Recombinant Mycoplasma pneumoniae Uncharacterized protein MG255 homolog (MPN_358)
Description
Overview
Recombinant Mycoplasma pneumoniae Uncharacterized protein MG255 homolog (MPN_358) is a protein derived from the bacterium Mycoplasma pneumoniae, also known as Mycoplasmoides pneumoniae . M. pneumoniae is a microorganism lacking a cell wall that causes chronic respiratory infections in humans . MPN_358 is a protein of currently unknown function, but research suggests its potential involvement in bacterial processes .
Basic Information
Production and Characteristics
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Expression: MPN_358 is often produced recombinantly in E. coli . The recombinant protein includes a Histidine tag (His-tag) at the N-terminus to facilitate purification .
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Formulation: Recombinant MPN_358 is typically available as a lyophilized powder .
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Reconstitution: The protein can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . Adding glycerol to a final concentration of 5-50% is recommended for long-term storage at -20°C/-80°C .
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Storage: Store at -20°C or -80°C upon receipt, with aliquoting to avoid repeated freeze-thaw cycles . Working aliquots can be stored at 4°C for up to one week . Liquid form is generally stable for 6 months at -20°C/-80°C, while lyophilized form is stable for 12 months at -20°C/-80°C .
Potential Functions and Research Applications
While MPN_358 is currently annotated as an uncharacterized protein, its identification and recombinant production allows for research into its potential roles within Mycoplasma pneumoniae . Research has shown that M. pneumoniae can induce inflammatory responses in macrophages, and identifying the proteins involved is vital for understanding the pathogenesis of infections .
Potential research avenues include:
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Protein Interactions: Identifying interacting partners of MPN_358 within M. pneumoniae to elucidate its functional role. Techniques like GST pull-down assays combined with mass spectrometry can be employed .
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Structural Studies: Determining the three-dimensional structure of MPN_358, which could provide insights into its function.
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Functional Assays: Developing and performing in vitro and in vivo assays to assess the protein's involvement in essential bacterial processes.
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Role in Pathogenesis: Investigating the potential role of MPN_358 in the infection process, including adhesion, motility, and immune modulation.
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order notes for customized fulfillment.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires advance notice and incurs additional charges.
The tag type is determined during production. If you require a specific tag type, please inform us, and we will prioritize its development.
Target Background
KEGG: mpn:MPN358
Q&A
What is MPN_358 and what are its basic structural characteristics?
MPN_358 is an uncharacterized protein from Mycoplasma pneumoniae, a minimal prokaryotic organism capable of independent survival without a host cell . The protein consists of 534 amino acids in its full-length form . As an uncharacterized protein, its three-dimensional structure has not been fully elucidated through crystallography or other structural biology techniques.
Methodological approach for structural characterization:
Researchers should consider employing a combination of computational prediction tools (such as AlphaFold2 or RoseTTAFold) alongside experimental approaches including X-ray crystallography, NMR spectroscopy, or cryo-EM. For initial characterization, secondary structure prediction using circular dichroism spectroscopy can provide valuable insights into the protein's folding patterns. Expression optimization in E. coli systems has been successful for recombinant production .
What expression systems are most effective for producing recombinant MPN_358?
The most documented expression system for MPN_358 is E. coli . The protein has been successfully expressed as a His-tagged recombinant protein with the full sequence (amino acids 1-534).
Methodological approach for optimizing expression:
When designing an experimental protocol for expressing MPN_358:
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Utilize the pET expression system in E. coli BL21(DE3) or similar strains
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Consider codon optimization for improved expression
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Test multiple induction conditions (IPTG concentration, temperature, duration)
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Evaluate solubility and employ solubilization strategies if inclusion bodies form
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Optimize purification using immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography
A systematic approach to expression optimization might include the following experimental conditions:
| Parameter | Test Conditions | Evaluation Method |
|---|---|---|
| E. coli strain | BL21(DE3), Rosetta(DE3), Arctic Express | SDS-PAGE, Western blot |
| Induction temperature | 16°C, 25°C, 37°C | Protein yield, solubility analysis |
| IPTG concentration | 0.1mM, 0.5mM, 1.0mM | Densitometry of protein bands |
| Induction time | 4h, 8h, overnight | Time-course sampling |
What experimental approaches are most suitable for determining the function of an uncharacterized protein like MPN_358?
As MPN_358 remains uncharacterized, a multi-faceted approach is necessary to elucidate its function.
