Recombinant Lactobacillus plantarum Ribosome-recycling factor (frr)

What makes Lactobacillus plantarum suitable as a recombinant protein expression system?

Lactobacillus plantarum is particularly suitable as an expression host due to its status as a food-grade organism widely found in human gut, saliva, and fermented foods. It possesses robust survival mechanisms, including tolerance to both acid and bile salts, enabling it to survive passage through the gastrointestinal tract . The organism has well-established immunomodulating properties, increasing anti-inflammatory cytokines . For researchers, L. plantarum offers several advantages including: (1) availability of multiple constitutive promoters of varying strengths, (2) compatible high and low copy number plasmid systems, and (3) ability to be transformed with non-methylated DNA, eliminating the need for intermediate hosts like E. coli for plasmid construction . The complete genome sequence of L. plantarum WCFS1 has been determined, comprising 3,308,274 bp with 3,052 predicted protein-encoding genes, providing a solid genetic foundation for recombinant work .

What are the key genetic elements to consider when designing expression systems for frr in L. plantarum?

When designing expression systems for frr in L. plantarum, researchers must consider three main genetic elements that control expression levels:

• Promoter selection: Different constitutive promoters show varying strengths. The P11 promoter has been confirmed as strong for constitutive expression, while Ptuf33 and Ptuf34 provide additional options with different expression profiles .

• Plasmid copy number: Expression levels can be modulated through the choice between high and low copy number plasmid backbones. The pCDLbu-1ΔEc constructs containing origins of replication from L. buchneri CD034 plasmid pCD034-1 demonstrate approximately twofold higher product yields compared to low-copy alternatives .

• Translation efficiency optimization: The space between the Shine-Dalgarno sequence (ribosomal binding site) and the start codon significantly impacts protein production. Optimal spacing ranges from 5-12 nucleotides, with research showing that carefully designed RBS regions can substantially improve translational efficiency .

For frr expression specifically, these elements should be balanced to achieve sufficient expression without overburdening cellular machinery, as the ribosome-recycling factor plays a critical role in protein synthesis.

What is the most efficient protocol for transforming L. plantarum with frr expression constructs?

The most efficient transformation protocol for L. plantarum involves direct transformation with non-methylated DNA. L. plantarum CD033 has been specifically described as feasible for highly efficient transformation with non-methylated DNA, allowing direct transfer of ligation mixes or assembled PCR fragments . This approach eliminates the need for intermediate hosts such as L. lactis or E. coli for high-yield plasmid production.

The recommended procedure involves:

• Culture preparation: Grow L. plantarum cells to early-mid log phase (OD600 0.4-0.6) in MRS broth supplemented with 1% glycine

• Cell harvesting: Centrifuge at 4,000 × g for 10 minutes at 4°C

• Cell washing: Wash cells twice with ice-cold electroporation buffer (0.5M sucrose, 7mM potassium phosphate, 1mM MgCl2, pH 7.4)

• Resuspension: Resuspend cells in electroporation buffer at 1/100th the original culture volume

• Electroporation: Mix 50μL of cell suspension with 1-5μL DNA (50-500ng) in a pre-chilled electroporation cuvette (0.2cm gap)

• Pulse conditions: Apply pulse at 2.0 kV, 25 μF, and 400 Ω

• Recovery: Immediately add 950μL of recovery medium (MRS with 0.5M sucrose and 20mM MgCl2) and incubate at 30°C for 2-3 hours

• Plating: Plate on selective media containing appropriate antibiotics

This method enables very fast plasmid construction and manipulation, ideal for testing multiple genetic element combinations when optimizing frr expression .

How can I verify successful surface display of frr in recombinant L. plantarum?

Verification of successful surface display of frr in recombinant L. plantarum requires multiple complementary techniques:

• Western blotting: Similar to the approach used for gp85 protein verification, prepare cell lysates of the recombinant L. plantarum, separate proteins by SDS-PAGE, transfer to membrane, and probe with specific antibodies against frr . The expected molecular weight of your fusion protein should be calculated based on the combination of frr with any surface-display motifs (similar to the pgsA-gp85 fusion that produced a 69 kDa protein) .

