Recombinant Listeria monocytogenes serotype 4b UPF0356 protein LMOf2365_1049 (LMOf2365_1049)

What is the structural characterization of Recombinant Listeria monocytogenes serotype 4b UPF0356 protein LMOf2365_1049?

The Recombinant Listeria monocytogenes serotype 4b UPF0356 protein LMOf2365_1049 belongs to a family of proteins with unknown function (UPF0356). While the specific structure of LMOf2365_1049 is not fully characterized, related proteins from this serotype typically feature specific structural motifs. Similar recombinant proteins from this organism, such as LMOf2365_1590, contain 153 amino acids with specific membrane-associated domains .

For proper structural characterization, researchers should employ a combination of techniques:

• Begin with SDS-PAGE analysis to confirm protein purity (>90% is standard for research applications)

• Perform circular dichroism (CD) spectroscopy to assess secondary structure

• Use X-ray crystallography or NMR spectroscopy for tertiary structure determination

• Apply computational prediction methods to identify functional domains

What are the optimal storage and reconstitution conditions for this recombinant protein?

Based on similar recombinant proteins from Listeria monocytogenes serotype 4b, the following storage and reconstitution protocol is recommended:

Storage conditions:

• Store lyophilized protein at -20°C/-80°C upon receipt

• Aliquot reconstituted protein to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

Reconstitution protocol:

• Centrifuge the vial briefly before 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% for long-term storage

• The reconstituted protein is typically stable in Tris/PBS-based buffer, 6% Trehalose, pH 8.0

How can researchers validate the authenticity and activity of the recombinant protein upon receipt?

To ensure the received protein meets research standards, follow this validation workflow:

• Purity assessment:

  • Perform SDS-PAGE analysis and compare to the manufacturer's certificate of analysis

  • Confirm purity is greater than 90% as specified in quality control documentation

• Identity confirmation:

  • Western blot analysis using anti-His tag antibodies (if His-tagged) or specific antibodies against the protein

  • Mass spectrometry to confirm the molecular weight matches the expected size

• Activity testing:

  • Design functional assays specific to the predicted function of the UPF0356 protein family

  • Compare activity to positive controls and manufacturer's specifications

• Structural integrity:

  • Circular dichroism to confirm proper folding

  • Dynamic light scattering to assess aggregation state

What are the key considerations for designing experiments involving this recombinant protein?

When designing experiments with Recombinant Listeria monocytogenes serotype 4b UPF0356 protein LMOf2365_1049, apply these fundamental experimental design principles:

• Variable definition:

  • Clearly define independent variables (e.g., protein concentration, incubation time) and dependent variables (e.g., binding affinity, enzymatic activity)

  • Control variables must be rigorously maintained throughout experiments

• Experimental controls:

  • Include positive controls (known functional proteins)

  • Include negative controls (buffer-only, inactive protein variants)

  • Consider including related proteins from other Listeria serotypes for comparison

• Sample size determination:

  • Conduct power analysis prior to experiments to determine appropriate replication

  • Ensure sufficient technical and biological replicates

• Data collection planning:

  • Pre-determine data collection timepoints and methods

  • Establish clear criteria for data inclusion/exclusion

How should researchers approach experimental design when investigating potential functions of the UPF0356 protein family?

Given the "unknown function" designation of the UPF0356 family, a systematic approach to functional characterization is necessary:

• Comparative genomic analysis:

  • Align LMOf2365_1049 with homologous proteins from related bacteria

  • Identify conserved domains and motifs that suggest function

  • Map genomic context to identify operons or functional clusters

• Structural prediction and analysis:

  • Use computational tools to predict binding pockets or catalytic sites

  • Conduct docking simulations with potential substrates

  • Generate structural models to guide mutagenesis studies

• Systematic functional screening:

  • Develop a tiered experimental approach:

• Knockout/complementation studies:

  • Generate knockout strains in Listeria

  • Perform complementation with the recombinant protein

  • Compare phenotypes under various stress conditions

What methodological approaches can researchers use to investigate protein-protein interactions involving LMOf2365_1049?