Methodological approach for functional characterization:
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Comparative genomics: Analyze homologs in related species, particularly MG255 in M. genitalium, to identify conserved domains and potential functions
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Protein-protein interaction (PPI) studies: Employ yeast two-hybrid, pull-down assays, or proximity labeling approaches to identify interaction partners
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Gene knockout/knockdown: Generate CRISPR-Cas9 knockouts or antisense RNA to assess phenotypic changes
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Transcriptomic analysis: Compare gene expression profiles between wild-type and MPN_358-deficient strains
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Biochemical assays: Test for common enzymatic activities (kinase, phosphatase, protease, etc.)
For experimental design, researchers should consider the following structure:
| Approach | Specific Method | Expected Outcome | Controls |
|---|---|---|---|
| Interactome analysis | BioID proximity labeling | Identification of protein complexes | BirA* alone, non-relevant protein |
| Loss-of-function | CRISPR interference | Growth phenotype, morphological changes | Non-targeting sgRNA |
| Transcriptomics | RNA-seq of knockout vs. WT | Differentially expressed genes | Multiple biological replicates |
| Domain mapping | Truncation analysis | Functional domains identified | Full-length protein |
How can researchers effectively design experiments to study MPN_358's potential role in Mycoplasma pneumoniae pathogenesis?
M. pneumoniae is a significant pathogen causing community-acquired pneumonia, particularly in pediatric populations . Understanding MPN_358's potential role in pathogenesis requires specialized experimental approaches.
Methodological approach for pathogenesis studies:
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Adherence assays: Test whether MPN_358 influences bacterial attachment to respiratory epithelial cells
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Infection models: Compare wild-type and MPN_358-mutant strains in cell culture and animal models
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Immune response assays: Measure cytokine production and immune cell activation in response to purified MPN_358
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Localization studies: Determine subcellular localization and potential surface exposure
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Secretome analysis: Assess whether MPN_358 is secreted or membrane-associated
When designing infection experiments, consider the following structure:
| Experimental System | Parameters to Measure | Time Points | Analysis Method |
|---|---|---|---|
| Human bronchial epithelial cells | Adherence, invasion, cytotoxicity | 2h, 4h, 24h | Immunofluorescence, CFU counting |
| Mouse model | Lung colonization, inflammatory response | Days 1, 3, 7, 14 | Histopathology, qPCR, ELISA |
| Cytokine profiling | IL-6, TNF-α, IL-1β, IL-8 | 6h, 12h, 24h | Multiplex cytokine assay |
| Transcriptional response | Host gene expression changes | 2h, 8h, 24h | RNA-seq, pathway analysis |
How can contradictory findings regarding MPN_358's function be reconciled through rigorous experimental design?
When studying uncharacterized proteins like MPN_358, researchers may encounter conflicting results across different experimental systems or laboratories.
Methodological approach for resolving contradictions:
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Standardize experimental conditions: Ensure consistent protein preparations, cell lines, and assay conditions
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Multi-method validation: Confirm findings using orthogonal techniques
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Collaboration and replication: Establish inter-laboratory validation studies
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Meta-analysis: Systematically review all available data to identify patterns
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Context-dependent function: Investigate whether conflicting results stem from different physiological contexts
For example, if contradictory results emerge regarding MPN_358's subcellular localization, researchers should:
| Localization Method | Advantages | Limitations | Controls Needed |
|---|---|---|---|
| Immunofluorescence | In situ visualization | Antibody specificity issues | Pre-immune serum, knockout strain |
| Cell fractionation | Biochemical validation | Potential contamination | Multiple fractionation methods |
| GFP fusion | Live-cell imaging | Tag interference | Multiple tag positions, functional validation |
| Mass spectrometry | Unbiased approach | Sample preparation biases | Multiple extraction methods |
What computational strategies can predict MPN_358's function when experimental data is limited?
For uncharacterized proteins like MPN_358, computational approaches can provide critical insights to guide experimental design.
Methodological approach for computational prediction:
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Sequence-based analysis: Employ PSI-BLAST, HHpred, and HMMER to identify distant homologs
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Structural prediction: Use AlphaFold2 to generate structural models and identify structural homologs
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Functional domain prediction: Apply InterProScan and SMART to identify conserved domains
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Molecular docking: Predict potential ligands or interaction partners
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Genomic context analysis: Examine operonic structure and gene neighborhood
A systematic computational workflow might include:
| Computational Approach | Tools | Expected Insights | Validation Method |
|---|---|---|---|
| Homology detection | HHpred, PSI-BLAST | Potential functional homologs | Experimental testing of predicted activities |
| Structural prediction | AlphaFold2, I-TASSER | Fold classification, binding sites | Circular dichroism, limited proteolysis |
| Network analysis | STRING, GeneMANIA | Functional associations | Co-immunoprecipitation |
| Evolutionary analysis | ConSurf, Rate4Site | Conserved residues | Site-directed mutagenesis |
How can multi-omics approaches be leveraged to understand MPN_358's role in Mycoplasma pneumoniae biology?
