Recombinant Lactobacillus plantarum UPF0398 protein lp_1753 (lp_1753)

What is the UPF0398 protein lp_1753 in Lactobacillus plantarum?

UPF0398 protein lp_1753 is a protein of interest from Lactobacillus plantarum with the designation lp_1753 in its genome. The "UPF" prefix (Uncharacterized Protein Family) indicates that while the protein has been identified and sequenced, its complete biological function remains to be fully characterized. The protein can be produced recombinantly in expression systems such as E. coli for research purposes . Researchers working with this protein should note that L. plantarum has recently been reclassified taxonomically as Lactiplantibacillus plantarum in scientific literature, though both nomenclatures remain in use .

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

While the UPF0398 protein lp_1753 can be expressed in E. coli systems , research on recombinant L. plantarum proteins demonstrates that both heterologous (non-native) and homologous (native) expression systems can be employed. For homologous expression, L. plantarum itself can serve as the expression host, as demonstrated in studies with other recombinant proteins. For example, the SARS-CoV-2 spike protein has been successfully expressed in L. plantarum using the pSIP411 expression vector system . E. coli remains the predominant heterologous expression system due to its well-established protocols and high yield capabilities. The methodological approach should be determined based on the intended application, with homologous expression in L. plantarum being particularly valuable when studying probiotic applications or developing oral vaccine candidates .

What are the typical yields of recombinant L. plantarum proteins when expressed in E. coli?

Recombinant L. plantarum proteins expressed in E. coli typically yield in the milligram range per liter of culture. Specifically for the UPF0398 protein lp_1753, commercial preparations are available in quantities of 0.02 mg . Yield optimization requires careful consideration of induction conditions, media composition, and purification strategies. For comparison, when expressing recombinant proteins in L. plantarum itself, studies with the SARS-CoV-2 spike protein demonstrated that optimal induction involved 50 ng/mL SppIP at 37°C for 6-10 hours . This methodology provided significant protein expression with approximately 37.5% of bacterial cells showing positive expression compared to 2.5% in the parental strain .

How can codon optimization enhance the expression of recombinant L. plantarum proteins?

Codon optimization is a critical methodology for enhancing heterologous protein expression. For recombinant L. plantarum proteins, including UPF0398 protein lp_1753, codon optimization should be performed according to the codon usage bias of the expression host. Research has demonstrated that when expressing viral proteins in L. plantarum, codon optimization significantly improved expression efficiency . The methodological approach involves analyzing the frequency of codon usage in the expression host and modifying the gene sequence accordingly while maintaining the amino acid sequence. For instance, when expressing the SARS-CoV-2 spike protein in L. plantarum, researchers optimized codons according to L. plantarum's codon usage bias, which resulted in efficient expression and high antigenicity . This method is particularly important when the target protein originates from organisms with significantly different GC content or codon preferences compared to the expression host.

What purification strategies are most effective for recombinant L. plantarum proteins?

Effective purification of recombinant L. plantarum proteins requires a multi-step approach tailored to the protein's properties. For tagged recombinant proteins like the UPF0398 protein lp_1753, affinity chromatography represents the primary methodology. When designing expression constructs, researchers frequently incorporate histidine (His) or hemagglutinin (HA) tags to facilitate purification. Evidence from studies with other recombinant L. plantarum proteins demonstrates the effectiveness of this approach, with researchers successfully using anti-HA tag antibodies for detection and purification of recombinant spike protein . The methodological workflow typically involves:

• Cell lysis using appropriate buffer systems

• Initial clarification by centrifugation (12,000-15,000 g for 20-30 minutes)

• Affinity chromatography using the appropriate matrix

• Secondary purification through size exclusion or ion-exchange chromatography

• Quality assessment through SDS-PAGE and Western blotting

Protein purity should be verified through both Coomassie staining and Western blot analysis, with detection using specific antibodies against the target protein or incorporated tags .

How can researchers optimize induction conditions for maximum protein yield?

Optimization of induction conditions is a critical methodology for maximizing recombinant protein yield. For lp_1753 and other L. plantarum proteins, researchers should systematically evaluate multiple parameters:

Research with other recombinant L. plantarum proteins demonstrates that optimal conditions may vary by protein. For the spike protein expressed in L. plantarum, maximum yield was achieved with 50 ng/mL SppIP induction at 37°C for 6-10 hours . After optimization, researchers should verify protein expression through Western blot analysis using appropriate antibodies and quantify yield using Bradford or BCA protein assays .

What methodologies are recommended for assessing the stability of recombinant L. plantarum proteins?

