Recombinant Mycoplasma pneumoniae Uncharacterized protein MG280 homolog (MPN_399)

What is MPN_399 and what is its relationship to MG280?

MPN_399 is an uncharacterized protein encoded by the Mycoplasma pneumoniae genome. It is classified as a homolog of the MG280 protein from Mycoplasma genitalium, showing approximately 61% sequence identity . The protein has a full length of 287 amino acids and is identified by UniProt ID P75384. The gene has several synonyms in databases, including F11_orf287 and MP439 .

The homology between MPN_399 and MG280 suggests potential functional conservation between these two minimal genome bacteria. This relationship is particularly significant as both M. pneumoniae and M. genitalium are human pathogens with reduced genomes, indicating that conserved proteins between them may serve essential functions. Studying MPN_399 may therefore provide insights into the core functional requirements of Mycoplasma species.

How can researchers express and purify recombinant MPN_399?

Based on established protocols, researchers can express and purify recombinant MPN_399 using the following methodology:

Expression System Selection:

E. coli has been successfully used as an expression host for MPN_399, with the full-length protein (amino acids 1-287) expressed with an N-terminal His tag .

Expression Protocol:

• Clone the MPN_399 gene into an appropriate E. coli expression vector containing an N-terminal His tag sequence

• Transform the expression construct into E. coli (BL21(DE3) or similar strains are typically used for recombinant protein expression)

• Grow transformed bacteria in suitable media (such as LB with appropriate antibiotics)

• Induce protein expression with IPTG at optimal conditions

• Harvest cells by centrifugation

Purification Strategy:

• Lyse cells using appropriate buffer systems (typically Tris or phosphate-based buffers)

• Clarify lysate by centrifugation

• Purify using nickel-affinity chromatography (Ni-NTA or similar matrices)

• Consider additional purification steps such as ion-exchange or size-exclusion chromatography if higher purity is required

• Assess protein purity using SDS-PAGE (>90% purity is achievable)

Storage and Handling:

• For short-term storage: Buffer containing Tris/PBS with 6% Trehalose at pH 8.0

• For long-term storage: Lyophilize the protein or store in solution with 50% glycerol at -20°C/-80°C

• Avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

Reconstitution Protocol:

• Briefly centrifuge product vial before opening

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

• Add glycerol (5-50% final concentration) for long-term storage

• Aliquot appropriately to minimize freeze-thaw cycles

This protocol has been shown to produce recombinant MPN_399 with >90% purity suitable for various research applications.

Sequence-Based Functional Predictions:

• The hydrophobic regions in the N-terminus and throughout the sequence strongly suggest a membrane-associated protein, potentially involved in:

  • Cell envelope structure maintenance

  • Transport processes

  • Host-pathogen interactions

• The similarity to MG280 from M. genitalium (61% identity) suggests functional conservation, though MG280 is also uncharacterized .

Contextual Functional Hypotheses:

• Genomic context: Analysis of neighboring genes in the M. pneumoniae genome might provide clues about functional associations

• M. pneumoniae pathogenesis: Given that M. pneumoniae is a respiratory pathogen, MPN_399 could potentially be involved in:

  • Adherence to respiratory epithelium

  • Immune evasion mechanisms

  • Adaptation to host environment

• Potential involvement in antigenic variation: Some M. pneumoniae proteins participate in antigenic variation mechanisms involving RepMP elements , and MPN_399 might play a role in this process if it is surface-exposed.

Experimental Approaches to Determine Function:

• Gene knockout/knockdown studies to observe phenotypic effects

• Protein localization experiments to confirm membrane association

• Protein-protein interaction studies to identify binding partners

• Comparative genomics across Mycoplasma species to identify conservation patterns

Without experimental validation, these remain hypotheses that require further investigation to establish the true biological role of MPN_399.

What expression systems are most effective for producing recombinant MPN_399?

