Recombinant Listeria monocytogenes serotype 4b UPF0348 protein LMOf2365_2080 (LMOf2365_2080)

What is the structural characterization of Recombinant Listeria monocytogenes serotype 4b UPF0348 protein LMOf2365_2080?

Recombinant Listeria monocytogenes serotype 4b UPF0348 protein LMOf2365_2080 (aa 1-390) belongs to the UPF0348 protein family. The complete amino acid sequence spans positions 1-390 of the native protein. The protein maintains its structural integrity when expressed as a recombinant form, and its three-dimensional configuration is crucial for its biological activity .

The methodological approach to structural characterization typically involves:

• X-ray crystallography to determine precise atomic structure

• Circular dichroism (CD) spectroscopy to assess secondary structure elements

• Mass spectrometry for molecular weight confirmation

• SDS-PAGE analysis for purity assessment and apparent molecular weight determination

Researchers should note that recombinant expression may introduce minor conformational differences compared to the native protein, particularly when using heterologous expression systems.

What expression systems are optimal for producing Recombinant Listeria monocytogenes serotype 4b UPF0348 protein LMOf2365_2080?

The choice of expression system significantly impacts protein yield, folding, and biological activity. For Recombinant Listeria monocytogenes serotype 4b UPF0348 protein LMOf2365_2080, several systems have been validated with varying advantages :

The methodological recommendation is to begin with E. coli for initial characterization studies. If protein activity requires post-translational modifications, progress to eukaryotic systems. For vaccine development and immunological studies, mammalian or insect cell expression may provide proteins with more native-like epitope presentation .

How can researchers optimize purification protocols for Recombinant Listeria monocytogenes serotype 4b UPF0348 protein LMOf2365_2080?

Purification of Recombinant Listeria monocytogenes serotype 4b UPF0348 protein LMOf2365_2080 requires a multi-step approach. The recommended methodology includes:

• Initial capture using affinity chromatography (if expressed with a tag)

• Intermediate purification using ion exchange chromatography

• Polishing step with size exclusion chromatography

Critical parameters to monitor include:

• Buffer composition: Optimize pH and salt concentration to maintain protein stability

• Temperature: Maintain 4°C throughout purification to minimize degradation

• Protease inhibitors: Include throughout early purification steps

• Reducing agents: Add if the protein contains disulfide bonds

For long-term storage, a buffer containing 50% glycerol in Tris-based solution is recommended, with storage at -20°C for regular use or -80°C for extended periods .

What are the immunological properties of Recombinant Listeria monocytogenes serotype 4b UPF0348 protein LMOf2365_2080 in vaccine development?

Recombinant Listeria monocytogenes serotype 4b LMOf2365_2080 protein has significant potential in vaccine development due to the unique immunological properties of Listeria monocytogenes. The protein can be utilized in several vaccine strategies:

• As a purified subunit vaccine component

• As an expressed antigen in recombinant Listeria monocytogenes vectors

• As part of multi-epitope vaccine constructs

The immunological mechanism leverages Listeria monocytogenes' ability to access the host cell cytosol, allowing expressed proteins to enter the major histocompatibility complex (MHC) class I antigen processing pathway . This cytosolic access is particularly valuable for developing CD8+ T cell responses, which are critical for protection against intracellular pathogens.

For optimal vaccine development, researchers should:

• Assess protein immunogenicity through epitope mapping

• Determine dosage requirements through dose-response studies

• Evaluate adjuvant requirements for enhanced immunogenicity

• Monitor both humoral and cell-mediated immune responses

Evidence from related studies demonstrates that recombinant Listeria monocytogenes expressing heterologous antigens can confer protection against virulent pathogens, as shown in lymphocytic choriomeningitis virus (LCMV) challenge models .

How does the genetic manipulation of Listeria monocytogenes serotype 4b affect UPF0348 protein LMOf2365_2080 expression and function?

