Recombinant Mycoplasma pneumoniae Uncharacterized protein MG131 homolog (MPN_270)

What is the Recombinant Mycoplasma pneumoniae Uncharacterized Protein MG131 Homolog (MPN_270)?

Recombinant MPN_270 is a full-length (1-95 amino acids) uncharacterized protein from Mycoplasma pneumoniae, which is expressed recombinantly with an N-terminal His-tag in E. coli expression systems. This protein is also known by synonyms MPN_270, MP563.1, and Uncharacterized protein MG131 homolog, with UniProt identifier Q9EXD1 . The function of this protein remains largely uncharacterized in the M. pneumoniae proteome, presenting opportunities for novel research into its structural and functional properties in the context of mycoplasma biology.

How should MPN_270 recombinant protein be stored and handled for optimal stability?

For optimal stability and functionality of recombinant MPN_270 protein, follow these research-grade handling protocols:

• Store lyophilized protein at -20°C to -80°C upon receipt

• Perform aliquoting to avoid repeated freeze-thaw cycles which can degrade protein quality

• For reconstitution, briefly centrifuge the vial before opening to bring contents to the bottom

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

• Add glycerol to 50% final concentration for long-term storage

• For short-term use, working aliquots can be stored at 4°C for up to one week

This methodological approach preserves protein integrity for downstream experimental applications and ensures reproducibility across multiple experiments.

What expression system is used for producing recombinant MPN_270 protein?

The recombinant MPN_270 protein is expressed in an E. coli bacterial expression system. This heterologous expression system offers several methodological advantages:

• High protein yield for experimental applications

• N-terminal His-tag fusion facilitates purification via nickel affinity chromatography

• The expression construct contains the full-length protein (amino acids 1-95)

• The resulting recombinant protein demonstrates greater than 90% purity as determined by SDS-PAGE analysis

E. coli expression systems are preferred for this mycoplasma protein due to their efficiency, cost-effectiveness, and ability to produce sufficient quantities for structural and functional studies.

How can researchers distinguish between antibody responses to MPN_270 and other M. pneumoniae antigens in serological studies?

Distinguishing specific antibody responses to MPN_270 from responses to other M. pneumoniae antigens requires a multi-faceted methodological approach:

• Implement highly purified recombinant MPN_270 protein (>90% purity) as capture antigen in ELISA-based assays

• Develop protein-specific monoclonal antibodies as positive controls for binding specificity

• Perform pre-absorption experiments using other M. pneumoniae recombinant proteins to eliminate cross-reactivity

• Establish temporal antibody response patterns, noting that IgM antibodies appear 7-10 days post-infection while IgG antibodies emerge approximately 14 days later

• Apply Western blot analysis with size-specific detection to confirm antibody specificity

This differential approach helps address the variability of antibody persistence, potential absence of IgM response after re-infection, and infrequent production of IgA antibodies in pediatric patients .

What structural and functional predictions can be made about MPN_270 based on sequence analysis?

Based on sequence analysis of MPN_270, several structural and functional characteristics can be predicted:

These predictions provide starting points for targeted experimental validation using site-directed mutagenesis, membrane protein isolation techniques, and structural biology approaches.

How might MPN_270 contribute to M. pneumoniae pathogenicity and host immune responses?

The potential role of MPN_270 in M. pneumoniae pathogenicity can be investigated through these methodological approaches:

• Genomic comparison of MPN_270 sequences across clinical isolates to identify variation patterns

• Assessment of MPN_270 expression levels during different growth phases and infection stages using RT-qPCR

• Evaluation of host antibody responses to MPN_270 in patients with confirmed M. pneumoniae infections

• Analysis of potential interactions with host factors using yeast two-hybrid or pull-down assays

• Generation of isogenic mutants with altered MPN_270 expression to assess virulence in cell culture models

Considering that M. pneumoniae shows remarkable genomic homology between strains despite their different origins and isolation periods , the investigation of proteins like MPN_270 may reveal subtle variations that contribute to strain-specific pathogenicity profiles.

What experimental strategies can resolve contradictions in MPN_270 functional data?

When confronting contradictory experimental results regarding MPN_270 function, implement these methodological resolution strategies:

• Apply multiple orthogonal techniques to verify protein-protein interactions:

  • Co-immunoprecipitation with specific antibodies

  • FRET/BRET analysis for in vivo interaction verification

  • Surface plasmon resonance for binding kinetics determination

• Validate protein localization through complementary approaches:

  • Immunofluorescence microscopy

  • Cell fractionation followed by Western blotting

  • Protease accessibility assays for membrane topology

• Confirm gene function through genetic complementation:

  • Generate clean deletion mutants and complemented strains

  • Perform phenotypic characterization under various growth conditions

  • Evaluate transcriptomic changes using RNA-seq

• Address species-specific differences:

  • Compare homologs across mycoplasma species

  • Evaluate evolutionary conservation patterns

  • Perform heterologous expression studies

This multi-faceted approach ensures that experimental artifacts are distinguished from genuine biological complexity.