The choice of expression system for MPN_399 depends on research objectives, desired protein properties, and downstream applications. Based on available data and general recombinant protein expression principles:

coli Expression System:

As demonstrated in published research, E. coli has been successfully used to express recombinant MPN_399 . This system offers several advantages:

• High yield potential

• Cost-effectiveness

• Rapid growth and protein production

• Well-established protocols for His-tagged protein purification

• Demonstrated success with full-length MPN_399 (1-287 amino acids)

• Potential improper folding of complex proteins

• Lack of post-translational modifications present in native Mycoplasma

• Challenges with expressing membrane proteins (though successful for MPN_399)

Alternative Expression Systems to Consider:

• Insect Cell Expression (BEVS):

  • May provide better folding environment for complex proteins

  • More suitable for proteins requiring disulfide bond formation

  • Often yields higher amounts of properly folded membrane proteins

  • Could be considered if E. coli-expressed protein shows functional limitations

• Cell-Free Protein Synthesis:

  • Allows direct manipulation of the reaction environment

  • Eliminates toxicity concerns for the host

  • Particularly advantageous for membrane proteins

  • Enables incorporation of non-natural amino acids if desired for functional studies

• Mycoplasma-Based Expression:

  • Though technically challenging, expression in a related Mycoplasma species would provide the most native-like post-translational modifications and folding environment

Optimization Considerations:

E. coli remains the most practical first-choice expression system for MPN_399, with alternatives considered if specific experimental goals require properties not achievable in bacterial systems .

What experimental techniques are most appropriate for studying MPN_399 function?

Given the uncharacterized nature of MPN_399, a multi-faceted experimental approach is recommended to elucidate its function:

Localization Studies:

• Immunofluorescence microscopy using antibodies against recombinant MPN_399

• Fractionation of M. pneumoniae cells followed by Western blot analysis

• GFP/fluorescent protein tagging if genetic manipulation systems are available

• Membrane topology mapping using protease accessibility assays

Protein Interaction Studies:

• Pull-down assays using His-tagged recombinant MPN_399

• Co-immunoprecipitation followed by mass spectrometry

• Bacterial two-hybrid systems

• Surface plasmon resonance (SPR) or microscale thermophoresis to measure binding affinities with candidate partners

• Crosslinking approaches to capture transient interactions

Genetic Approaches:

• Gene knockout or knockdown (if feasible in M. pneumoniae)

• Complementation studies

• Overexpression phenotypic analysis

• Comparative genomics across clinical isolates

Functional Assays:

• If predicted to be membrane-associated:

  • Lipid binding assays

  • Membrane fusion/stability assays

  • Ion conductance measurements

• Host cell interaction assays:

  • Adhesion to respiratory epithelial cells

  • Immunomodulation assays

  • Cytotoxicity assays

Structural Studies:

• Circular dichroism for secondary structure assessment

• X-ray crystallography or cryo-EM for high-resolution structure (challenging for membrane proteins)

• Hydrogen-deuterium exchange mass spectrometry for solvent accessibility

• NMR studies of soluble domains

Biochemical Characterization:

• Enzymatic activity screening

• Lipid binding assays

• Post-translational modification analysis by mass spectrometry

• Stability and folding studies

Similar approaches have been successfully applied to study other M. pneumoniae proteins like MPN229, a single-stranded DNA-binding protein involved in DNA recombination , providing a methodological framework that could be adapted for MPN_399.

How can researchers design experiments to identify potential binding partners of MPN_399?

Identifying interaction partners of MPN_399 would provide crucial insights into its biological function. Several complementary approaches can be employed:

Affinity Purification-Mass Spectrometry (AP-MS):

• Express and purify His-tagged MPN_399 as described in previous protocols

• Immobilize purified protein on an affinity matrix

• Incubate with M. pneumoniae lysate under various conditions

• Wash extensively to remove non-specific interactions

• Elute bound proteins and identify by mass spectrometry

• Validate hits with reciprocal pull-downs

Proximity-Based Labeling:

• Express MPN_399 fused with a proximity labeling enzyme (BioID or APEX2)

• Allow in vivo biotinylation of proximal proteins

• Purify biotinylated proteins using streptavidin

• Identify by mass spectrometry

Crosslinking Mass Spectrometry (XL-MS):

• Use chemical crosslinkers in live M. pneumoniae or with purified components

• Digest crosslinked complexes and analyze by specialized mass spectrometry

• Identify crosslinked peptides to map interaction interfaces

• Particularly useful for membrane proteins like MPN_399

Protein Microarrays:

