Recombinant Mycoplasma pneumoniae Uncharacterized protein MG423 homolog (MPN_621)

What expression systems are recommended for recombinant MPN_621 production?

For recombinant production of MPN_621, E. coli has been successfully employed as an expression host system for the full-length protein (1-561 amino acids) with an N-terminal His-tag . When using E. coli as your expression system, consider the following methodological approaches:

• Vector selection: pET vectors with T7 promoter systems are commonly used for high-level expression.

• E. coli strain optimization: BL21(DE3) or Rosetta strains may improve expression of mycoplasma proteins which often have different codon usage.

• Expression conditions: Optimize temperature (often lower temperatures like 18-25°C improve solubility), IPTG concentration, and induction time.

• Solubility enhancement: Consider fusion partners such as SUMO, MBP, or GST if solubility issues arise.

The recombinant protein expression in E. coli has been shown to yield sufficient quantities for downstream applications including SDS-PAGE analysis .

What purification methods are most effective for His-tagged MPN_621?

Based on the available information on His-tagged MPN_621, the following purification workflow is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins is the primary method for His-tagged proteins.

• Buffer optimization: Use Tris/PBS-based buffer at pH 8.0 with 6% Trehalose for optimal stability .

• Polishing step: Consider size exclusion chromatography or ion exchange chromatography to achieve >90% purity as determined by SDS-PAGE .

• Quality control: Verify protein purity by SDS-PAGE and confirm protein identity through Western blotting or mass spectrometry.

For formulation of the final product, the protein has been successfully maintained in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 . This buffer composition helps maintain protein stability during storage and handling.

How should researchers store and handle recombinant MPN_621?

Proper storage and handling of MPN_621 is critical for maintaining protein integrity and activity. The following guidelines are recommended:

• Long-term storage: Store at -20°C/-80°C upon receipt. Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles .

• Working aliquots: Store at 4°C for up to one week .

• Reconstitution protocol:

  • Briefly centrifuge the vial prior to 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 a final concentration of 5-50% (50% is recommended) and aliquot for long-term storage at -20°C/-80°C

• Stability considerations: Repeated freezing and thawing is not recommended as it may lead to protein degradation or loss of activity .

What is known about post-translational modifications of MPN_621?

While the search results don't specifically mention phosphorylation of MPN_621, the context of phosphoproteome studies in Mycoplasma pneumoniae provides insight into potential approaches for investigating post-translational modifications of this protein.

Mycoplasma pneumoniae has a relatively small number of proteins that undergo phosphorylation. Studies have identified 58 phosphoproteins in M. pneumoniae through two-dimensional gel electrophoresis and mass spectrometry approaches . These studies used Pro-Q Diamond staining to detect phosphorylated proteins and Flamingo fluorescent dye to detect total proteins .

To investigate whether MPN_621 undergoes phosphorylation or other post-translational modifications, researchers should consider:

• Phosphoproteome analysis: Using the methodology described in the literature - two-dimensional gel electrophoresis with isoelectric focusing in two pH ranges (4-7 and 6-11) followed by staining with Pro-Q Diamond .

• Mass spectrometry verification: Phosphorylation sites can be identified through mass spectrometry analysis of tryptic peptides.

• Kinase/phosphatase mutant studies: Comparing protein modifications in wild-type strains versus mutants defective in protein kinases (HPrK, PrkC) or the protein phosphatase (PrpC) .

Research has shown that the protein kinase PrkC in M. pneumoniae phosphorylates several cell adhesion and surface proteins. To determine if MPN_621 is a substrate of PrkC, researchers could compare its phosphorylation status in wild-type and prkC mutant strains, as done for other proteins .

How might MPN_621 function in the context of Mycoplasma pneumoniae biology?

Although MPN_621 remains uncharacterized, we can make educated hypotheses about its potential function based on what is known about other characterized proteins in Mycoplasma pneumoniae:

• Cellular adhesion: Several proteins phosphorylated by PrkC in M. pneumoniae, such as HMW3, P41, and MPN474, are involved in cell adhesion. If MPN_621 is also a substrate of PrkC, it may play a role in adhesion to host cells .