To systematically investigate protein-protein interactions of LMOf2365_1049, researchers should follow this comprehensive workflow:

• In silico prediction:

  • Apply computational tools to predict potential interaction partners based on:

    • Structural complementarity

    • Co-expression patterns

    • Genomic context

• Primary interaction screening:

  • Yeast two-hybrid screening with a Listeria prey library

  • Affinity purification coupled with mass spectrometry (AP-MS)

  • Protein arrays using the recombinant protein as bait

• Validation of interactions:

  • Co-immunoprecipitation studies

  • Bioluminescence resonance energy transfer (BRET)

  • Förster resonance energy transfer (FRET)

  • Surface plasmon resonance (SPR) for quantitative binding parameters

• Functional characterization of interactions:

  • Mutagenesis studies targeting predicted interaction interfaces

  • Competition assays with predicted binding partners

  • Structural studies of protein complexes

• Data analysis framework:

How can researchers integrate multi-omics approaches to contextualize the role of LMOf2365_1049 in Listeria biology?

A comprehensive multi-omics strategy provides deeper insights into the biological context of LMOf2365_1049:

• Experimental design considerations:

  • Apply a systematic approach that integrates multiple data types

  • Design experiments with compatible sample preparation for cross-omics analysis

  • Include appropriate controls for each omics platform

  • Implement rigorous data management practices

• Multi-omics workflow:

• Data integration strategy:

  • Apply computational methods to integrate heterogeneous data types

  • Use machine learning approaches to identify patterns across datasets

  • Develop visualization tools for multi-dimensional data representation

• Validation experiments:

  • Design targeted experiments to test hypotheses generated from multi-omics data

  • Use genome editing (CRISPR-Cas9) to manipulate LMOf2365_1049 expression

  • Measure phenotypic outcomes across multiple scales (molecular to organismal)

What statistical and computational approaches are most appropriate for analyzing data from experiments involving recombinant LMOf2365_1049?

When analyzing experimental data involving recombinant LMOf2365_1049, researchers should implement these methodological approaches:

• Experimental design for robust statistical analysis:

  • Implement factorial designs to assess multiple variables simultaneously

  • Use randomization and blinding where appropriate

  • Include sufficient replication to achieve adequate statistical power

• Data preprocessing workflow:

  • Quality control assessment of raw data

  • Normalization appropriate to the experimental technique

  • Outlier detection and handling

• Statistical analysis framework:

• Advanced computational approaches:

  • Machine learning for pattern recognition in complex datasets

  • Network analysis to contextualize protein function

  • What-If Analysis for predictive modeling of protein behavior under varying conditions

  • Simulation studies to predict functional outcomes

• Visualization best practices:

  • Select appropriate visualization methods for different data types

  • Ensure visualizations accurately represent statistical significance

  • Use consistent formatting across related figures

How should researchers formulate research questions when investigating the structural-functional relationship of LMOf2365_1049?

Formulating effective research questions is critical for investigating LMOf2365_1049. Following the FINERMAPS framework , researchers should develop questions that are:

• Feasible - Addressable with available techniques and resources

• Interesting - Contributes novel insights to protein function understanding

• Novel - Explores unexplored aspects of the protein

• Ethical - Considers biosafety implications of working with Listeria proteins

• Relevant - Connects to broader understanding of bacterial protein function

• Manageable - Can be addressed within a reasonable research timeframe

• Appropriate - Matches available methodologies and expertise

Structured approach to research question formulation:

• Define knowledge gaps:

  • Assess current literature on UPF0356 family proteins

  • Identify unanswered questions about structure-function relationships

• Formulate primary research questions:

  • Focus on the relationship between specific structural elements and functional outcomes

  • Example: "How do conserved residues in the N-terminal domain of LMOf2365_1049 contribute to its biochemical function?"

• Develop supporting sub-questions:

  • Break down primary questions into testable components

  • Example: "What effect does site-directed mutagenesis of residue X have on the binding affinity to potential substrates?"

