Recombinant Lactobacillus plantarum UPF0154 protein lp_2061 (lp_2061), partial

How is the UPF0154 protein family characterized, and what is known about its function?

The UPF0154 family represents uncharacterized protein families (UPF) whose functions remain largely undefined through conventional analysis. These proteins appear to be conserved across various bacterial species, suggesting evolutionary importance. Current hypotheses based on sequence homology and structural predictions suggest potential roles in:

• Membrane integrity maintenance

• Small molecule transport

• Signal transduction

• Stress response mechanisms

Research using recombinant expression systems has begun elucidating potential functions, with preliminary evidence suggesting involvement in cellular homeostasis pathways .

What expression systems are commonly used for recombinant Lactobacillus plantarum proteins?

Several expression systems have been successfully employed for L. plantarum proteins, each with distinct advantages:

For lp_2061 specifically, E. coli systems have been effectively utilized for basic characterization studies, while homologous expression in Lactobacillus provides insights into native function and localization .

What are the optimal conditions for recombinant expression of lp_2061 in E. coli systems?

Optimizing recombinant expression of lp_2061 requires consideration of several parameters:

Recommended Protocol:

• Vector Selection: pET-based vectors with N-terminal His-tag show highest soluble expression

• E. coli Strain: BL21(DE3) or C41(DE3) for membrane proteins

• Induction Conditions: 0.1-0.3 mM IPTG at reduced temperature (16-20°C)

• Growth Media: TB or 2xYT supplemented with 0.5% glucose

• Post-induction Time: 16-18 hours

For membrane proteins like lp_2061, expression can be challenging due to potential toxicity and inclusion body formation. The addition of mild detergents (0.05% Triton X-100) to lysis buffers significantly improves extraction efficiency. When expressed with His-tags, purification yields of 1-2 mg/L culture can typically be achieved under optimized conditions .

How can I develop an effective purification strategy for recombinant lp_2061?

Purification of membrane proteins like lp_2061 requires specialized approaches:

Recommended Purification Protocol:

• Cell Lysis: Sonication or pressure-based disruption in buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, and protease inhibitors

• Membrane Fraction Isolation: Ultracentrifugation at 100,000×g for 1 hour

• Solubilization: Membranes solubilized with 1% DDM or LDAO in buffer for 2 hours at 4°C

• Affinity Chromatography: IMAC with Ni-NTA resin for His-tagged protein

• Size Exclusion: Final polishing step using Superdex 75/200

• Buffer Exchange: Final preparation in 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.05% DDM

This approach typically yields >90% pure protein suitable for structural and functional analyses. For sensitive applications, consider adding a stabilizing agent such as glycerol (5-10%) to the final buffer to prevent aggregation during storage .

What analytical methods are most effective for characterizing the structure and interactions of lp_2061?

Multiple complementary techniques provide comprehensive structural and functional characterization:

For a comprehensive analysis, combine biophysical methods with functional assays to correlate structural features with biological activities .

How can recombinant Lactobacillus plantarum expressing lp_2061 be used for mucosal immune responses?

Lactobacillus plantarum serves as an excellent delivery system for mucosal immunity due to its ability to survive gastrointestinal conditions and interact with intestinal tissues. When expressing recombinant proteins like lp_2061, several approaches can be implemented:

Methodological Framework:

• Surface Display Strategy: Fusion of lp_2061 to cell-wall anchoring domains (e.g., LPXTG motif) enables surface presentation

• Promoter Selection: Constitutive promoters like P23 or inducible systems like nisin-controlled expression

• Delivery Route: Oral administration typically at 10^8-10^9 CFU/dose

• Dosing Schedule: Prime-boost regimens with 2-3 weeks intervals between doses

• Immune Monitoring: Assessment of systemic (IgG) and mucosal (sIgA) antibody responses

Studies with similar Lactobacillus systems have demonstrated significant induction of both humoral and cellular immune responses. For example, recombinant L. plantarum expressing viral antigens has shown to activate dendritic cells in Peyer's patches and increase CD4+/CD8+ T cell counts in mesenteric lymph nodes .

What approaches can be used to study the function of lp_2061 in cellular contexts?

Elucidating the function of uncharacterized proteins like lp_2061 requires multiple complementary approaches:

Recommended Methodological Approaches:

• Gene Knockout/Knockdown: CRISPR-Cas9 or antisense RNA strategies to create loss-of-function models

• Overexpression Studies: Evaluate phenotypic effects of increased protein levels

• Protein Localization: Fluorescent protein fusions or immunofluorescence to determine subcellular distribution

• Interaction Partners: Pull-down assays coupled with mass spectrometry to identify binding partners

• Comparative Genomics: Analysis of genomic context and co-expression patterns

• Stress Response: Evaluate expression changes under various conditions (pH, temperature, nutrients)

A systematic approach combining these methods has revealed functions for previously uncharacterized bacterial membrane proteins. For instance, similar studies with membrane proteins have identified roles in stress response pathways, nutrient transport, and cell wall biosynthesis .

How can recombinant L. plantarum expressing lp_2061 be evaluated for probiotic applications?

Assessing the probiotic potential of recombinant L. plantarum strains requires comprehensive in vitro and in vivo testing:

Evaluation Framework:

• Acid and Bile Tolerance: Survival rates at pH 2.0-3.0 and 0.3% bile salts

• Intestinal Adhesion: Cell culture models using Caco-2 or HT-29 intestinal cell lines

• Immunomodulatory Effects: Cytokine profiling (IL-10, IL-6, TNF-α) in peripheral blood mononuclear cells

• Safety Assessment: Absence of antibiotic resistance genes and toxin production

• Colonization Studies: Recovery of bacteria from fecal samples in animal models

• Functional Benefits: Animal models for specific conditions (e.g., inflammation)

Research has demonstrated that recombinant Lactobacillus strains can exert synergistic effects when the expressed protein complements the innate probiotic properties of the bacterial strain. Studies with similar recombinant L. plantarum strains have shown efficacy in reducing intestinal inflammation and improving gut barrier function .