• Metabolism: Many phosphoproteins in M. pneumoniae are involved in metabolic processes. Analysis of MPN_621's sequence for enzymatic domains or motifs may provide clues to a potential metabolic function.

• Virulence factor: Given that protein phosphorylation by PrkC is implicated in virulence in other bacteria, MPN_621 could potentially contribute to M. pneumoniae pathogenicity .

To investigate these hypotheses, researchers could:

• Generate MPN_621 knockout mutants and assess phenotypic changes

• Perform comparative proteomics between wild-type and MPN_621 mutant strains

• Conduct protein-protein interaction studies to identify binding partners

• Assess changes in virulence or host cell adhesion properties in MPN_621 mutant strains

What experimental approaches can be used to identify protein-protein interactions of MPN_621?

To elucidate the function of an uncharacterized protein like MPN_621, identifying its interaction partners is crucial. Several complementary approaches can be employed:

• Co-immunoprecipitation (Co-IP): Using antibodies against MPN_621 or its tag (His-tag) to pull down the protein along with its binding partners from M. pneumoniae lysates. The protein complexes can then be analyzed by mass spectrometry.

• Bacterial two-hybrid system: Adapting yeast two-hybrid methodology for bacterial proteins to screen for potential interaction partners.

• Crosslinking mass spectrometry (XL-MS): Chemical crosslinking of proteins in vivo or in vitro, followed by mass spectrometry analysis to identify proximal proteins.

• Protein array screening: Using purified MPN_621 to probe arrays containing other M. pneumoniae proteins to identify binding partners.

• Pull-down assays: Immobilizing purified His-tagged MPN_621 on Ni-NTA resin and incubating with bacterial lysates to capture interaction partners.

The search results mention that techniques such as yeast two hybrid, co-IP, and pull-down have been used to detect protein interactions in M. pneumoniae , making these appropriate methodologies for studying MPN_621 interactions.

How can researchers determine if MPN_621 is phosphorylated and identify its phosphorylation sites?

Based on the phosphoproteome studies of M. pneumoniae, the following experimental workflow can be used to investigate MPN_621 phosphorylation:

• Two-dimensional gel electrophoresis: Separate proteins from M. pneumoniae wild-type and kinase/phosphatase mutant strains (particularly the prpC phosphatase mutant where phosphorylation is often enhanced) .

• Phosphoprotein detection: Stain gels with Pro-Q Diamond for phosphorylated proteins and Flamingo fluorescent dye for total proteins .

• Protein identification: Excise spots of interest and identify proteins by mass spectrometry.

• Phosphopeptide enrichment: Use titanium dioxide (TiO2) or immobilized metal affinity chromatography (IMAC) to enrich phosphopeptides prior to mass spectrometry analysis.

• Mass spectrometry analysis: Employ collision-induced dissociation (CID) or electron transfer dissociation (ETD) for phosphopeptide sequencing to identify exact phosphorylation sites.

• Validation of phosphorylation sites: Create site-directed mutants (Ser/Thr to Ala) to confirm the functional significance of identified phosphorylation sites.

The phosphoproteome analysis of the prpC mutant revealed enhanced phosphorylation of several proteins not readily detected in the wild type . Similar approaches could be used to detect potential low-abundance phosphorylation of MPN_621.

What functional assays would be appropriate for characterizing the biochemical activities of MPN_621?

Without knowing the specific function of MPN_621, a systematic approach to functional characterization should include:

• Bioinformatic analysis: Identify conserved domains, motifs, or structural similarities to proteins with known functions.

• Enzymatic activity screening: Test for common enzymatic activities (kinase, phosphatase, protease, glycosidase, etc.) using commercial assay kits.

• Binding assays: Assess binding to various substrates including:

  • Nucleic acids (DNA/RNA binding assays)

  • Carbohydrates (glycan arrays)

  • Lipids (lipid binding assays)

  • Small molecules (thermal shift assays to identify stabilizing ligands)

• Structural studies: X-ray crystallography or cryo-EM to determine protein structure, potentially providing functional insights.