• Probe arrays containing M. pneumoniae or host proteins with labeled MPN_399

• Alternatively, immobilize MPN_399 and probe with labeled potential partners

• Detect interactions using fluorescence or other detection methods

Surface Plasmon Resonance (SPR):

• Immobilize MPN_399 on a sensor chip

• Flow potential binding partners over the surface

• Measure real-time binding and determine kinetic parameters

• Particularly useful for validating interactions identified by other methods

Yeast Two-Hybrid (Y2H) or Bacterial Two-Hybrid:

• Create fusion constructs of MPN_399 (considering membrane topology)

• Screen against a library of M. pneumoniae proteins

• Focus on soluble domains if working with membrane-associated regions

Experimental Design Considerations:

These approaches have been successfully applied to other bacterial membrane proteins and should provide valuable insights into MPN_399 function and its role in M. pneumoniae biology.

What bioinformatic approaches can help predict the function of MPN_399?

Bioinformatic analysis offers powerful tools for generating functional hypotheses about uncharacterized proteins like MPN_399. A comprehensive strategy would include:

Sequence-Based Analysis:

• Remote homology detection using PSI-BLAST, HHpred, or HMMER

• Identification of conserved domains using InterPro, Pfam, or SMART

• Transmembrane topology prediction using TMHMM, Phobius, or MEMSAT

• Signal peptide prediction with SignalP

• Functional site prediction using ScanProsite or MotifFinder

Structural Prediction:

• Tertiary structure prediction using AlphaFold2 or RoseTTAFold

• Template-based modeling if homologous structures exist

• Analysis of predicted binding pockets using CASTp or FTsite

• Molecular docking simulations with potential ligands

• Electrostatic surface analysis to identify potential interaction sites

Genomic Context Analysis:

• Examination of gene neighborhood in M. pneumoniae genome

• Comparison with syntenic regions in related Mycoplasma species

• Identification of gene fusion events in other organisms

• Co-occurrence patterns across bacterial genomes

• Phylogenetic profiling to identify functionally related genes

Network-Based Approaches:

• Protein-protein interaction prediction using STRING database

• Co-expression network analysis if transcriptomic data is available

• Pathways enrichment analysis for homologs with known functions

• Integration of multiple data sources using machine learning

Specific Tools for Mycoplasma Research:

• MycoBrowser or MycoBase databases for Mycoplasma-specific information

• Comparative analysis with MG280 from M. genitalium

• Integration with proteogenomic mapping data available for M. pneumoniae

Implementation Workflow:

• Begin with basic sequence analysis to identify domains and motifs

• Generate structural predictions to visualize potential functional sites

• Perform comparative genomics across Mycoplasma species

• Integrate with existing experimental data on M. pneumoniae

• Formulate testable hypotheses based on combined evidence

This integrative bioinformatic approach has proven valuable for functional annotation of hypothetical proteins in minimal genomes and would provide direction for experimental studies of MPN_399.

How does MPN_399 relate to the antigenic variation mechanisms in M. pneumoniae?

While direct evidence linking MPN_399 to antigenic variation is not established in the current literature, its potential involvement can be explored through several analytical and experimental approaches:

Background on M. pneumoniae Antigenic Variation:

M. pneumoniae employs antigenic variation mechanisms involving RepMP elements, particularly affecting the major surface protein P1. This variation occurs through homologous recombination between RepMP sequences located throughout the genome .

Potential Relationships of MPN_399 to Antigenic Variation:

• As a Target of Variation:

  • If MPN_399 is surface-exposed (suggested by its hydrophobic domains), it might itself be subject to antigenic variation

  • Analysis of MPN_399 sequences across clinical isolates could reveal polymorphisms indicative of immune selection

• As a Participant in Recombination:

  • MPN_399 could potentially participate in the recombination machinery, similar to MPN229 (a single-stranded DNA-binding protein that stimulates RecA-promoted recombination)

  • Its membrane association could potentially facilitate localization of recombination machinery to specific cellular regions

Experimental Approaches to Investigate Potential Involvement:

• Sequence Variation Analysis:

  • Compare MPN_399 sequences across multiple clinical isolates

  • Analyze selection pressure using dN/dS ratio calculations

  • Identify potential RepMP-like elements near the MPN_399 gene

• Interaction Studies:

  • Test for physical interactions between MPN_399 and known components of the recombination machinery (e.g., RecA, MPN229)

  • Assess binding to single-stranded DNA using electrophoretic mobility shift assays

• Functional Assays:

  • Examine if MPN_399 affects recombination rates using reporter systems

  • Test if MPN_399 expression changes during conditions that promote antigenic variation

• Comparative Genomics:

  • Analyze the conservation and genomic context of MPN_399 across Mycoplasma species with different antigenic variation mechanisms

  • Compare with M. genitalium MG280 in the context of recombination genes

Understanding any potential role of MPN_399 in antigenic variation would provide insights into M. pneumoniae pathogenesis and potentially identify new targets for therapeutic intervention.

What structural analysis techniques are most suitable for uncharacterized proteins like MPN_399?

Given the hydrophobic nature and potential membrane association of MPN_399, a combination of computational and experimental structural biology approaches is recommended:

Computational Structure Prediction:

• AI-based prediction: AlphaFold2 or RoseTTAFold can predict tertiary structure with increasing accuracy

• Comparative modeling: If structural homologs exist, tools like SWISS-MODEL or Phyre2 can generate models

• Topology prediction: TMHMM, MEMSAT, or Phobius can predict membrane-spanning regions

• Molecular dynamics simulations: To understand flexibility and behavior in membrane environments

High-Resolution Structural Techniques:

• X-ray crystallography: If the protein can be crystallized (challenging for membrane proteins)

• Cryo-electron microscopy (cryo-EM): Increasingly powerful for membrane proteins, especially in complexes

• Nuclear magnetic resonance (NMR) spectroscopy: For smaller soluble domains or in detergent micelles

Specialized Approaches for Membrane Proteins:

• Detergent screening: Systematic testing of detergents for optimal solubilization

• Lipid cubic phase crystallization: Alternative method for membrane protein crystallization

• Nanodiscs or lipid bilayer reconstitution: To study the protein in a more native-like environment

• Hydrogen-deuterium exchange mass spectrometry: To identify solvent-accessible regions

• Site-directed spin labeling combined with EPR: To determine distances between labeled sites

Integrative Structural Biology:

• Cross-linking mass spectrometry: To identify spatial proximity between residues

• Limited proteolysis: To identify domain boundaries and flexible regions

• Combination of multiple techniques: Integration of low and high-resolution data

Experimental Design Table for MPN_399 Structural Analysis:

This multi-technique approach acknowledges the challenges of membrane protein structural biology while providing complementary information to build a comprehensive structural understanding of MPN_399.

How can researchers develop antibodies against MPN_399 for experimental studies?

Developing specific antibodies against MPN_399 is crucial for many experimental applications. A systematic approach includes:

Full-Length Protein Approach:

• Use purified recombinant His-tagged MPN_399 as described in commercial sources

• Ensure proper folding and high purity (>90%) for immunization

• Consider detergent solubilization to maintain native-like conformation

Peptide-Based Approach:

• Identify hydrophilic, surface-exposed regions using sequence analysis and structural predictions

• Design 15-20 amino acid peptides from these regions

• Synthesize peptides and conjugate to carrier proteins (KLH or BSA)

• Multiple peptides from different regions increase chances of successful antibody production

Polyclonal Antibody Production:

• Immunize rabbits with purified protein or peptide conjugates

• Follow standard immunization schedule with multiple boosts

• Collect serum and purify IgG fraction

• Consider affinity purification against the immunizing antigen

Monoclonal Antibody Development:

• Immunize mice with the antigen

• Harvest B cells and fuse with myeloma cells to create hybridomas

• Screen hybridoma supernatants for specific binding

• Select and expand positive clones

• Purify antibodies from culture supernatant

Recombinant Antibody Methods:

• Display libraries (phage, yeast, or mammalian)

• Selection against the purified protein

• Affinity maturation if needed

Antibody Validation:

Special Considerations for MPN_399:

• If membrane topology predictions are available, target extracellular loops for antibodies intended for live-cell applications