Genetic manipulation of Listeria monocytogenes serotype 4b to modify UPF0348 protein LMOf2365_2080 expression requires careful consideration of several factors:

• Integration site selection: Stable site-specific integration of expression cassettes into the Listeria monocytogenes genome is critical for reliable expression

• Promoter selection: Constitutive versus inducible promoters affect expression timing and levels

• Signal sequence optimization: For efficient secretion of the protein

A methodological approach includes:

• Construction of integration vectors containing the UPF0348 protein LMOf2365_2080 gene

• Transformation into Listeria monocytogenes through electroporation

• Selection of stable integrants using appropriate antibiotics

• Verification of integration through PCR and sequencing

• Quantification of protein expression through Western blotting

Researchers should be aware that genetic modifications may affect bacterial virulence and growth characteristics. Comparative analysis between wild-type and recombinant strains is essential to identify any unintended consequences of genetic manipulation .

What analytical methods are most effective for characterizing host immune responses to Recombinant Listeria monocytogenes serotype 4b UPF0348 protein LMOf2365_2080?

Comprehensive characterization of host immune responses to Recombinant Listeria monocytogenes serotype 4b UPF0348 protein LMOf2365_2080 requires multiple analytical approaches:

• T cell response analysis:

  • Intracellular cytokine staining (ICS) for IFN-γ, TNF-α, and IL-2

  • ELISPOT assays for enumeration of antigen-specific T cells

  • T cell proliferation assays using CFSE dilution

  • Tetramer staining for identification of antigen-specific CD8+ T cells

• Antibody response analysis:

  • ELISA for detection of antigen-specific antibodies

  • Neutralization assays to assess functional antibody activity

  • Isotype analysis (IgG, IgM, IgA) to determine antibody class switching

• In vivo assessment:

  • Challenge studies to evaluate protective efficacy

  • In vivo depletion of specific immune cell populations (e.g., CD8+ T cells) to determine their contribution to protection

For data integration, multiparameter flow cytometry combined with computational analysis provides the most comprehensive assessment of immune responses. This allows for correlation between T cell functionality and protective efficacy .

How should researchers design experiments to compare UPF0348 protein expression across different Listeria monocytogenes serotypes?

When designing experiments to compare UPF0348 protein expression across different Listeria monocytogenes serotypes, researchers should implement:

• Standardized growth conditions:

  • Define precise media composition

  • Maintain consistent temperature (typically 37°C)

  • Standardize growth phase for harvest (mid-log phase recommended)

  • Control oxygen levels (facultative anaerobic conditions)

• Expression analysis methodology:

  • Quantitative real-time PCR for mRNA expression

  • Western blotting with densitometry for protein quantification

  • Mass spectrometry for absolute protein quantification

  • Flow cytometry for single-cell expression analysis

• Experimental controls:

  • Include housekeeping genes/proteins as internal controls

  • Compare with related UPF family proteins (e.g., UPF0316)

  • Include different growth conditions to assess regulatory mechanisms

• Statistical considerations:

  • Perform at least three biological replicates

  • Conduct power analysis to determine appropriate sample size

  • Apply appropriate statistical tests (ANOVA with post-hoc tests recommended)

This experimental design enables robust comparison across serotypes while controlling for variables that might influence expression levels .

What are the critical parameters for optimizing recombinant UPF0348 protein folding and stability?

Optimizing folding and stability of recombinant UPF0348 protein requires careful attention to several critical parameters:

• Expression temperature:

  • Lower temperatures (15-25°C) often improve folding by slowing protein synthesis

  • Temperature optimization should be system-specific (e.g., 15°C for E. coli, 27°C for insect cells)

• Buffer composition:

  • pH optimization typically within 6.5-8.0 range

  • Salt concentration (typically 100-500 mM NaCl)

  • Addition of stabilizing agents (glycerol, sucrose, arginine)

• Redox environment:

  • Addition of reducing agents (DTT, β-mercaptoethanol) for proteins with free cysteines

  • Oxidizing conditions for proteins requiring disulfide bonds

• Co-expression strategies:

  • Molecular chaperones (GroEL/GroES, DnaK/DnaJ)

  • Foldases (protein disulfide isomerases)

• Storage conditions:

  • 50% glycerol recommended for long-term storage

  • Avoid repeated freeze-thaw cycles

  • Store at -20°C for routine use or -80°C for extended storage

A systematic approach testing these parameters individually and in combination will yield optimal conditions for protein folding and stability.

How can researchers resolve discrepancies in experimental results when working with Recombinant Listeria monocytogenes serotype 4b UPF0348 protein?

