Recombinant Lactobacillus plantarum UPF0210 protein lp_2507 (lp_2507)

What expression systems are suitable for producing Recombinant L. plantarum UPF0210 protein lp_2507?

Recombinant UPF0210 protein lp_2507 can be expressed and purified from multiple host systems, each offering distinct advantages:

• E. coli expression systems: Provide high yields and shorter turnaround times, ideal for initial structural studies .

• Yeast expression systems: Offer good yields with some post-translational modifications .

• Baculovirus expression systems: Provide more complex eukaryotic post-translational modifications necessary for correct protein folding .

• Mammalian cell expression systems: Offer the most extensive post-translational modifications, potentially retaining higher protein activity .

The selection of an appropriate expression system should be based on your specific research requirements, including needed protein yield, folding requirements, and post-translational modifications essential for function.

What are the common purification methods for recombinant lp_2507?

The purification strategy for recombinant lp_2507 typically involves:

• Cell lysis: Using mechanical disruption methods like homogenization, particularly effective for bacterial cell walls .

• Initial separation: Cell wall debris containing the protein can be isolated through centrifugation.

• Heat treatment: The sample can be subjected to 100°C for 5 minutes when using Western blot analysis .

• Affinity chromatography: If expressed with an affinity tag (commonly His-tag), this allows for selective purification.

• Size exclusion chromatography: For further purification based on molecular size.

For surface-displayed proteins, specialized extraction techniques may be necessary to efficiently isolate the protein from the cell wall components.

What detection methods can confirm successful expression of recombinant lp_2507?

Multiple complementary detection methods can be employed:

• Western blot analysis: The primary method to confirm protein expression and determine molecular weight. Anti-His tag antibodies are commonly used if the protein contains a His-tag .

• Fluorescence microscopy: If the protein is fused with GFP or FITC, direct visualization of expression and localization is possible .

• Flow cytometry: Can quantify expression efficiency in a population of recombinant bacteria. Studies with similar recombinant proteins have shown expression rates of 37.5% in modified strains compared to 2.5% in parental strains .

• Transmission electron microscopy (TEM): Useful for visualizing surface-displayed proteins and assessing whether protein expression affects bacterial morphology .

How can expression conditions be optimized to maximize yield and functionality of recombinant lp_2507?

Optimization requires systematic evaluation of multiple parameters:

Studies with similar recombinant proteins in L. plantarum have shown that:

• Highest protein yields were obtained with 50 ng/mL inducer concentration

• Optimal induction time was 6-10 hours at 37°C

• Early passages (1-5) maintained consistent expression levels, while later passages showed variable results

When optimizing, it's crucial to assess not only quantity but also quality (folding, activity) of the expressed protein through functional assays specific to lp_2507.

What strategies can mitigate the metabolic burden associated with recombinant lp_2507 expression in L. plantarum?

Recombinant protein production imposes metabolic stress on host organisms at multiple levels:

• Transcriptional burden: Even transcription without translation creates significant metabolic burden . Consider using:

  • Tightly regulated promoters to control expression timing

  • Lower copy number plasmids to reduce burden

  • Optimized codon usage according to L. plantarum preferences

• Translational burden: Can contribute to metabolic stress, especially when:

  • Protein folding issues arise (e.g., inclusion body formation)

  • High-level expression depletes cellular resources

• Post-translational processing: Surface display systems like pgsA may increase burden

Mitigation strategies:

• Use truncated anchor proteins (e.g., pgsA' instead of full-length pgsA) which has shown superior expression efficiency

• Implement fed-batch cultivation to maintain optimal nutrient availability

• Consider inducible systems that allow biomass accumulation before protein expression

• Monitor growth curves to identify optimal harvest points before growth inhibition becomes severe

Recombinant proteins may encounter folding issues that affect functionality:

• Computational analysis and rational design:

  • Analyze the lp_2507 sequence for potential folding obstacles

  • Identify domains that might benefit from flexible linkers

  • Remove or modify hydrophobic regions prone to aggregation

• Co-expression strategies:

  • Express with molecular chaperones to assist folding

  • Consider fusion partners known to enhance solubility (e.g., thioredoxin, SUMO)

• Expression conditions modifications:

  • Lower expression temperature (30°C instead of 37°C) to slow folding

  • Adjust media composition to provide necessary co-factors

  • Optimize inducer concentration to prevent overwhelming cellular machinery

• Post-expression processing:

  • In vitro refolding protocols if the protein forms inclusion bodies

  • On-column refolding during purification

Success indicators should include not only solubility but functional assays to confirm proper folding has occurred.

What are the most effective protocols for studying lp_2507 interactions with host immune cells?