Understanding an uncharacterized protein requires integrating multiple types of -omics data to build a comprehensive functional profile.
Methodological approach for multi-omics integration:
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Transcriptomics: Analyze co-expression patterns of MPN_358 with known genes
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Proteomics: Identify post-translational modifications and interaction partners
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Metabolomics: Detect metabolic changes in MPN_358 mutants
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Phenomics: Assess growth, morphology, and stress response phenotypes
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Systems biology modeling: Integrate data into predictive network models
A comprehensive multi-omics experimental design would include:
| Omics Layer | Technique | Sample Comparison | Integration Method |
|---|---|---|---|
| Transcriptomics | RNA-seq | WT vs. knockout, different growth conditions | Co-expression network analysis |
| Proteomics | LC-MS/MS | Pulldown vs. control, temporal dynamics | Protein-protein interaction networks |
| Metabolomics | Untargeted metabolomics | WT vs. knockout | Pathway enrichment analysis |
| Phenomics | High-content screening | Growth, morphology under stress | Clustering analysis |
What are the most rigorous approaches for validating hypothesized functions of MPN_358?
Validation is critical when studying uncharacterized proteins to avoid propagating incorrect functional annotations.
Methodological approach for function validation:
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Complementation studies: Restore function in knockout strains with wild-type and mutant variants
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Biochemical assays: Directly test predicted enzymatic activities with purified protein
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Site-directed mutagenesis: Mutate predicted functional residues to confirm importance
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Heterologous expression: Express in different bacterial species to assess function conservation
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In vivo relevance: Determine phenotypic consequences in infection models
A validation framework should include:
| Validation Approach | Experimental Design | Success Criteria | Alternative Explanations |
|---|---|---|---|
| Complementation | Knockout + plasmid with WT or mutant MPN_358 | Restoration of phenotype with WT but not mutant | Polar effects, expression levels |
| Biochemical validation | Purified protein with predicted substrates | Substrate conversion, binding constants | Non-physiological conditions |
| Structure-function | Mutants of predicted catalytic residues | Loss of function in specific mutants | Protein misfolding |
| In vivo significance | Animal model with WT vs. mutant strains | Attenuated virulence, changed colonization | Compensatory mechanisms |
What are the optimal conditions for storing and handling recombinant MPN_358 to maintain its stability and activity?
Proper storage and handling of recombinant proteins is crucial for experimental reproducibility.
Methodological approach for protein stability:
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Buffer optimization: Test multiple buffer compositions for maximal stability
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Storage conditions: Determine optimal temperature, concentration, and additives
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Freeze-thaw sensitivity: Assess activity loss after multiple freeze-thaw cycles
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Long-term stability: Monitor degradation over time under different conditions
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Activity preservation: Identify stabilizing agents that preserve function
A systematic approach to optimization might include:
| Parameter | Test Conditions | Evaluation Method | Expected Outcome |
|---|---|---|---|
| Buffer composition | pH 6.0-8.0, NaCl 50-500mM | Thermal shift assay, DLS | Optimal stability conditions |
| Storage temperature | 4°C, -20°C, -80°C | Activity assay after storage | Recommended storage temperature |
| Freeze-thaw cycles | 0, 1, 3, 5 cycles | Size exclusion chromatography | Maximum allowable cycles |
| Additives | Glycerol, sucrose, arginine | Aggregation monitoring | Effective stabilizing agents |
What are the most sensitive analytical techniques for detecting interactions between MPN_358 and potential binding partners?
Detecting protein-protein interactions for uncharacterized proteins requires sensitive and specific methods.
Methodological approach for interaction analysis:
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Surface Plasmon Resonance (SPR): Measure real-time binding kinetics
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Isothermal Titration Calorimetry (ITC): Determine binding thermodynamics
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Microscale Thermophoresis (MST): Assess interactions in solution with minimal protein consumption
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Biolayer Interferometry (BLI): Monitor association and dissociation rates
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Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Map interaction interfaces
When designing interaction studies, consider this framework:
| Technique | Advantage | Sample Requirements | Data Output | Limitations |
|---|---|---|---|---|
| SPR | Real-time kinetics | Immobilized protein (μg) | ka, kd, KD values | Surface effects |
| ITC | Label-free, in solution | 0.1-1 mg protein | ΔH, ΔS, ΔG, KD | High protein consumption |
| MST | Low sample amount | nM-μM concentration | KD values | Fluorescent labeling |
| BLI | Real-time, high-throughput | Immobilized protein (μg) | ka, kd, KD values | Surface effects |
| HDX-MS | Structural information | 10-100 μg protein | Binding interface | Complex data analysis |
