Recombinant Mycoplasma pneumoniae Uncharacterized protein MG123 homolog (MPN_262)

What is MPN_262 and why is it significant for research?

MPN_262 is an uncharacterized protein in Mycoplasma pneumoniae, a minimal bacterial pathogen responsible for respiratory infections. The significance of studying MPN_262 lies in understanding its potential role in M. pneumoniae pathogenicity and basic cellular functions. As M. pneumoniae has one of the smallest self-replicating genomes, each protein potentially serves critical functions, making uncharacterized proteins valuable targets for expanding our knowledge of minimal cellular systems . Research on MPN_262 contributes to our understanding of reduced genome organisms and may reveal novel mechanisms of bacterial pathogenesis.

What expression systems are recommended for producing recombinant MPN_262?

E. coli BL21(DE3) remains the preferred expression system for MPN_262 due to its established protocols and high yield. The recommended methodology includes:

• Transformation of the MPN_262 sequence into an expression vector (pGEX for GST-tagged or pET for His-tagged constructs)

• Expression induction using IPTG (0.6 mM)

• Growth at 37°C for 12 hours

• Cell harvesting and lysis via sonication

• Protein purification using affinity chromatography (Ni-NTA for His-tagged constructs)

For challenging expression, consider using specialized strains designed for membrane or toxic proteins, or alternative systems like insect cells if E. coli yields are insufficient.

What purification strategies yield the highest purity MPN_262 protein?

A multi-step purification approach ensures highest purity for MPN_262:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged MPN_262)

• Intermediate purification via ion exchange chromatography

• Polishing step with size exclusion chromatography

• Quality assessment using 12% SDS-PAGE

This approach typically yields >95% pure protein suitable for functional and structural studies. Maintaining buffer conditions at pH 7.4 with 150 mM NaCl helps preserve protein stability throughout the purification process.

How can researchers verify the identity and integrity of purified MPN_262?

Identity and integrity verification requires multiple complementary approaches:

These methods collectively provide comprehensive validation of purified MPN_262 before proceeding with functional studies .

What bioinformatic approaches can predict potential functions of MPN_262?

A comprehensive bioinformatic pipeline for MPN_262 functional prediction should include:

• Sequence homology analysis using PSI-BLAST and HHpred against multiple databases (UniProt, PDB, CDD)

• Structural prediction using AlphaFold2 or RoseTTAFold

• Domain and motif analysis using InterProScan and SMART

• Co-expression analysis using transcriptomic databases

• Genomic context analysis examining neighboring genes and operons

This multi-layered approach compensates for the limitations of individual methods when analyzing uncharacterized proteins. Integration of results from different algorithms and databases increases prediction confidence and generates testable hypotheses about protein function.

How can researchers determine if MPN_262 undergoes post-translational modifications?

A systematic approach to identifying post-translational modifications (PTMs) in MPN_262 includes:

• In silico prediction of potential modification sites using specialized algorithms (for palmitoylation, use IFS-Palm predictor with reported 90.65% accuracy)

• Mass spectrometry analysis of the native protein from M. pneumoniae lysates using:

  • Enrichment strategies for specific PTMs (e.g., TiO₂ for phosphorylation)

  • Multiple fragmentation methods (CID, HCD, ETD)

  • Modified residue mapping against the MPN_262 sequence

• Site-directed mutagenesis of predicted modification sites followed by functional assays

• Western blot with PTM-specific antibodies (when available)

When analyzing results, consider that bacterial proteins like MPN_262 may exhibit unique modification patterns compared to eukaryotic proteins, and that certain PTMs may be condition-dependent or substoichiometric .

What experimental approaches can identify potential binding partners of MPN_262?