• Establish experimental approach:

  • Match each question with appropriate methodologies

  • Create a logical sequence of experiments that build upon each other

• Research question validation matrix:

What are the best practices for expression and purification of functional recombinant LMOf2365_1049?

To optimize expression and purification of functional LMOf2365_1049, researchers should follow this methodology:

• Expression system selection:

  • E. coli is commonly used for recombinant Listeria proteins

  • Consider BL21(DE3) or Rosetta strains for optimal expression

  • Evaluate alternative systems (yeast, insect cells) if E. coli yields poor results

• Expression optimization matrix:

• Purification strategy:

  • Implement immobilized metal affinity chromatography (IMAC) for His-tagged protein

  • Follow with size exclusion chromatography for higher purity

  • Consider ion exchange chromatography as an additional step

  • Evaluate protein folding and activity after each purification step

• Quality control checkpoints:

  • Purity assessment via SDS-PAGE (target >90%)

  • Western blot confirmation of identity

  • Activity assays to confirm functionality

  • Endotoxin testing for downstream applications

• Storage optimization:

  • Test stability in different buffer compositions

  • Evaluate lyophilization vs. solution storage

  • Add stabilizers such as trehalose (6%) when appropriate

  • Monitor activity retention over time under different storage conditions

How can researchers design effective control experiments when studying the biochemical properties of LMOf2365_1049?

Robust control experiments are essential for reliable interpretation of results:

• Positive control strategy:

  • Include well-characterized proteins from the same family

  • Use proteins with known activity in the same assay system

  • Consider homologous proteins from related bacterial species

• Negative control framework:

  • Include buffer-only controls in all experiments

  • Use heat-inactivated or denatured protein samples

  • Generate and test non-functional mutants (e.g., catalytic site mutations)

• Systematic control experiment design:

• Validation experiments:

  • Dose-response curves to establish concentration-dependent effects

  • Time-course experiments to determine optimal reaction times

  • Competition assays to confirm specificity of interactions

• Specialized controls for specific techniques:

  • For binding studies: non-specific binding controls

  • For enzymatic assays: substrate-only and enzyme-only controls

  • For structural studies: properly folded and misfolded protein comparisons

What approaches can researchers use to investigate post-translational modifications of LMOf2365_1049?

To comprehensively characterize post-translational modifications (PTMs) of LMOf2365_1049:

• PTM prediction and prioritization:

  • Apply computational tools to predict potential modification sites

  • Prioritize evolutionarily conserved sites for experimental validation

  • Consider bacterial PTMs common in Listeria (phosphorylation, acetylation, etc.)

• Analytical workflow for PTM identification:

• Site-specific validation strategies:

  • Mutagenesis of putative modification sites

  • Antibodies specific to the modified form

  • Chemical or enzymatic treatments to remove specific modifications

• Functional significance assessment:

  • Compare activity of modified vs. unmodified protein

  • Evaluate structural changes induced by modifications

  • Investigate differential PTMs under various physiological conditions

• Biological context experiments:

  • Determine when and where modifications occur in vivo

  • Identify enzymes responsible for adding/removing modifications

  • Map PTM-dependent protein-protein interactions

What data analysis pipeline is recommended for interpreting structural studies of LMOf2365_1049?

A comprehensive data analysis pipeline for structural studies should include:

• Raw data processing workflow:

  • Implement quality control metrics specific to each structural technique

  • Apply appropriate noise reduction and signal enhancement methods

  • Ensure data normalization consistent with field standards

• Structural data analysis approach:

• Structure validation framework:

  • Geometric validation (bond lengths, angles, Ramachandran plots)

  • Energy minimization to optimize structures

  • Comparison with homologous structures

  • Assessment of experimental data fit

• Functional annotation pipeline:

  • Identify conserved structural motifs

  • Map functional residues and domains

  • Assess surface properties (electrostatics, hydrophobicity)

  • Predict ligand binding sites and interaction surfaces

• Advanced computational analyses:

  • Molecular dynamics simulations to assess structural flexibility

  • Normal mode analysis to identify functional movements

  • In silico docking to predict binding partners

  • Machine learning approaches for structure-function prediction

How should researchers address solubility and stability challenges when working with recombinant LMOf2365_1049?

