Recombinant Mycoplasma pneumoniae UPF0134 protein MPN_038 (MPN_038)
Description
Genomic Context of M. pneumoniae Proteins
The M. pneumoniae genome is highly conserved, with limited horizontal gene transfer but notable recombination hotspots in adhesion-associated genes ( ). Key findings include:
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Adhesin proteins (e.g., P1, P30) are critical for host cell attachment and immune evasion .
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Repetitive elements (RepMPs) drive recombination in genes like MPN141 (P1) and MPN142, influencing strain variability and adaptation .
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Type I restriction-modification systems involve variable hsdS genes, which encode specificity subunits that may contribute to epigenetic regulation .
Functional Annotation of Hypothetical Proteins
UPF0134 is a conserved domain (Pfam: UPF0134) often associated with uncharacterized proteins. In M. pneumoniae, hypothetical proteins with repetitive domains are frequently linked to:
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Immune modulation: Example: DUF16 activates the NOD2/RIP2/NF-κB pathway to induce inflammation .
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Host-pathogen interactions: Proteins like CARDS toxin and hydrogen peroxide-producing enzymes mediate cytotoxicity .
Comparative Genomic Analysis
A study of 15 M. pneumoniae strains revealed:
| Gene/Protein | Function | Variability | Recombination Frequency |
|---|---|---|---|
| P1 adhesin (MPN141) | Host cell attachment | High | 0.18–0.24 blocks/strain |
| MPN366–MPN371 | Unknown | Moderate | 1.3 kb/event |
| hsdS genes | Restriction-modification systems | High | Tandem repeats |
While MPN_038 is not listed, genes like MPN_RS02085 and MPN_RS02055 in subtype 2 strains show elevated recombination rates, suggesting roles in adaptation .
Research Gaps and Future Directions
The absence of explicit data on MPN_038 highlights opportunities for further investigation:
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Proteomic studies: Targeted mass spectrometry could identify interactions between MPN_038 and host pathways.
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Structural analysis: Resolving its tertiary structure may clarify whether UPF0134 domains mediate enzymatic or ligand-binding activities.
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Knockout models: Assessing MPN_038 deletion mutants for changes in virulence or immune evasion.
Product Specs
Q&A
What is Mycoplasma pneumoniae UPF0134 protein MPN_038?
MPN_038 is a hypothetical protein encoded in the genome of Mycoplasma pneumoniae, one of the smallest known self-replicating bacteria with fewer than 700 predicted proteins. It belongs to the UPF0134 protein family, a group of proteins with conserved sequences but poorly characterized functions. While its existence has been confirmed in proteomic studies, its specific biological role remains largely uncharacterized. Alternative gene names include MPN038 and B01_orf116L as documented in protein databases .
Why is MPN_038 classified as a "hypothetical protein"?
The designation "hypothetical protein" indicates that while genome sequencing has identified the MPN_038 gene, and proteomic studies may have detected the expressed protein, its biological function remains unknown or unverified experimentally. M. pneumoniae has undergone extensive proteome analysis with approximately 90% of its predicted proteome (about 620 proteins) experimentally identified . The persistence of MPN_038 in the minimal genome of M. pneumoniae suggests it likely serves an important function, despite our current limited understanding.
How does MPN_038 relate to M. pneumoniae biology?
While specific functions of MPN_038 are not well-documented, understanding its role requires context within M. pneumoniae biology. M. pneumoniae is a human pathogen that causes respiratory infections and has a minimal genome resulting from reductive evolution. The bacterium has distinctive features including gliding motility and an attachment organelle essential for pathogenesis . As part of a minimal genome, MPN_038 may contribute to essential cellular processes, potentially in ways unique to this highly specialized pathogen.
What expression systems are most effective for recombinant production of MPN_038?
Recombinant MPN_038 can be expressed in multiple host systems including E. coli, yeast, baculovirus-infected insect cells, and mammalian cells . Each system offers distinct advantages:
| Expression System | Advantages | Disadvantages | Recommended Application |
|---|---|---|---|
| E. coli | Rapid growth, high yield, cost-effective | Potential misfolding, lacks PTMs | Initial structural studies |
| Yeast | Some PTMs, secretion possible | Different glycosylation patterns | Functional studies requiring basic PTMs |
| Baculovirus/Insect | Complex PTMs, better folding | Time-consuming, moderate yield | Interaction studies |
| Mammalian | Native-like PTMs, optimal folding | Expensive, lowest yield | Studies where authentic PTMs are critical |
The choice depends on research objectives - structural studies may prioritize yield (E. coli), while functional studies might require proper folding and post-translational modifications (mammalian systems).
What purification strategies yield the highest purity for recombinant MPN_038?
Standard purification protocols can achieve ≥85% purity as determined by SDS-PAGE . An effective purification strategy typically involves:
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Affinity chromatography using appropriate tags (His-tag, GST)
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Size-exclusion chromatography to separate monomeric protein from aggregates
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Ion-exchange chromatography for removal of remaining contaminants
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Optional: Tag removal using specific proteases if the tag interferes with function
When designing a purification strategy, researchers should consider the downstream applications, as some experiments may require higher purity than others.
How can researchers design experiments to elucidate the function of MPN_038?