• Consider generating antibodies against both N and C-terminal regions to determine orientation in the membrane

• For immunofluorescence, optimize fixation protocols that preserve membrane protein epitopes

• Validate specificity against the M. genitalium homolog (MG280) to assess cross-reactivity

Applications of MPN_399-Specific Antibodies:

• Cellular localization studies

• Expression analysis under different growth conditions

• Immunoprecipitation for interaction partner identification

• Functional studies using neutralizing antibodies (if applicable)

• Detection in clinical samples

Well-characterized antibodies against MPN_399 would serve as valuable tools for investigating its function, localization, and role in M. pneumoniae pathogenesis.

What are the optimal buffer conditions for handling recombinant MPN_399?

Based on available information about recombinant MPN_399 and general principles for handling membrane-associated proteins, the following buffer recommendations can be made:

Storage Buffer Composition:

Commercial recombinant MPN_399 is typically provided in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 . This formulation offers several advantages:

• Tris buffer (pH 8.0): Provides good buffering capacity in the physiological pH range

• Trehalose (6%): Acts as a protein stabilizer, particularly effective during freeze-thaw cycles

• Optional glycerol (up to 50%): Recommended for long-term storage to prevent freeze damage

Reconstitution and Working Buffers:

*Only if required for solubilization; specific detergent should be determined empirically

Handling Recommendations:

• Thawing: Thaw frozen aliquots rapidly at 37°C followed by immediate transfer to ice

• Concentration: If concentration is required, use centrifugal concentrators with appropriate molecular weight cutoff (10 kDa)

• Temperature: Handle at 4°C when possible to minimize degradation

• Avoid: Repeated freeze-thaw cycles, vigorous vortexing, and prolonged exposure to room temperature

• Aliquoting: Store in small single-use aliquots at -80°C for long-term storage

Buffer Optimization for Specific Applications:

• Binding assays: Include 0.005-0.01% detergent and 0.1-0.5% BSA to reduce non-specific binding

• Enzymatic assays: Buffer composition may need optimization based on specific activity

• Structural studies: Higher protein concentrations (1-10 mg/mL) may require additional stabilizers

• Cell-based assays: Use physiological buffers without components toxic to cells

Stability Assessment:

• Monitor protein stability using methods such as dynamic light scattering, thermal shift assays, or activity assays

• Optimize buffer conditions empirically for specific applications

• Consider using protein thermal shift assays to identify stabilizing buffer components

These recommendations provide a starting point for handling recombinant MPN_399. Specific applications may require further optimization based on empirical testing.

How does the amino acid sequence of MPN_399 compare to other bacterial proteins?

Comparative sequence analysis of MPN_399 reveals several important relationships with other bacterial proteins and provides insights into its potential function:

Homology to M. genitalium MG280:

The most direct relationship is with MG280 from M. genitalium, showing approximately 61% sequence identity . This high conservation between these minimal genome bacteria suggests an important functional role.

Hydrophobic Domains:

The N-terminal region (MLLFINRFAKTIILLFGMLVFLVLLGLGGAALYFKD) and C-terminal segment (AVSGGVLAVLIVSTVMTFLGSKK) show characteristic patterns of membrane-spanning domains. These regions likely form transmembrane helices and are commonly found in bacterial membrane proteins.

Conserved Motifs:

Several motifs within MPN_399 show conservation across different bacterial species:

• The LYFKDNAAKLYIDTRKS motif shows similarity to membrane protein interaction domains

• The SSSKFSVESINK region contains potential phosphorylation sites that are conserved in related proteins

Phylogenetic Distribution:

This distribution pattern suggests MPN_399 plays a specialized role in Mycoplasma biology rather than serving a universal bacterial function.

Structural Homologs:

While direct structural information is lacking, secondary structure predictions and hydrophobicity profiles suggest MPN_399 shares features with:

• Bacterial adhesins, particularly those with multiple membrane-spanning regions

• Transport proteins with multiple transmembrane domains

• Membrane anchors for larger protein complexes

Functional Implications:

The conservation pattern of MPN_399 suggests several functional possibilities:

• Involvement in Mycoplasma-specific membrane organization

• Possible role in host-pathogen interactions (common for conserved surface proteins)

• Potential involvement in essential cellular processes (given its conservation in minimal genomes)

These comparative sequence analyses provide valuable clues about MPN_399 function and establish a framework for experimental studies to define its role in M. pneumoniae biology.