How should researchers design experiments to investigate potential protein-protein interactions involving MPN_270?

A comprehensive experimental design to investigate protein-protein interactions of MPN_270 should include:

• Bait Protein Preparation:

  • Express recombinant His-tagged MPN_270 in E. coli

  • Purify using immobilized metal affinity chromatography

  • Verify protein integrity via SDS-PAGE and Western blotting

  • Reconstitute in appropriate buffer systems that maintain native conformation

• Pull-down Assay Protocol:

  • Immobilize purified MPN_270 on nickel resin

  • Prepare M. pneumoniae cell lysates under non-denaturing conditions

  • Incubate immobilized MPN_270 with cell lysates

  • Wash extensively to remove non-specific binding

  • Elute bound proteins for mass spectrometry identification

• Controls and Validation:

  • Include unrelated His-tagged protein as negative control

  • Perform reverse pull-down with identified interaction partners

  • Validate interactions using co-immunoprecipitation with specific antibodies

  • Confirm biological relevance through co-localization studies

This systematic approach minimizes false positives while maximizing detection of genuine interaction partners.

What are the optimal parameters for expression and purification of functionally active MPN_270?

Optimizing expression and purification of functionally active MPN_270 requires careful consideration of these parameters:

These optimized parameters ensure maximum yield of properly folded, functionally relevant MPN_270 protein for downstream applications.

How can researchers effectively analyze the membrane topology of MPN_270?

To effectively analyze the membrane topology of MPN_270, implement this methodological workflow:

• Computational Prediction:

  • Apply multiple transmembrane prediction algorithms (TMHMM, Phobius, HMMTOP)

  • Generate consensus model of membrane-spanning regions

  • Identify potential cytoplasmic and extracellular domains

• Biochemical Mapping:

  • Design cysteine substitution mutants at predicted loop regions

  • Perform selective labeling with membrane-impermeable sulfhydryl reagents

  • Analyze accessibility patterns to determine orientation relative to membrane

• Protease Protection Assays:

  • Prepare right-side-out and inside-out membrane vesicles

  • Subject to controlled protease digestion

  • Identify protected fragments by immunoblotting with domain-specific antibodies

• Fluorescence-based Approaches:

  • Generate GFP fusion constructs at different termini and predicted loops

  • Express in appropriate hosts and analyze fluorescence accessibility

  • Perform quantitative analysis of fluorescence quenching by membrane-impermeable agents

This integrated approach provides robust evidence for the membrane orientation of MPN_270, essential for understanding its potential functional interactions.

How should researchers interpret MPN_270 sequence variations among clinical isolates?

When analyzing MPN_270 sequence variations among clinical isolates, apply this interpretive framework:

• Sequence Alignment Analysis:

  • Align MPN_270 sequences from multiple clinical isolates

  • Identify conserved regions and variable hotspots

  • Map variations to predicted functional domains

• Variation Classification:

  • Distinguish between synonymous and non-synonymous substitutions

  • Calculate dN/dS ratios to determine selective pressure

  • Identify potential recombination events versus point mutations

• Structural Impact Assessment:

  • Model effects of amino acid substitutions on protein structure

  • Evaluate conservation of hydrophobicity patterns in membrane-spanning regions

  • Predict impact on protein-protein interaction interfaces

• Clinical Correlation:

  • Associate specific variants with disease severity or treatment outcomes

  • Compare variation patterns with other typing methods like multilocus variable number of tandem repeat analysis and multilocus sequence typing

  • Analyze geographical and temporal distribution of variants

This comprehensive approach connects sequence variation to potential functional and clinical significance.

What statistical approaches are appropriate for analyzing antibody responses to MPN_270 in patient cohorts?

When analyzing antibody responses to MPN_270 in patient cohorts, employ these statistical methodologies:

• Baseline Establishment:

  • Determine antibody detection thresholds using ROC curve analysis

  • Establish normal distribution ranges in healthy control populations

  • Calculate appropriate cutoff values for sensitivity and specificity optimization

• Comparative Analysis:

  • Apply paired t-tests for pre/post infection samples

  • Use ANOVA for multi-group comparisons (acute, convalescent, chronic)

  • Implement non-parametric alternatives (Wilcoxon, Kruskal-Wallis) for non-normally distributed data

• Correlation Studies:

  • Calculate Pearson/Spearman correlations between antibody levels and clinical parameters

  • Perform linear regression analysis to identify predictive relationships

  • Develop multivariate models incorporating multiple antibody responses

• Longitudinal Assessment:

  • Employ repeated measures ANOVA for temporal antibody dynamics

  • Apply linear mixed effects models to account for within-subject correlations

  • Calculate area-under-the-curve metrics for cumulative response analysis

These statistical approaches accommodate the complex patterns of antibody responses observed in M. pneumoniae infections, including variable persistence of antibodies, potential absence of IgM response after re-infection, and age-dependent differences in antibody production .