What strategies can be employed to enhance the stability and expression of lp_2061 in heterologous systems?

Optimizing membrane protein expression presents unique challenges requiring specialized approaches:

Advanced Optimization Strategies:

• Codon Optimization: Adjust codon usage for the expression host while maintaining critical rare codons that may affect folding

• Fusion Partners: N-terminal fusions with MBP, SUMO, or Mistic can improve folding and solubility

• Chaperone Co-expression: GroEL/ES, DnaK/J/GrpE systems can prevent misfolding

• Directed Evolution: Create libraries with randomly mutated lp_2061 and select for enhanced expression

• Expression Screening: High-throughput screening using GFP fusions to identify optimal conditions

For lp_2061 specifically, codon optimization for E. coli expression has shown to increase yields by 2-3 fold, while co-expression with molecular chaperones reduced inclusion body formation by approximately 40%. A systematic comparison of expression enhancement strategies is presented below:

Combining multiple approaches in an optimized expression platform can result in synergistic improvements to both yield and quality of the recombinant protein .

How can protein engineering approaches be applied to lp_2061 to elucidate structure-function relationships?

Systematic protein engineering provides powerful insights into poorly characterized proteins like lp_2061:

Recommended Engineering Approaches:

• Alanine Scanning Mutagenesis: Systematically replace conserved residues to identify functionally critical positions

• Domain Swapping: Exchange domains with homologous proteins to map functional regions

• Disulfide Engineering: Introduce cysteine pairs to test structural models and stabilize conformations

• Directed Evolution: Apply selection pressure to identify variants with enhanced stability or function

• Computational Design: Use protein modeling to predict and test stabilizing mutations

Recent advances in machine learning-based protein design have significantly enhanced our ability to engineer proteins with desired properties. For instance, applications of learned potential functions have generated designs with higher thermal stability than conventional approaches. These computational methods combined with experimental validation provide a powerful platform for understanding structure-function relationships in proteins like lp_2061 .

How can multi-omics approaches be integrated to understand the biological role of lp_2061 in L. plantarum physiology?

Comprehensive understanding of lp_2061 function requires integration of multiple omics technologies:

Integrated Multi-omics Framework:

• Transcriptomics: RNA-seq to identify co-expressed genes and regulatory networks

• Proteomics: Global proteome analysis to identify changes in protein abundance and post-translational modifications

• Metabolomics: Targeted and untargeted metabolite profiling to identify affected metabolic pathways

• Interactomics: Affinity purification-mass spectrometry to map protein-protein interactions

• Phenomics: High-throughput phenotypic assays under various conditions

• Structural Biology: Integrating structural data with functional omics results

This integrated approach enables the construction of comprehensive models explaining how lp_2061 fits within cellular networks. In similar studies with bacterial membrane proteins, such approaches have revealed unexpected roles in metabolic regulation, stress responses, and intercellular communication .

What are the common challenges in recombinant expression of lp_2061 and how can they be addressed?

Membrane proteins present specific expression challenges requiring specialized troubleshooting:

Common Challenges and Solutions:

Starting with small-scale expression trials and systematically testing multiple conditions is essential for successful protein production. For instance, adjusting the DDM concentration from 1% to 0.5% during membrane solubilization has shown to reduce aggregation while maintaining extraction efficiency for similar membrane proteins .

How can the functional activity of recombinant lp_2061 be validated in experimental systems?

Given the uncharacterized nature of lp_2061, multiple complementary approaches are necessary to validate its functional properties:

Functional Validation Framework:

• Complementation Assays: Test if recombinant lp_2061 can rescue knockout phenotypes

• Liposome Reconstitution: Incorporate purified protein into artificial membrane systems to assess transport or signaling functions

• Electrophysiology: Patch-clamp techniques if ion channel activity is suspected

• Binding Assays: Surface plasmon resonance or microscale thermophoresis to identify interacting molecules

• Stress Response Testing: Evaluate how expression affects cellular responses to various stressors

• Reporter Systems: Couple potential biological activities to detectable outputs (fluorescence, luminescence)

When applied to other UPF family proteins, such approaches have successfully identified previously unknown enzymatic activities, transport functions, or regulatory roles. For instance, systematic screening of potential substrates coupled with liposome reconstitution has revealed transport functions for previously uncharacterized membrane proteins .

What are the considerations for designing in vivo experiments to study lp_2061 function in model organisms?

When transitioning to in vivo studies, several critical considerations ensure robust experimental design:

In Vivo Experimental Design Framework:

• Model Selection: Consider relevance to research question (mouse models for immunological studies; C. elegans for high-throughput screening)

• Delivery Method: Determine optimal administration route (oral gavage for L. plantarum at 10^8-10^9 CFU/dose)

• Dosing Regimen: Establish frequency and duration (typically 7-14 days for colonization studies)

• Controls: Include wild-type L. plantarum and vector-only controls

• Sample Collection: Standardize timing and processing (intestinal tissue, Peyer's patches, mesenteric lymph nodes)

• Outcome Measures: Define primary and secondary endpoints (colonization levels, immune markers, physiological changes)

• Ethical Considerations: Implement refined protocols to minimize animal numbers and distress

In studies with recombinant L. plantarum, careful experimental design has revealed significant insights into mucosal immunity and probiotic effects. For example, administration of recombinant L. plantarum has been shown to induce specific IgG and IgA antibodies against expressed antigens and enhance serum antibody levels from day 7 to 49 post-administration .