When facing solubility and stability issues with LMOf2365_1049:

• Systematic solubility optimization:

  • Implement a buffer screening approach:

• Expression strategy modifications:

  • Consider fusion tags known to enhance solubility (MBP, SUMO, GST)

  • Test low-temperature expression to slow folding

  • Co-express with chaperones to assist proper folding

  • Use cell-free expression systems for difficult proteins

• Refolding approaches for inclusion bodies:

  • Develop a step-wise dialysis protocol to gradually remove denaturants

  • Test rapid dilution vs. dialysis methods

  • Include stabilizing additives during refolding

  • Monitor refolding efficiency with activity assays

• Stability enhancement strategies:

  • Identify and mutate destabilizing residues

  • Add stabilizing co-factors or ligands

  • Optimize formulation with excipients like trehalose

  • Evaluate chemical crosslinking to stabilize quaternary structure

What strategies should researchers employ when encountering contradictory results in functional studies of LMOf2365_1049?

When confronted with contradictory experimental results:

• Systematic error identification:

  • Review experimental protocols for procedural differences

  • Validate reagent quality and consistency

  • Assess equipment calibration and performance

  • Examine environmental variables (temperature, humidity)

• Biological variability assessment:

  • Check protein batch consistency

  • Evaluate expression system variations

  • Consider post-translational modification differences

  • Test for presence of contaminating proteins or activities

• Methodological reconciliation approach:

• Reproducibility enhancement:

  • Increase sample size and replication

  • Blind analysis to reduce experimenter bias

  • Pre-register experimental protocols and analysis plans

  • Collaborate with independent laboratories for validation

• Data integration framework:

  • Apply meta-analysis techniques to synthesize contradictory results

  • Develop hypotheses that account for seemingly contradictory data

  • Design critical experiments to directly test competing hypotheses

  • Use What-If Analysis to model different scenarios that could explain contradictions

How can researchers effectively design experiments to elucidate the role of LMOf2365_1049 in pathogenesis?

To investigate the potential role of LMOf2365_1049 in Listeria pathogenesis:

• Experimental design foundation:

  • Clearly define research questions related to pathogenesis

  • Establish appropriate in vitro and in vivo model systems

  • Design experiments with appropriate controls

  • Ensure ethical considerations for pathogenesis studies

• Comparative virulence assessment:

• Host-pathogen interaction studies:

  • Investigate protein interactions with host factors

  • Assess impact on host cell signaling pathways

  • Evaluate effects on immune response

  • Determine contribution to intracellular survival

• Mechanistic dissection:

  • Structure-function analysis of domains involved in virulence

  • Identification of critical residues through site-directed mutagenesis

  • Time-course studies to position function in infection process

  • Cross-species comparison with homologs from other pathogens

• Translational considerations:

  • Evaluate potential as therapeutic target

  • Assess conservation across clinical isolates

  • Investigate immunogenicity and vaccine potential

  • Consider diagnostic applications

How can researchers best integrate findings about LMOf2365_1049 into the broader understanding of Listeria biology?

To effectively contextualize research on LMOf2365_1049:

• Knowledge integration framework:

  • Map findings to existing understanding of Listeria biology

  • Identify connections to known pathways and processes

  • Position results within evolutionary context of UPF0356 family

  • Develop models that incorporate new functional insights

• Collaborative research opportunities:

  • Engage with experts in complementary techniques

  • Establish consortia focused on functional annotation of UPF proteins

  • Leverage comparative biology approaches across bacterial species

  • Integrate findings with broader microbiology and protein science fields

• Future research directions:

  • Develop targeted experiments to address remaining knowledge gaps

  • Apply emergent technologies to challenging questions

  • Explore translational applications of basic research findings

  • Investigate environmental and clinical relevance

• Research communication strategies:

  • Publish in appropriate peer-reviewed journals

  • Present at relevant scientific conferences

  • Contribute data to appropriate databases and repositories

  • Engage with broader scientific community through reviews and commentaries