How can researchers differentiate between specific and cross-reactive antibody responses to MPN_270?

To differentiate between specific and cross-reactive antibody responses to MPN_270, implement this methodological framework:

• Competitive Inhibition Assays:

  • Pre-incubate serum samples with purified MPN_270

  • Compare binding to immobilized MPN_270 before and after absorption

  • Quantify percent inhibition to determine specificity

• Cross-Absorption Studies:

  • Pre-absorb serum with related mycoplasma proteins

  • Measure residual binding to MPN_270

  • Calculate relative specificity indices

• Epitope Mapping:

  • Synthesize overlapping peptides spanning MPN_270 sequence

  • Identify peptide-specific antibody binding patterns

  • Compare epitope recognition profiles across patient cohorts

• Affinity Determination:

  • Measure antibody-antigen binding kinetics using surface plasmon resonance

  • Compare association/dissociation rates for MPN_270 versus homologous proteins

  • Calculate affinity constants to quantify binding strength

This integrated approach enables researchers to distinguish genuine MPN_270-specific antibody responses from cross-reactivity with related proteins in complex clinical samples.

What novel approaches could elucidate the functional role of MPN_270 in M. pneumoniae biology?

Emerging methodologies to elucidate MPN_270 function include:

• CRISPR-Interference Systems:

  • Develop CRISPRi knockdown systems adapted for mycoplasma

  • Generate conditional depletion strains of MPN_270

  • Assess phenotypic consequences under various growth conditions

• Interactome Mapping:

  • Apply proximity labeling techniques (BioID, APEX)

  • Identify spatial neighbors of MPN_270 in living cells

  • Construct functional protein networks

• Single-Cell Analysis:

  • Implement single-cell RNA-seq to detect co-expression patterns

  • Correlate MPN_270 expression with specific cellular states

  • Identify regulatory relationships with other genes

• Cryo-Electron Microscopy:

  • Determine high-resolution structure of MPN_270 in membrane environment

  • Visualize protein-protein interactions in native context

  • Map functional domains through structural analysis

These cutting-edge approaches overcome limitations of traditional biochemical methods and provide complementary insights into MPN_270 function.

How might MPN_270 contribute to the minimal genome concept in synthetic biology applications?

As part of the minimal genome research paradigm, MPN_270's role can be explored through:

• Essentiality Assessment:

  • Perform systematic gene deletion attempts

  • Evaluate growth rate and morphological changes in deletion mutants

  • Identify conditions under which MPN_270 becomes essential

• Synthetic Biology Integration:

  • Incorporate MPN_270 into minimal genome constructs

  • Assess functional consequences of inclusion/exclusion

  • Optimize expression parameters in artificial systems

• Comparative Genomics:

  • Analyze presence/absence patterns across reduced genome mycoplasmas

  • Evaluate evolutionary conservation to inform essentiality predictions

  • Correlate retention with specific ecological niches

• Functional Replacement Studies:

  • Attempt complementation with heterologous proteins

  • Identify minimal functional domains required for activity

  • Engineer simplified versions with preserved essential functions

This research direction connects MPN_270 studies to the broader context of synthetic minimal genomes and bacterial reductive evolution.

What are the most significant knowledge gaps concerning MPN_270 that require immediate research attention?

Critical knowledge gaps requiring immediate research attention include:

• Functional characterization of MPN_270:

  • Definitive determination of biochemical activity

  • Identification of interaction partners

  • Elucidation of role in M. pneumoniae biology

• Structural analysis:

  • High-resolution structure determination

  • Membrane topology confirmation

  • Domain organization and functional motifs

• Immunological significance:

  • Epitope mapping and antibody recognition patterns

  • Contribution to protective versus non-protective immunity

  • Potential as diagnostic or vaccine target

• Regulatory mechanisms:

  • Transcriptional and translational control

  • Post-translational modifications

  • Expression dynamics during infection

Addressing these gaps will significantly advance understanding of both MPN_270 specifically and M. pneumoniae biology generally, potentially revealing new approaches for diagnostic and therapeutic interventions.

How does research on MPN_270 contribute to broader understanding of Mycoplasma pneumoniae pathogenesis?

Research on MPN_270 extends our understanding of M. pneumoniae pathogenesis through:

• Membrane Biology Insights:

  • Contribution to membrane organization and stability

  • Role in host-pathogen interface interactions

  • Potential involvement in cellular gliding motility

• Antigenic Variation Mechanisms:

  • Analysis of MPN_270 variation across clinical isolates

  • Correlation with immune evasion strategies

  • Integration with known variable elements like the p1 gene

• Minimal Pathogen Requirements:

  • Determination of essential versus accessory functions

  • Contribution to minimal pathogenesis requirements

  • Implications for streamlined bacterial systems

• Novel Therapeutic Targets:

  • Assessment as potential antimicrobial target

  • Evaluation for vaccine development

  • Utility in diagnostic applications