To systematically identify MPN_262 interaction partners, researchers should employ multiple complementary techniques:

• Pull-down assays: Use purified His-tagged MPN_262 as bait with M. pneumoniae lysates, followed by mass spectrometry identification of captured proteins

• Yeast two-hybrid screening: Construct a library from M. pneumoniae genes to screen against MPN_262 bait

• Cross-linking mass spectrometry (XL-MS): Apply chemical cross-linkers to stabilize transient interactions prior to analysis

• Co-immunoprecipitation: Generate specific antibodies against MPN_262 for native complex isolation

• Proximity labeling: Use BioID or APEX2 fusions to identify proteins in close proximity to MPN_262 in living cells

Each technique has distinct advantages and limitations, and the integration of results from multiple methods provides higher confidence in identified interactions .

How can researchers determine if MPN_262 plays a role in M. pneumoniae adhesion to host cells?

Investigating MPN_262's potential role in adhesion requires a multi-faceted experimental design:

• Generate recombinant MPN_262 and test its direct binding to host cells using fluorescently labeled protein and flow cytometry

• Perform adhesion inhibition assays using:

  • Anti-MPN_262 antibodies

  • Recombinant MPN_262 as a competitor

  • Peptides derived from MPN_262 sequences

• Create gene knockdown or knockout M. pneumoniae strains (if technically feasible) and assess adhesion capacity

• Screen potential host receptors using modified virus overlay protein binding assay (VOPBA) with MPN_262 as probe

• Confirm direct interactions with candidate receptors using surface plasmon resonance or microscale thermophoresis

When interpreting results, consider that adhesion may involve multiple bacterial factors, and MPN_262 might play an auxiliary rather than primary role in the process .

What approaches can evaluate if MPN_262 belongs to the DNA damage response pathway in M. pneumoniae?

To investigate MPN_262's potential involvement in DNA damage response pathways:

• Co-expression analysis: Determine if MPN_262 expression correlates with known DNA damage response genes under genotoxic stress conditions

• Protein localization: Use fluorescent protein fusions or immunofluorescence to track MPN_262 localization before and after DNA damage induction

• Interaction studies: Perform pull-down assays using known DNA repair proteins as bait to identify if MPN_262 is captured

• Functional assays:

  • Measure DNA repair efficiency in cells with altered MPN_262 expression

  • Assess sensitivity to DNA-damaging agents when MPN_262 levels are modulated

• Post-translational modification analysis: Determine if MPN_262 undergoes phosphorylation or other modifications following DNA damage

Integration of these approaches provides a comprehensive assessment of MPN_262's potential role in DNA damage response pathways .

How can researchers investigate the structural characteristics of MPN_262?

A comprehensive structural characterization strategy for MPN_262 includes:

When pursuing structural studies, consider starting with computational models from AlphaFold2 to guide experimental design and interpret low-resolution data. For challenging crystallization, employ surface entropy reduction mutations or fusion partners to promote crystal contacts .

What cellular localization methods are most effective for studying MPN_262 in M. pneumoniae?

Due to the small size of M. pneumoniae cells, specialized approaches for accurate localization include:

• Super-resolution microscopy:

  • STORM or PALM imaging using photoswitchable fluorophore-conjugated antibodies

  • Structured illumination microscopy (SIM) for live-cell imaging

  • Resolution of 20-50 nm allows precise localization within the limited cellular space

• Correlative light and electron microscopy (CLEM):

  • Combines fluorescence specificity with ultrastructural context

  • Particularly valuable for membrane-associated proteins

• Fractionation approaches:

  • Carefully optimized protocols for membrane, cytosolic, and DNA-associated fractions

  • Western blot analysis of fractions with marker controls for each compartment

• Proximity labeling:

  • APEX2 or BioID fusions for in situ biotinylation of proximal proteins

  • Provides functional context through identification of neighboring proteins

These methods overcome the challenges posed by M. pneumoniae's minimal cell structure and provide complementary data for conclusive localization .

What are the critical considerations when designing cell-based assays to study MPN_262 function?