Stability assessment of recombinant L. plantarum proteins, including lp_1753, requires evaluation under various environmental conditions relevant to potential applications. A comprehensive stability analysis methodology includes:

• Temperature stability: Expose purified protein to temperature range (4-50°C) for defined periods (20-60 minutes), then assess structural integrity and activity. Research with recombinant spike protein in L. plantarum demonstrated stability at temperatures up to 50°C for 20 minutes .

• pH stability: Subject protein to buffers ranging from acidic (pH 1.5) to neutral/alkaline (pH 7-9) for 30-60 minutes. Studies with other L. plantarum recombinant proteins showed remarkable stability even at pH 1.5 for 30 minutes .

• Salt concentration stability: Test protein in varying salt concentrations (0-500 mM NaCl).

• Bile salt tolerance: Evaluate stability in 0-0.5% bile salt concentrations for 2-3 hours. Previous research has shown that some recombinant proteins in L. plantarum actually exhibited increased expression when exposed to 0.2% bile salt .

• Storage stability: Assess activity retention after storage at different temperatures (-80°C, -20°C, 4°C) over time (days to months).

Following exposure to these conditions, researchers should analyze protein integrity by SDS-PAGE, Western blotting, and functional assays relevant to the protein's biological activity .

How can researchers evaluate the immunogenicity of recombinant L. plantarum proteins?

Immunogenicity evaluation of recombinant L. plantarum proteins requires a systematic approach combining in vitro and in vivo methodologies:

In vitro methods:

• Antigenicity assessment: Use enzyme-linked immunosorbent assay (ELISA) with specific antibodies or sera from immunized animals to detect epitope recognition.

• Dendritic cell activation: Measure upregulation of surface markers (CD80, CD86, MHC-II) and cytokine production after dendritic cell exposure to the recombinant protein.

• T-cell proliferation assays: Assess lymphocyte proliferation in response to protein stimulation using CFSE dilution or tritiated thymidine incorporation.

In vivo methods:

• Immunization protocol: Administer recombinant L. plantarum orally to mice (typically 10^9 CFU) in a prime-boost regimen. A typical protocol involves initial immunization on days 1-3, followed by booster immunizations on days 10-12 and 21-23 .

• Antibody response measurement: Collect serum samples to quantify IgG, IgG1, and other antibody isotypes by ELISA .

• Mucosal immunity assessment: Analyze fecal samples for secretory IgA (sIgA) levels .

• Cellular immunity analysis: Perform flow cytometry to quantify CD4+ T cells and IgA+ B cells in gut-associated lymphoid tissues .

Research with recombinant L. plantarum expressing viral proteins has demonstrated significant increases in serum IgG/IgG1 and fecal sIgA, along with enrichment of CD4+ T cells and IgA+ B cells, confirming the immunomodulatory potential of these recombinant bacteria .

How does recombinant L. plantarum modulate gut microbiota composition and function?

Recombinant L. plantarum strains have demonstrated significant effects on gut microbiota composition and function through several mechanisms. The methodological approach to investigating these effects includes:

These methodologies have revealed that recombinant L. plantarum can significantly reshape gut microbiota composition while enhancing beneficial metabolic and immunoregulatory functions .

What are the optimal methods for developing L. plantarum as a delivery vehicle for heterologous proteins?

Developing L. plantarum as a delivery vehicle for heterologous proteins requires a systematic methodology addressing expression system design, protein localization, and delivery efficiency:

• Vector selection: The pSIP411 expression vector system has demonstrated high efficiency for protein expression in L. plantarum. This vector contains the sakacin P (spp) promoter, which is inducible with the SppIP peptide pheromone .

• Signal peptide optimization: For surface display, researchers should clone the target gene with an endogenous signal peptide, such as signal peptide 1320 (ALX04_001320) from L. plantarum. This approach facilitates protein translocation to the cell surface .

• Anchoring domain selection: C-terminal anchoring domains can enhance surface display. Research has utilized target peptide D (DCpep: FYPSYHSTPQRP) and HA tags for both anchoring and detection purposes .

• Codon optimization: Target gene codons should be optimized according to L. plantarum codon usage bias to maximize expression efficiency .

• Transformation protocol: Electroporation remains the standard method for introducing recombinant plasmids into L. plantarum. Competent cells should be prepared according to established protocols, and transformation confirmed by colony PCR and sequencing .

• Expression verification: Surface display should be verified through multiple techniques:

  • Western blot analysis using antibodies against the target protein or incorporated tags

  • Transmission electron microscopy to visualize surface-displayed proteins

  • Indirect immunofluorescence assay to confirm accessibility

  • Flow cytometry to quantify the percentage of bacteria expressing the target protein

This methodological approach has successfully produced recombinant L. plantarum expressing viral proteins with expression rates of approximately 37.5% compared to 2.5% in parental strains .