What post-translational modifications have been observed or are predicted for MPN_399?

While specific post-translational modifications (PTMs) of MPN_399 have not been directly documented in the literature provided, potential modifications can be predicted based on sequence analysis and general knowledge of bacterial PTMs:

Phosphorylation:

• Several serine, threonine, and tyrosine residues in MPN_399 could serve as phosphorylation sites

• Key predicted sites include:

  • Serine residues at positions 61, 62, 63 (cluster of serines: SSSKFS)

  • Threonine residues at positions 128, 146

  • Tyrosine residues at positions 58, 228

Lipid Modifications:

• The N-terminal region contains motifs that could potentially undergo lipidation

• Specifically, the hydrophobic N-terminus might be subject to lipid attachment, which could anchor the protein to the membrane

Other Potential Modifications:

• Methylation of lysine residues (positions 68, 81, 85, 86)

• Acetylation of the N-terminus or internal lysines

• Glycosylation (although less common in bacteria than eukaryotes)

Experimental Approaches to Identify PTMs:

Functional Significance of Predicted PTMs:

• Regulation of Activity: Phosphorylation often serves as a regulatory switch in bacterial proteins

• Membrane Localization: Lipid modifications can influence membrane association and localization

• Protein-Protein Interactions: PTMs can create or disrupt interaction interfaces

• Stability and Turnover: Some modifications affect protein half-life in the cell

Mycoplasma-Specific Considerations:

• M. pneumoniae has a reduced genome, potentially limiting the repertoire of enzymes capable of adding PTMs

• The minimal PTM machinery likely focuses on essential modifications

• Comparison with PTMs identified in other Mycoplasma proteins could provide insights

Experimental Validation Strategy:

• Express and purify native MPN_399 from M. pneumoniae

• Perform comprehensive PTM analysis using mass spectrometry

• Compare PTM profile between different growth conditions

• Generate site-directed mutants of modified residues to assess functional impact

While these predictions provide a starting point for investigation, experimental validation remains essential to determine the actual PTMs present on MPN_399 and their functional significance.

What role might MPN_399 play in M. pneumoniae pathogenesis?

Although direct evidence for the role of MPN_399 in pathogenesis is not presented in the provided literature, several hypotheses can be formulated based on its predicted properties and the broader context of M. pneumoniae infection:

Membrane Localization and Adhesion:

• The hydrophobic regions in MPN_399 suggest membrane association

• Many surface-exposed proteins in M. pneumoniae contribute to adhesion to respiratory epithelium

• MPN_399 could potentially function as an adhesin or accessory protein for the adhesion complex

Immune Evasion:

• If exposed on the cell surface, MPN_399 might participate in antigenic variation mechanisms

• M. pneumoniae uses variation of surface proteins to escape host immune recognition

• The homology to MG280 in the genital pathogen M. genitalium suggests possible conserved virulence functions

Host Cell Manipulation:

• Membrane proteins often mediate interactions with host cellular components

• MPN_399 could potentially affect host cell signaling or membrane integrity

• Possible involvement in the characteristic cytopathic effects of M. pneumoniae infection

Experimental Approaches to Investigate Pathogenic Roles:

Clinical Relevance:

• Understanding MPN_399 function could potentially lead to:

  • New diagnostic approaches targeting this protein

  • Vaccine development if the protein proves immunogenic

  • Novel therapeutic approaches if it serves an essential function

Contextual Evidence:

• M. pneumoniae causes respiratory infections with significant morbidity

• The minimal genome of this pathogen suggests most proteins serve important functions

• Conservation with M. genitalium (another human pathogen) strengthens the case for a role in pathogenesis

• Homologous proteins in related species may offer clues about function

While these remain hypotheses until experimentally validated, investigating the role of MPN_399 in pathogenesis represents an important direction for future research that could contribute to our understanding of M. pneumoniae infections and potential intervention strategies.