To assess immunomodulatory properties of lp_2507, similar to studies with other L. plantarum surface proteins:

• Co-culture experiments:

  • Prepare RAW264.7 macrophage cells or primary immune cells

  • Co-culture with recombinant L. plantarum expressing lp_2507

  • Include appropriate controls: non-recombinant L. plantarum, empty vector controls

• Cytokine profiling:

  • Measure expression of key cytokines (IL-1β, IL-6, TNF-α, IFN-γ)

  • Assess NF-κB p65 activation as a marker of immune response

  • Use qRT-PCR for transcript analysis and ELISA for protein quantification

• Flow cytometry analysis:

  • Assess lymphocyte differentiation (CD3+CD4+ and CD3+CD8+ T cells)

  • Measure maturation markers on dendritic cells

  • Quantify intracellular cytokine production

• Stimulation experiments:

  • Pre-stimulate immune cells with immune activators (LPS, Poly(I:C))

  • Assess the capacity of lp_2507 to modulate the inflammatory response

  • Compare with known immunomodulatory proteins like IL-10

These methodologies can determine whether lp_2507 has immunostimulatory or immunoregulatory properties and characterize its mechanism of action.

How can the stability and storage conditions for recombinant lp_2507 be systematically evaluated?

A comprehensive stability assessment should include:

• Temperature stability testing:

  • Expose purified protein to temperature range (4°C, 25°C, 37°C, 50°C)

  • Test at multiple timepoints (24h, 48h, 72h, 1 week)

  • Assess remaining activity through functional assays

  • Analyze structural integrity via circular dichroism or thermal shift assays

• pH stability assessment:

  • Test stability at pH range (1.5-8.0)

  • Important for applications involving gastrointestinal conditions

  • Quantify activity retention at each pH point

• Salt concentration effects:

  • Evaluate stability in varying ionic strengths

  • Assess protein aggregation tendencies

• Freeze-thaw stability:

  • Subject protein to multiple freeze-thaw cycles

  • Quantify activity loss per cycle

  • Determine optimal cryoprotectant formulations

• Long-term storage optimization:

  • Compare different storage buffers

  • Test additives (glycerol, trehalose, albumin)

  • Establish shelf-life under optimized conditions

Results should be tabulated with specific activity retention percentages under each condition to develop evidence-based storage recommendations.

What strategies can effectively characterize the function of UPF0210 protein lp_2507 given its uncharacterized ("UPF") status?

As an uncharacterized protein family member, systematic approaches are needed:

• Bioinformatic analysis:

  • Perform sequence alignments with characterized proteins

  • Identify conserved domains that suggest function

  • Use structure prediction algorithms (AlphaFold2) to model 3D structure

  • Apply molecular dynamics simulations to predict binding pockets

• Protein interaction studies:

  • Conduct pulldown assays with cell lysates to identify binding partners

  • Perform yeast two-hybrid screening

  • Use protein arrays to identify possible interactions

  • Validate interactions with co-immunoprecipitation

• Activity screening:

  • Test for enzymatic activities common to bacterial proteins

  • Assess effects on cell signaling pathways

  • Examine impacts on bacterial physiology when overexpressed

• Loss-of-function studies:

  • Create knockout strains using CRISPR-Cas systems

  • Perform phenotypic characterization under various growth conditions

  • Conduct transcriptome analysis to identify affected pathways

• Localization studies:

  • Use fluorescence microscopy with GFP-lp_2507 fusions

  • Perform subcellular fractionation to determine natural localization

  • Assess temporal expression patterns during bacterial growth phases

The combination of these approaches can provide converging evidence about the function of this previously uncharacterized protein.

How can researchers troubleshoot low expression yields of recombinant lp_2507?

When encountering low expression yields, a systematic approach is necessary:

Specific recommendations for lp_2507:

• Consider using a truncated anchor system (pgsA') which has shown improved expression compared to full-length systems

• Empirically determine optimal induction parameters (time, temperature, inducer concentration)

• Monitor protein expression at different growth phases to identify ideal harvest points

What are the most effective approaches for distinguishing between native and recombinant lp_2507 in analytical assays?

This distinction is critical for accurate characterization:

• Tag-based differentiation:

  • Express recombinant lp_2507 with affinity tags (His, FLAG, HA)

  • Use tag-specific antibodies for selective detection

  • Employ tag-based purification to isolate only recombinant protein

• Mass spectrometry approaches:

  • Compare peptide mass fingerprints between native and recombinant proteins

  • Identify post-translational modifications that differ

  • Use stable isotope labeling to differentiate recombinant protein

• Specific antibody development:

  • Generate antibodies against unique epitopes present only in recombinant versions

  • Use epitope mapping to confirm specificity

  • Develop sandwich ELISA systems with tag-specific capture and protein-specific detection

• Expression level analysis:

  • Compare expression levels in recombinant vs. wild-type strains

  • Quantitative Western blotting with density analysis

  • qRT-PCR to distinguish expression levels at mRNA level

These approaches enable researchers to accurately quantify recombinant protein expression and distinguish it from background levels of native protein.