When designing cell-based assays for MPN_262 functional studies:

• Cell line selection: Use respiratory epithelial cell lines (BEAS-2B) for physiological relevance or RAW264.7 for immune response studies

• Verification of mycoplasma contamination: Critical to avoid background effects from endogenous mycoplasma proteins

• Exposure conditions optimization:

  • Concentration range testing (typically 1-10 μg/ml for recombinant MPN_262)

  • Time course determination (4-72 hours)

  • Treatment in serum-free media to avoid protein interactions with serum components

• Appropriate controls:

  • Heat-inactivated protein

  • Unrelated recombinant protein of similar size

  • Buffer-only treatment

• Readout selection:

  • Direct binding assays using labeled protein

  • Functional consequences (cytokine production, adhesion, cytotoxicity)

  • Transcriptional responses via RNA-seq

Meticulous optimization of these parameters ensures reproducibility and physiological relevance of obtained results .

How should researchers approach solubility issues with recombinant MPN_262?

When encountering solubility challenges with MPN_262:

• Expression optimization:

  • Lower induction temperature (16-25°C)

  • Reduce IPTG concentration (0.1-0.5 mM)

  • Use auto-induction media for gradual protein expression

• Solubility enhancement strategies:

  • Fusion tags: MBP, SUMO, or TrxA tags often increase solubility

  • Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Addition of mild detergents (0.05-0.1% Triton X-100) during lysis

• Buffer optimization:

  • Screen pH ranges (pH 6.0-9.0)

  • Test various salt concentrations (100-500 mM NaCl)

  • Add stabilizing agents (5-10% glycerol, 1-5 mM DTT)

• Refolding approaches (if inclusion bodies form):

  • Solubilization in 8M urea or 6M guanidine-HCl

  • Step-wise dialysis for gradual denaturant removal

  • On-column refolding during affinity purification

Systematic testing of these approaches, often in factorial design experiments, can identify conditions that yield soluble, functional MPN_262 .

What strategies can overcome challenges in generating antibodies against MPN_262?

To address challenges in antibody generation against MPN_262:

• Epitope selection strategy:

  • Utilize bioinformatic tools to identify surface-exposed, antigenic regions

  • Target multiple distinct epitopes (typically 15-20 amino acids)

  • Avoid regions with high sequence conservation among mycoplasma species unless specificity is not required

• Production approaches:

  • Synthetic peptide conjugation to carrier proteins (KLH or BSA)

  • Recombinant protein fragments expressed with high solubility tags

  • DNA immunization with codon-optimized MPN_262 sequence

• Host animal selection:

  • Rabbits for polyclonal antibodies with sufficient serum volume

  • Mice or rats for monoclonal antibody development

  • Consider chickens for IgY production when mammalian protein homology is a concern

• Screening and validation:

  • ELISA against immunizing antigen and full-length protein

  • Western blot against recombinant protein and M. pneumoniae lysates

  • Immunoprecipitation efficiency testing

  • Immunofluorescence microscopy validation

A combination of these approaches increases the likelihood of generating functional antibodies for MPN_262 research applications .

How can contradictory results about MPN_262 function from different experimental approaches be reconciled?

When faced with conflicting data about MPN_262 function:

• Methodological examination:

  • Evaluate differences in protein preparation (tags, purity, folding)

  • Assess cell types or experimental conditions used

  • Consider detection methods and their sensitivity/specificity

• Context-dependent function analysis:

  • Test if MPN_262 exhibits different activities under varying conditions

  • Examine potential post-translational modifications affecting function

  • Consider if MPN_262 functions as part of different complexes

• Integration approaches:

  • Weight evidence based on methodological robustness

  • Develop testable hypotheses that could explain apparent contradictions

  • Design experiments specifically to address discrepancies

• Computational modeling:

  • Use systems biology approaches to integrate contradictory data

  • Simulate different conditions that might explain contextual differences

The integration of multiple experimental approaches provides a more comprehensive understanding than reliance on any single method, particularly for multifunctional proteins .

What bioinformatic tools are most effective for predicting if MPN_262 belongs to known protein families despite low sequence homology?