When encountering discrepancies in experimental results with Recombinant Listeria monocytogenes serotype 4b UPF0348 protein, researchers should implement a structured troubleshooting approach:

• Protein quality assessment:

  • Verify protein identity through mass spectrometry

  • Assess purity through SDS-PAGE and size exclusion chromatography

  • Confirm protein conformation through circular dichroism or fluorescence spectroscopy

  • Check for batch-to-batch variation

• Experimental variables analysis:

  • Document all experimental conditions precisely

  • Assess reagent quality and preparation methods

  • Review equipment calibration and maintenance records

  • Verify cell line authentication and passage number

• Statistical approach:

  • Increase sample size to improve statistical power

  • Apply appropriate statistical tests for the data distribution

  • Conduct outlier analysis using established statistical methods

  • Consider blinded experimental design to reduce bias

• Cross-validation strategies:

  • Employ alternative experimental methodologies

  • Verify key findings in different laboratories

  • Compare results with related UPF proteins

  • Consult published literature for conflicting or supporting evidence

This systematic approach helps identify sources of variability and resolve experimental discrepancies .

What gene editing approaches are most effective for studying UPF0348 protein function in Listeria monocytogenes?

For studying UPF0348 protein function in Listeria monocytogenes, several gene editing approaches have proven effective:

• Homologous recombination:

  • Traditional method requiring selection markers

  • Typically used for gene knockouts and replacements

  • Requires long homology arms (500-1000 bp)

• CRISPR-Cas9 system:

  • More precise and efficient than traditional methods

  • Allows for marker-less gene editing

  • Can be used for gene knockouts, knock-ins, and point mutations

  • Requires optimization of guide RNA design for Listeria monocytogenes

• Site-specific recombination systems:

  • Integrase-based systems (e.g., phage integrase)

  • Allows for stable site-specific integration of expression cassettes

  • Useful for complementation studies and heterologous protein expression

• Inducible gene expression systems:

  • Tetracycline-responsive promoters

  • IPTG-inducible systems

  • Enables temporal control of gene expression

The methodological recommendation is to use CRISPR-Cas9 for initial gene knockout studies, followed by complementation with wild-type or mutant genes using site-specific recombination systems. This combination provides both gene deletion and functional complementation capabilities.

How can mass spectrometry be optimized for comprehensive characterization of UPF0348 protein post-translational modifications?

Comprehensive characterization of UPF0348 protein post-translational modifications (PTMs) requires optimized mass spectrometry (MS) approaches:

• Sample preparation strategies:

  • Enrichment methods for specific PTMs (e.g., phosphopeptide enrichment using TiO2)

  • Multiple proteolytic enzymes (trypsin, chymotrypsin, Glu-C) for improved sequence coverage

  • Offline fractionation (SCX, HILIC) to reduce sample complexity

• MS acquisition methods:

  • Data-dependent acquisition (DDA) for discovery

  • Parallel reaction monitoring (PRM) for targeted analysis

  • Data-independent acquisition (DIA) for comprehensive PTM mapping

  • Electron transfer dissociation (ETD) or electron capture dissociation (ECD) for labile modifications

• Data analysis workflow:

  • Search against multiple PTM databases

  • Apply false discovery rate control at both peptide and PTM site levels

  • Implement site localization algorithms (e.g., PTM score, Ascore)

  • Quantify PTM stoichiometry using label-free or labeled approaches

• Validation strategies:

  • Targeted MS methods for confirmatory analysis

  • Biochemical assays for functional validation

  • Site-directed mutagenesis to confirm PTM sites

This integrated approach provides comprehensive characterization of PTMs, their stoichiometry, and their biological significance .

What bioinformatic approaches should be used to analyze UPF0348 protein evolution across Listeria species?