How can researchers assess whether recombinant lp_2507 maintains its native conformation and functionality?

Confirming proper folding and functionality requires multiple complementary approaches:

• Structural analysis:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Nuclear magnetic resonance (NMR) for structural characterization

  • X-ray crystallography for high-resolution structure determination

  • Comparative analysis with computational structure predictions

• Functional assays:

  • Develop activity assays based on predicted function

  • Compare activity between recombinant and native protein (if available)

  • Assess dose-response relationships to confirm specific activity

• Binding studies:

  • Surface plasmon resonance (SPR) to measure binding kinetics with potential partners

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters

  • Microscale thermophoresis for interaction analysis

• Stability assessment:

  • Thermal shift assays to determine melting temperature

  • Limited proteolysis to assess structural integrity

  • Size exclusion chromatography to evaluate aggregation state

• Cell-based assays:

  • Compare cellular responses to recombinant vs. native protein

  • Assess physiological effects in relevant model systems

  • Analyze signaling pathway activation patterns

The integration of these methods provides a comprehensive assessment of whether recombinant lp_2507 faithfully recapitulates the structure and function of the native protein.

How should researchers interpret contradictory results between different expression systems for lp_2507?

When faced with contradictory results across expression systems:

• Systematic comparison framework:

  • Create a standardized analytical pipeline for all expression systems

  • Include positive controls that perform consistently across systems

  • Document all variables between systems (media, growth conditions, extraction methods)

• Expression system-specific considerations:

  • E. coli: Evaluate effects of inclusion body formation and refolding

  • Yeast/insect cells: Assess impact of different post-translational modifications

  • Mammalian cells: Consider species-specific chaperone interactions

• Analytical approaches to resolve contradictions:

  • Perform parallel structural analysis (CD spectroscopy, thermal shift) across all versions

  • Develop multiple independent functional assays to triangulate true activity

  • Use mass spectrometry to identify system-specific modifications

• Biological relevance assessment:

  • Determine which system best recapitulates the native bacterial environment

  • Consider the research question when selecting the most appropriate data

  • Validate key findings in the most relevant model system

What statistical approaches are most appropriate for analyzing variability in recombinant lp_2507 expression across experimental replicates?

• Descriptive statistics:

  • Calculate means, standard deviations, and coefficients of variation

  • Generate box plots to visualize distribution of expression levels

  • Identify outliers using established statistical methods (Grubb's test)

• Inferential statistics for comparing conditions:

  • For normally distributed data: t-tests (paired or unpaired) or ANOVA for multiple conditions

  • For non-normally distributed data: Mann-Whitney U test or Kruskal-Wallis test

  • Post-hoc tests (Tukey's, Bonferroni) for multiple comparisons

• Variance components analysis:

  • Identify sources of variability (between batches, days, operators)

  • Perform nested ANOVA to quantify contribution of each factor

  • Use results to focus optimization efforts on highest-impact variables

• Power analysis:

  • Calculate appropriate sample sizes needed for statistical significance

  • Determine minimum detectable differences based on observed variability

  • Adjust experimental design accordingly

• Multivariate approaches:

  • Principal component analysis to identify patterns across multiple parameters

  • Multiple regression for modeling relationships between expression conditions and yields

  • Response surface methodology for optimizing multiple parameters simultaneously

These statistical approaches help distinguish meaningful effects from experimental noise and guide efficient optimization of expression conditions.

How can researchers design experiments to definitively determine whether lp_2507 has effects on host cell metabolism or immune responses?

Rigorous experimental design is crucial for establishing causal relationships:

• Controlled comparison framework:

  • Wild-type L. plantarum vs. lp_2507 knockout strain

  • Empty vector control vs. lp_2507 overexpression

  • Dose-response relationships with purified protein

  • Time-course experiments to distinguish primary from secondary effects

• Comprehensive metabolic analysis:

  • Metabolomics to assess global metabolic changes

  • Isotope labeling to track specific metabolic pathways

  • Respirometry to measure oxygen consumption and metabolic activity

  • Growth curve analysis under various nutrient conditions

• Immune response characterization:

  • Cytokine profiling (IL-1β, IL-6, TNF-α, IFN-γ)

  • Transcriptional analysis of immune response genes

  • Cell surface marker changes on immune cells

  • NF-κB pathway activation assessment

• Mechanistic validation:

  • Inhibitor studies to block specific pathways

  • Genetic knockdowns of putative interaction partners

  • Rescue experiments to confirm specificity

  • Structure-function studies with protein variants

• Cross-validation in multiple systems:

  • Different cell types (macrophages, dendritic cells, epithelial cells)

  • Ex vivo tissue models

  • In vivo models where appropriate

  • Comparison with known immunomodulatory proteins as benchmarks