For detecting distant evolutionary relationships for MPN_262:

• Profile-based methods:

  • HHpred: Leverages hidden Markov model comparisons with >90% sensitivity

  • HMMER: Searches against profile databases like Pfam and SUPERFAMILY

  • PSIBLAST: Iterative searches to detect remote homologs

• Structure-based approaches:

  • AlphaFold2 predictions followed by structural similarity searches (DALI, TM-align)

  • Threading methods (RAPTOR, I-TASSER) that fit sequences to known folds

  • Analysis of predicted secondary structure patterns

• Machine learning integrators:

  • PSSM-based neural network approaches

  • Feature extraction from multiple prediction algorithms

  • Integration of co-evolution data with structural predictions

• Functional site conservation:

  • Active site template matching

  • Ligand binding site prediction and comparison

  • Conserved residue cluster identification

When sequence identity falls below 20-25%, these advanced methods significantly outperform standard BLAST searches for detecting meaningful relationships .

How should researchers integrate MPN_262 findings into the broader understanding of M. pneumoniae pathogenesis?

To contextualize MPN_262 research within M. pneumoniae pathogenesis:

• Pathway integration:

  • Map potential interactions with known virulence factors

  • Position findings within established pathogenicity mechanisms

  • Develop network models incorporating new MPN_262 data

• Comparative analysis:

  • Examine homologs in related mycoplasma species

  • Compare phenotypic effects with other characterized M. pneumoniae proteins

  • Context within minimal genome evolution

• Clinical correlation:

  • Assess MPN_262 conservation across clinical isolates

  • Investigate potential associations with disease severity

  • Examine immune responses to MPN_262 in patient samples

• Host-pathogen interface:

  • Determine if MPN_262 interacts with host receptors like vimentin

  • Assess effects on host signaling pathways

  • Evaluate contributions to immune evasion strategies

This multi-level integration approach places MPN_262 findings within the broader context of M. pneumoniae biology and host interactions, contributing to a more comprehensive understanding of pathogenesis mechanisms .

What emerging technologies could advance understanding of MPN_262 function?

Cutting-edge approaches with particular promise for MPN_262 characterization include:

• CryoEM tomography:

  • Visualization of MPN_262 in native cellular environment

  • 3D reconstruction at near-atomic resolution

  • Contextual information about protein complexes and localization

• AlphaFold-based interaction prediction:

  • Modeling potential protein-protein interfaces

  • Predicting binding modes with putative partners

  • Guiding mutagenesis for functional studies

• Micro-scale thermophoresis (MST):

  • Quantitative binding measurements in near-native conditions

  • Low protein consumption (nanomolar range)

  • Compatible with complex buffers and cell lysates

• CRISPR interference in minimal genomes:

  • Targeted gene repression in M. pneumoniae

  • Tunable expression for dosage studies

  • Circumvention of difficulties with complete gene deletion

These technologies offer new avenues for understanding MPN_262 function, particularly in the challenging context of minimal genome organisms .

How might knowledge of MPN_262 contribute to vaccine or therapeutic development against M. pneumoniae?

The translational potential of MPN_262 research includes:

• Vaccine candidate assessment:

  • Evaluation of conservation across clinical strains

  • Immunogenicity testing in animal models

  • Accessibility on bacterial surface for antibody recognition

  • Functional importance that would limit mutation escape

• Therapeutic targeting approaches:

  • Structure-based inhibitor design if enzymatic function is identified

  • Development of function-blocking antibodies

  • Peptide mimetics to disrupt essential interactions

• Diagnostic potential:

  • Assessment as biomarker for M. pneumoniae infection

  • Development of specific detection assays

  • Monitoring of immune responses in patients

• Platform technologies:

  • Potential use as protein scaffold or delivery system

  • Application in synthetic biology approaches

  • Biotechnological applications if unique properties are identified

While currently uncharacterized, the systematic study of MPN_262 may reveal properties valuable for clinical applications, particularly if it proves to have roles in adhesion, immune evasion, or essential cellular processes .