How can researchers assess the passage stability of recombinant L. plantarum strains?

Evaluating the passage stability of recombinant L. plantarum strains is essential for research applications requiring consistent protein expression over multiple generations. The recommended methodology includes:

• Serial passage protocol: Culture the recombinant strain in appropriate media (typically MRS broth) with selective antibiotics. Transfer a fixed volume (typically 1%) to fresh media every 18-24 hours for multiple passages (minimum 5-10 passages) .

• Plasmid retention analysis: At designated passages (e.g., 1, 3, 5, 10), plate dilutions on selective and non-selective media. Calculate the percentage of antibiotic-resistant colonies to determine plasmid retention rates.

• Expression level quantification: Analyze protein expression at each passage using:

  • Western blot analysis with densitometry to quantify relative protein levels

  • Flow cytometry to determine the percentage of bacteria expressing the target protein

• Induction efficiency assessment: Compare protein expression levels following standardized induction protocols across different passages.

• Genetic stability verification: Periodically sequence the expression vector from different passages to identify any mutations or rearrangements.

What are the potential applications of lp_1753 and other recombinant L. plantarum proteins in vaccine development?

Recombinant L. plantarum proteins, including potentially lp_1753, offer promising applications for vaccine development. L. plantarum's status as a Generally Recognized As Safe (GRAS) organism makes it particularly suitable for oral vaccine delivery systems. Methodological approaches for investigating vaccine applications include:

• Antigen selection and optimization: Target antigens should be selected based on immunogenicity and protective potential. The protein sequence may require optimization to enhance stability and immunogenicity when expressed in L. plantarum .

• Adjuvant co-expression: Research has demonstrated enhanced immune responses when target antigens are co-expressed with immune mediators. For example, fusion constructs combining viral proteins with cytokines like IL-33 have shown enhanced immunomodulatory effects .

• Delivery format development: Researchers should evaluate multiple delivery formats:

  • Recombinant L. plantarum expressing the antigen directly

  • Heat-killed recombinant bacteria

  • Purified antigen with L. plantarum as an adjuvant

• Stability testing under gastrointestinal conditions: Methodologies should include evaluating protein stability at low pH (1.5), in the presence of bile salts (0.2-0.5%), and at body temperature (37°C) for extended periods .

• Immunization protocol optimization: Animal studies should compare different dosing schedules, typically using prime-boost regimens with 10^9 CFU administered orally on consecutive days (days 1-3), followed by boosters on days 10-12 and 21-23 .

Research has demonstrated that recombinant L. plantarum expressing viral proteins remains stable under gastrointestinal conditions (pH 1.5, 50°C, bile salts) and can induce significant immune responses, including elevated serum IgG/IgG1 and mucosal sIgA, making it a promising food-grade oral vaccine platform .

How can researchers employ systems biology approaches to understand the impact of recombinant L. plantarum on host immunity?

Systems biology approaches offer comprehensive methodologies for understanding the complex interactions between recombinant L. plantarum, the gut microbiome, and host immunity:

• Multi-omics integration: Researchers should combine:

  • Metagenomics (16S rRNA sequencing) to profile gut microbiota composition

  • Metatranscriptomics to assess gene expression in the microbiome

  • Metabolomics to identify altered metabolic pathways

  • Host transcriptomics to evaluate immune gene expression

• Immune profiling methodology:

  • Flow cytometry to quantify immune cell populations (CD4+ T cells, IgA+ B cells)

  • Cytokine profiling in serum and intestinal tissue

  • Antibody quantification (IgG, IgG1, sIgA)

• Network analysis: Construct correlation networks between:

  • Microbial taxa and immune parameters

  • Metabolites and immune function

  • Gene expression patterns and immunological outcomes

• In silico modeling: Develop computational models predicting:

  • Antigen processing and presentation pathways

  • Host-microbe interaction networks

  • Immune response dynamics following intervention

• Gnotobiotic models: Use germ-free or defined-flora animals to dissect microbiota-dependent and independent effects of recombinant L. plantarum.

Research has demonstrated that recombinant L. plantarum can significantly modulate gut microbial diversity and structure, correlating with enhanced immune parameters including increased CD4+ T cells, IgA+ B cells, serum IgG/IgG1, and mucosal sIgA . These findings suggest a complex interplay between the recombinant bacteria, gut microbiota, and host immunity that requires sophisticated systems biology approaches for comprehensive understanding.