• Cell-based assays:

  • Adhesion assays with human epithelial cells

  • Cytotoxicity assays

  • Immunomodulation assays

If MPN_621 is phosphorylated by PrkC like other M. pneumoniae proteins involved in cell adhesion, researchers should prioritize adhesion assays to determine if it contributes to the bacterium's ability to adhere to host cells .

What approaches can be used to determine the three-dimensional structure of MPN_621?

Understanding the three-dimensional structure of MPN_621 would provide valuable insights into its function. Several complementary approaches can be employed:

For MPN_621 with 561 amino acids, X-ray crystallography or cryo-EM would be the most appropriate techniques for high-resolution structure determination.

How can researchers develop specific antibodies against MPN_621 for research applications?

Development of specific antibodies against MPN_621 would facilitate numerous research applications. Based on the approaches used for other Mycoplasma proteins , the following methodological workflow is recommended:

• Antigen preparation:

  • Use purified recombinant MPN_621 with His-tag

  • Alternatively, design synthetic peptides based on predicted epitopes (preferably from hydrophilic, surface-exposed regions)

  • Consider KLH conjugation for peptide antigens to enhance immunogenicity

• Immunization strategy:

  • Use BALB/c mice for monoclonal antibody development

  • Follow a prime-boost immunization schedule

  • For monoclonal antibodies, perform fusion of splenocytes with myeloma cell lines (SP2/0 or NS-1)

• Hybridoma screening:

  • Screen hybridomas by ELISA against the recombinant protein

  • Confirm specificity by Western blotting

  • Verify lack of cross-reactivity with other Mycoplasma proteins

• Antibody characterization:

  • Determine antibody isotype and affinity

  • Validate for different applications (Western blot, immunoprecipitation, immunofluorescence)

  • Assess performance in relevant assay conditions

The successful generation of monoclonal antibodies against Mycoplasma proteins has been demonstrated using similar approaches , making this a viable strategy for developing MPN_621-specific antibodies.

What genomic approaches can be used to investigate the role of MPN_621 in M. pneumoniae?

Given the small genome size of M. pneumoniae, several genomic approaches can be effectively applied to understand MPN_621 function:

• Gene knockout/knockdown strategies:

  • CRISPR-Cas9 genome editing (if established for M. pneumoniae)

  • Transposon mutagenesis

  • Antisense RNA approaches

• Complementation studies:

  • Re-introduction of wild-type or mutant MPN_621 into knockout strains

  • Analysis of phenotype restoration

• Transcriptomic analysis:

  • RNA-Seq comparing wild-type and MPN_621 mutant strains

  • Identification of genes co-regulated with MPN_621

• Comparative genomics:

  • Analysis of MPN_621 conservation across Mycoplasma species

  • Examination of syntenic regions and gene neighborhoods

• Regulatory element analysis:

  • Promoter characterization

  • Identification of transcription factor binding sites

This table summarizes the sequence homology of MPN_621 to its homologs in other Mycoplasma species:

Comparative genomic analysis would provide insights into the evolutionary conservation of MPN_621 and might suggest functional importance based on selection pressure across species.

How might MPN_621 contribute to M. pneumoniae pathogenicity?

Understanding how MPN_621 potentially contributes to pathogenicity requires integrating knowledge about M. pneumoniae virulence mechanisms with specific information about this protein:

• Cell adhesion hypothesis: If MPN_621 is phosphorylated by PrkC like other cell adhesion proteins (HMW3, P41, MPN474) , it may play a role in adherence to respiratory epithelium, which is a critical initial step in M. pneumoniae infection.

• Immunomodulation potential: Many surface proteins of pathogens interact with host immune receptors. MPN_621 could potentially modulate host immune responses if exposed on the bacterial surface.

• Metabolic adaptation: Uncharacterized proteins may contribute to the pathogen's ability to survive in the host environment by facilitating nutrient acquisition or metabolic adaptation.

• Cytotoxicity mechanisms: Some bacterial proteins contribute to host cell damage through direct cytotoxic effects or by triggering inflammatory responses.