A multi-dimensional approach is recommended for characterizing hypothetical proteins like MPN_038:
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Bioinformatic analysis: Sequence homology searches, structural predictions using tools like AlphaFold, and genomic context analysis to predict function .
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Protein-protein interaction studies: Identifying binding partners through techniques such as co-immunoprecipitation followed by mass spectrometry, similar to approaches used for other M. pneumoniae proteins where 178 soluble protein complexes have been successfully characterized .
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Localization studies: Determining subcellular localization using fluorescent protein fusions or immunofluorescence microscopy, particularly in relation to known structures like the terminal organelle.
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Gene disruption experiments: Creating knockout or knockdown strains to observe phenotypic effects, though techniques for targeted chromosomal knockouts in M. pneumoniae are challenging and relatively recent .
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Transcriptomic analysis: Examining expression patterns under various conditions to identify co-regulated genes and potential involvement in stress responses.
What are appropriate controls when studying MPN_038 function?
Robust controls are essential for meaningful functional studies:
| Control Type | Description | Purpose |
|---|---|---|
| Empty vector | Expression system without MPN_038 | Controls for effects of expression system |
| Inactive mutant | MPN_038 with mutations in predicted active sites | Confirms observed functions are specific to active protein |
| Related protein | Other UPF0134 family members | Establishes specificity vs. general family properties |
| Wild-type strain | M. pneumoniae without genetic manipulation | Baseline for phenotypic comparisons |
| Unrelated protein | Protein with different function/structure | Controls for non-specific effects |
The selection of appropriate controls should be guided by the specific experimental design and hypotheses being tested.
What structural analysis techniques are most informative for MPN_038 characterization?
Several complementary techniques can provide valuable structural insights:
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X-ray crystallography or Cryo-EM for high-resolution structural determination
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Circular dichroism spectroscopy for secondary structure analysis, as used successfully for other M. pneumoniae proteins like MPN387
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Analytical ultracentrifugation to determine oligomerization state
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Limited proteolysis to identify flexible regions and domains
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Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces
These techniques have proven effective for structural characterization of other M. pneumoniae proteins, as demonstrated in previous studies of proteins like MPN387, which was shown to form a dumbbell-shaped homodimer with a coiled-coil region .
What protein-protein interaction methods are most suitable for studying MPN_038's potential binding partners?
Based on successful approaches with other M. pneumoniae proteins, recommended methods include:
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Tandem affinity purification coupled with mass spectrometry, which has been used to isolate 178 soluble protein complexes from M. pneumoniae
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Yeast two-hybrid screening for binary interactions
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Proximity labeling methods (BioID, APEX) to identify neighboring proteins in vivo
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Crosslinking mass spectrometry to capture transient interactions
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Co-fractionation profiling across different biochemical separations
M. pneumoniae's relatively small proteome (approximately 700 proteins) makes comprehensive interaction mapping more feasible than in more complex organisms .
How does MPN_038 potentially relate to M. pneumoniae pathogenesis?
While direct evidence linking MPN_038 to pathogenesis is limited, several investigative approaches are warranted:
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Comparative expression analysis between virulent and avirulent strains
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Evaluation of MPN_038 expression during different stages of infection
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Assessment of potential interactions with host proteins, particularly in relation to respiratory epithelial cells
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Examination of potential involvement in immune evasion mechanisms
Research has shown that M. pneumoniae infections can lead to significant complications, including refractory M. pneumoniae pneumonia (RMPP) and extrapulmonary manifestations . Understanding the potential role of each protein, including MPN_038, is important for comprehensive pathogenesis models.
How might MPN_038 function in the context of M. pneumoniae's minimal genome?
M. pneumoniae has undergone extensive genome reduction during evolution, retaining primarily essential genes. Within this context:
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The retention of MPN_038 in a minimal genome suggests functional importance
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It may perform multiple functions (protein moonlighting) to compensate for the limited genome
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It could be involved in essential pathways unique to Mycoplasma biology
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The protein might participate in M. pneumoniae-specific adaptations to its ecological niche
Research has shown that M. pneumoniae's proteome is organized into approximately 178 protein complexes that interact to form larger assemblies , suggesting MPN_038 likely functions as part of one or more multimeric complexes.
How can systems biology approaches incorporate MPN_038 into models of M. pneumoniae function?
Systems biology offers powerful frameworks for understanding proteins of unknown function:
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Integration into genome-scale metabolic models to predict metabolic roles
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Network analysis to identify functional modules containing MPN_038
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Multi-omics data integration (proteomics, transcriptomics, metabolomics) to identify correlated changes
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Machine learning approaches to predict function based on multiple data types
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Comparative systems analysis across different Mycoplasma species
These approaches are particularly powerful for M. pneumoniae, which has been extensively studied as a model for systems biology due to its minimal genome .
What are the potential applications of MPN_038 research in understanding minimal genomes?
Research on MPN_038 contributes to broader questions in minimal genome biology:
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Defining the minimal set of proteins required for cellular life
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Understanding how proteins evolve new functions in reduced genomes
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Identifying novel essential functions not predicted by conventional annotations
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Developing synthetic biology applications based on minimal genome insights
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Informing therapeutic approaches targeting essential proteins in pathogens
M. pneumoniae serves as an excellent model organism for these studies due to its naturally reduced genome and the extensive experimental characterization of its proteome .
