microenvironment

  • Concentration-dependent effects with different outcomes at varying levels

  • Potential requirement for cofactors or binding partners

• Resolution approaches:

  • Perform dose-response studies across a wide concentration range

  • Test in multiple cell types reflecting different host environments

  • Examine time-course effects from minutes to days

  • Use domain mapping to identify regions responsible for distinct functions

  • Employ in vivo models to determine physiologically relevant functions

The multifaceted nature of M. pneumoniae interactions with host immune systems, including documented effects on cytokine production and T-cell populations, suggests MPN_143 may have complex, context-dependent activities .

How can sequence and structural analysis guide MPN_143 functional studies?

Bioinformatic analysis provides crucial guidance for experimental design:

• Sequence analysis approaches:

  • Homology searches against characterized proteins

  • Motif identification for functional domains

  • Disorder prediction identifying flexible regions

  • Post-translational modification site prediction

  • Signal peptide and transmembrane domain analysis

• Structural prediction value:

  • Secondary structure prediction guiding construct design

  • Homology modeling suggesting potential binding sites

  • Molecular docking with predicted interactors

  • Electrostatic surface analysis revealing potential interaction interfaces

  • Identification of conserved residues for mutagenesis studies

• Experimental validation:

  • Site-directed mutagenesis of predicted functional residues

  • Truncation constructs based on domain boundaries

  • Chimeric proteins to test domain-specific functions

  • Surface residue labeling to confirm structural predictions

Integration of computational predictions with experimental validation creates an iterative pathway to understanding MPN_143 function.