Experimental approaches to test these hypotheses include:

• Comparing the virulence of wild-type and MPN_621 knockout strains in appropriate model systems

• Analyzing host cell responses to purified MPN_621 protein

• Investigating interactions between MPN_621 and host cellular components

• Determining if anti-MPN_621 antibodies have protective effects against infection

The phosphorylation of cell adhesion proteins by PrkC and their dephosphorylation by PrpC appear to regulate M. pneumoniae virulence , suggesting that if MPN_621 is within this regulatory network, it may contribute to pathogenicity.

How can researchers study the immunogenicity of MPN_621 in the context of M. pneumoniae infection?

To investigate the immunogenicity of MPN_621 during M. pneumoniae infection, researchers can employ these methodological approaches:

• Serological analysis:

  • Screen sera from M. pneumoniae-infected patients for antibodies against recombinant MPN_621

  • Compare antibody titers between acute and convalescent samples

  • Assess correlation between anti-MPN_621 antibody levels and disease severity

• T cell response studies:

  • Identify potential T cell epitopes in MPN_621 using prediction algorithms

  • Test peripheral blood mononuclear cells (PBMCs) from patients for reactivity to MPN_621 peptides

  • Characterize the T cell subsets and cytokine profiles in response to MPN_621

• Vaccine potential assessment:

  • Evaluate protective efficacy of MPN_621 immunization in animal models

  • Determine correlates of protection

  • Compare recombinant protein versus DNA vaccination approaches

• Immunomodulatory effects:

  • Assess the impact of MPN_621 on dendritic cell maturation and function

  • Measure cytokine production by immune cells exposed to MPN_621

  • Determine if MPN_621 affects MHC expression or antigen presentation

The methodologies used to study immunogenicity of Mycoplasma proteins, including the development of monoclonal antibodies , provide a foundation for investigating MPN_621's potential role in immune recognition and response during infection.

How does MPN_621 compare to homologous proteins in other bacterial species?

A comprehensive comparative analysis of MPN_621 with its homologs can provide evolutionary insights and functional clues:

• Homology identification:

  • MPN_621 is known to be a homolog of MG423 from Mycoplasma genitalium

  • Further homology searches using BLAST or HHpred can identify additional homologs in other bacterial species

• Sequence conservation analysis:

  • Multiple sequence alignment to identify conserved residues or motifs

  • Conservation mapping onto predicted structural models

  • Identification of species-specific adaptations through sequence divergence

• Phylogenetic analysis:

  • Construction of phylogenetic trees to understand evolutionary relationships

  • Assessment of selective pressure through dN/dS ratio analysis

  • Identification of horizontal gene transfer events

• Comparative genomics:

  • Analysis of genomic context of MPN_621 homologs

  • Identification of conserved gene neighborhoods

  • Assessment of gene fusion/fission events

• Functional comparison:

  • Review of functional data for characterized homologs

  • Identification of conserved functional domains

  • Potential for functional prediction based on characterized homologs

The current information indicates that MPN_621 is homologous to MG423 in M. genitalium , suggesting functional conservation between these closely related species.

What computational methods can predict the function of MPN_621 based on sequence and structure?

For an uncharacterized protein like MPN_621, computational prediction methods can provide valuable functional hypotheses:

• Sequence-based function prediction:

  • InterPro, Pfam, or SMART for domain identification

  • BLAST and PSI-BLAST for homology detection

  • Motif analysis using PROSITE or ELM

  • Gene Ontology term prediction

• Structure-based function prediction:

  • AlphaFold2 or RoseTTAFold for structure prediction

  • Dali or VAST for structural homology detection

  • CASTp or POCASA for binding pocket identification

  • Molecular docking to predict potential ligands

• Integrated approaches:

  • SIFTER or COFACTOR for combined sequence and structure prediction

  • Protein-protein interaction network analysis

  • Integrative functional prediction with ProFunc or ProKnow

• Machine learning methods:

  • Deep learning approaches for function prediction

  • Feature-based classifiers for functional category assignment

  • Text mining of scientific literature for functional associations

• Evolutionary analyses:

  • Evolutionary trace method to identify functionally important residues

  • Coevolution analysis to identify potential interaction partners

  • Phylogenetic profiling to identify functionally related proteins

These computational approaches should be used to generate hypotheses that can then be tested experimentally to confirm the predicted functions of MPN_621.