Analyzing UPF0348 protein evolution across Listeria species requires a comprehensive bioinformatic approach:

• Sequence retrieval and alignment:

  • Collect UPF0348 protein sequences from all available Listeria species

  • Perform multiple sequence alignment using MUSCLE or MAFFT

  • Refine alignments manually to account for insertions/deletions

• Phylogenetic analysis:

  • Apply maximum likelihood (RAxML, IQ-TREE) and Bayesian inference (MrBayes) methods

  • Implement appropriate substitution models (LG, WAG, JTT)

  • Assess node support through bootstrap replication and posterior probabilities

  • Root trees using appropriate outgroups (related bacterial genera)

• Evolutionary rate analysis:

  • Calculate dN/dS ratios to identify selection pressures

  • Implement site-specific models to detect positively selected residues

  • Apply branch-site models to identify lineage-specific selection

• Structural mapping:

  • Map conserved and variable regions onto 3D protein structures

  • Identify functionally important domains through conservation analysis

  • Correlate evolutionary rates with structural features

• Comparative genomic context:

  • Analyze gene neighborhood conservation

  • Identify operon structures and potential co-evolving genes

  • Assess horizontal gene transfer events through anomalous GC content or codon usage

This comprehensive approach provides insights into UPF0348 protein evolution, functional constraints, and potential species-specific adaptations .

How can Recombinant Listeria monocytogenes serotype 4b UPF0348 protein be utilized in developing novel diagnostic methods for listeriosis?

Recombinant Listeria monocytogenes serotype 4b UPF0348 protein offers several approaches for developing novel diagnostic methods for listeriosis:

• Serological assays:

  • Development of ELISAs using purified UPF0348 protein as capture antigen

  • Lateral flow immunoassays for rapid point-of-care testing

  • Multiplexed bead-based assays combining UPF0348 with other Listeria antigens

• Molecular diagnostic approaches:

  • PCR primers targeting the UPF0348 gene for species and serotype identification

  • LAMP (Loop-mediated isothermal amplification) assays for field-deployable diagnostics

  • High-resolution melt curve analysis for strain differentiation

• Biosensor development:

  • Aptamer-based biosensors using UPF0348 protein as target

  • Surface plasmon resonance (SPR) systems for antibody detection

  • Electrochemical impedance spectroscopy for sensitive detection

• Multi-antigen approaches:

  • Combine UPF0348 protein with other serotype-specific markers

  • Develop serotype-specific fingerprinting methodology

  • Implement machine learning algorithms for pattern recognition

The methodological recommendation is to begin with ELISA development using purified recombinant protein, followed by validation against clinical samples. For point-of-care applications, lateral flow assays provide the best balance of sensitivity, specificity, and ease of use .

What considerations are critical when designing experiments to evaluate UPF0348 protein interaction with host immune receptors?

When designing experiments to evaluate UPF0348 protein interaction with host immune receptors, researchers should consider:

• Protein preparation:

  • Endotoxin removal is critical to prevent TLR4 activation artifacts

  • Protein folding verification through circular dichroism

  • Tagged versus untagged protein comparison to assess tag interference

  • Concentration range determination through dose-response studies

• Receptor interaction analysis:

  • Surface plasmon resonance (SPR) for binding kinetics

  • Bio-layer interferometry for real-time interaction analysis

  • Co-immunoprecipitation for complex identification

  • FRET/BRET for proximity assessment in living cells

• Cellular response assessment:

  • Reporter cell lines expressing specific pattern recognition receptors

  • Primary immune cell activation (DCs, macrophages, NK cells)

  • Cytokine profiling (Luminex, ELISA, intracellular cytokine staining)

  • Transcriptomic analysis to identify downstream signaling pathways

• In vivo validation:

  • Receptor knockout mouse models

  • Blocking antibody studies

  • Adoptive transfer experiments

  • Comparative analysis across different species

This comprehensive approach ensures robust identification of receptor interactions while accounting for potential experimental artifacts .

How should researchers approach the development of UPF0348 protein-based subunit vaccines against Listeria monocytogenes?

Development of UPF0348 protein-based subunit vaccines against Listeria monocytogenes requires a systematic approach:

• Antigen optimization:

  • Epitope mapping to identify immunodominant regions

  • Structure-based design to enhance stability and immunogenicity

  • Expression system selection for proper folding and post-translational modifications

  • Multivalent construct design incorporating multiple antigens

• Adjuvant selection:

  • TLR agonists (e.g., CpG, Poly I:C) for Th1-biased responses

  • Alum for enhanced antibody responses

  • Oil-in-water emulsions for balanced responses

  • Liposomal formulations for enhanced delivery

• Delivery system development:

  • Nanoparticle encapsulation for controlled release

  • Virus-like particles for enhanced immunogenicity

  • Needle-free delivery systems for mucosal immunity

  • Prime-boost strategies with varying delivery platforms

• Evaluation methodology:

  • In vitro antigen presentation assays

  • Ex vivo T cell activation studies

  • Challenge studies in appropriate animal models

  • Correlates of protection analysis

The recommended approach is to begin with a thorough epitope mapping of UPF0348 protein, followed by rational design of constructs incorporating immunodominant epitopes. Testing multiple adjuvant combinations is essential, with particular emphasis on those that promote strong CD8+ T cell responses, which are critical for protection against Listeria monocytogenes .

What emerging technologies show promise for enhancing recombinant UPF0348 protein expression and purification?

Several emerging technologies show significant promise for enhancing recombinant UPF0348 protein expression and purification:

• Cell-free protein synthesis systems:

  • Bypass cellular growth limitations

  • Rapid iteration of expression conditions

  • Direct incorporation of non-canonical amino acids

  • Elimination of cell lysis steps

• Continuous-flow protein production:

  • Integrated expression and purification

  • Reduced product degradation

  • Consistent protein quality

  • Scalable production capacity

• Machine learning-guided optimization:

  • Predictive modeling of expression parameters

  • Design of experiment (DoE) approaches

  • Process analytical technology integration

  • Real-time process adjustment

• Alternative affinity tags and purification methods:

  • Self-cleaving intein tags

  • Elastin-like polypeptide purification

  • Nanobody-based affinity chromatography

  • Continuous chromatography systems

• Synthetic biology approaches:

  • Genome-minimized expression hosts

  • Codon optimization algorithms

  • Ribosome binding site calculators

  • Synthetic promoter design

These technologies are expected to significantly improve protein yield, quality, and production efficiency in the coming years .

How might structural studies of UPF0348 protein contribute to understanding Listeria monocytogenes pathogenesis?

Structural studies of UPF0348 protein have significant potential to advance our understanding of Listeria monocytogenes pathogenesis through several avenues:

• Functional annotation:

  • Structure-based function prediction

  • Active site identification

  • Protein-protein interaction surfaces

  • Comparison with structurally similar proteins of known function

• Virulence mechanism insights:

  • Structural features that facilitate host cell interaction

  • Conformational changes associated with virulence

  • Potential involvement in secretion systems

  • Structural determinants of host specificity

• Drug target validation:

  • Identification of druggable pockets

  • Structure-based drug design

  • Rational design of inhibitors

  • Virtual screening for potential antimicrobials

• Host-pathogen interaction mapping:

  • Co-crystal structures with host proteins

  • Structural basis for immune evasion

  • Contribution to intracellular survival

  • Involvement in host cell modulation

The methodological approach should combine X-ray crystallography or cryo-electron microscopy with molecular dynamics simulations to understand both static structure and dynamic behavior. These insights could reveal previously unrecognized roles of UPF0348 protein in Listeria monocytogenes pathogenesis .

What computational approaches can predict novel functions and interactions for UPF0348 protein in Listeria monocytogenes?

Advanced computational approaches offer promising avenues for predicting novel functions and interactions for UPF0348 protein:

• Structure-based function prediction:

  • Threading and fold recognition

  • Active site prediction and comparison

  • Protein-protein docking simulations

  • Molecular dynamics to identify functional conformations

• Network-based approaches:

  • Protein-protein interaction network analysis

  • Guilt-by-association methods

  • Co-expression network integration

  • Pathway enrichment analysis

• Machine learning integration:

  • Deep learning for function prediction

  • Feature extraction from sequence and structure

  • Transfer learning from related proteins

  • Ensemble methods combining multiple predictors

• Comparative genomics:

  • Phylogenetic profiling across bacteria

  • Gene neighborhood analysis

  • Evolutionary covariance detection

  • Detection of horizontal gene transfer events

• Text mining and knowledge extraction:

  • Literature-based discovery

  • Semantic relationship extraction

  • Hypothesis generation systems

  • Knowledge graph construction and mining

These computational approaches, when combined, provide a comprehensive prediction landscape that can guide experimental validation of novel functions and interactions, particularly important for understudied proteins like UPF0348 .