• Flow cytometry: This technique provides quantitative assessment of surface display. The protocol involves:

  • Harvesting bacterial cells from culture

  • Washing cells with PBS

  • Incubating with primary antibody specific to frr

  • Washing to remove unbound antibody

  • Incubating with FITC-conjugated secondary antibody

  • Analyzing by flow cytometry to detect fluorescence intensity

Proper controls should include wild-type L. plantarum and L. plantarum containing the empty vector. The surface display is confirmed when cells with the recombinant construct show significantly greater fluorescence intensity than control cells . The quantification can be presented as a histogram showing the fluorescence intensity distribution.

• Immunofluorescence microscopy: This provides visual confirmation of surface localization and can complement flow cytometry data.

What promoter systems yield optimal expression levels for frr in L. plantarum?

Based on comparative studies of constitutive promoters in L. plantarum, several options exist for optimizing frr expression:

• P11 promoter: Confirmed to be highly effective for strong constitutive expression, particularly when combined with high copy number plasmid backbones. This is the recommended promoter when maximum frr expression is desired .

• Ptuf promoters: Both Ptuf33 and Ptuf34 (derived from elongation factor Tu genes) provide alternative expression profiles that may be more suitable if moderate expression is preferred. These promoters have been characterized in both L. plantarum and L. buchneri systems .

The optimal promoter choice depends on the specific research goals:

• For functional studies requiring high frr levels: P11 with high copy number plasmid

• For physiological studies where excessive expression might disrupt cell function: Ptuf promoters with lower copy number plasmids

• For comparative studies: Testing multiple promoters to find expression balance

The codon-adaptation index (CAI) should also be considered when designing the frr gene construct, as genes with higher CAI values tend to be more efficiently expressed in L. plantarum .

How does spacing between the Shine-Dalgarno sequence and start codon affect frr translation efficiency?

The spacing between the Shine-Dalgarno sequence (SDS) and the start codon significantly impacts translation efficiency of recombinant proteins like frr in L. plantarum. Research has shown that:

• Optimal spacing range: The distance between SDS and start codon can be varied between 5 and 12 nucleotides, with each spacing producing different translation efficiencies .

• Standard spacing: Most constructs typically use 9 nucleotides between the SDS and start codon, which provides good baseline expression .

• SDS optimization: Beyond spacing, matching the SDS more perfectly to the consensus sequence can further enhance translation. For example, using the RBS from highly expressed genes like the slpB gene from L. buchneri CD034 (which better matches the SDS core sequence) can improve translation efficiency .

For frr expression specifically, researchers should consider testing multiple SDS-to-start codon distances and selecting the optimal configuration based on protein yield measurements. When fine-tuning is required, experiments with spacing variants of 7, 8, 9, and 10 nucleotides are recommended as a starting point.

How can I assess the biological activity of recombinant frr expressed in L. plantarum?

Assessing the biological activity of recombinant frr expressed in L. plantarum requires multiple approaches:

• In vitro translation assays: Purified recombinant frr can be tested in cell-free translation systems to measure its ability to catalyze ribosome recycling. This involves:

  • Preparing post-termination ribosomal complexes

  • Adding purified frr protein

  • Measuring the dissociation of ribosomes from mRNA

  • Quantifying subsequent rounds of translation

• Growth curve analysis: Compare the growth characteristics of:

  • Wild-type L. plantarum

  • L. plantarum with empty vector

  • L. plantarum expressing recombinant frr

An active frr may provide growth advantages under certain stress conditions or when expressed in frr-deficient mutants.

• Polysome profiling: Analyze the distribution of polysomes (multiple ribosomes translating a single mRNA) using sucrose gradient centrifugation. Functional frr would be expected to increase the proportion of free ribosomes relative to polysomes by facilitating ribosome recycling.

• Complementation studies: Test whether the recombinant frr can complement temperature-sensitive frr mutants in model organisms like E. coli at non-permissive temperatures.

Each of these approaches provides different information about frr functionality, and combining multiple methods provides the most comprehensive assessment.

What immune responses are elicited by recombinant L. plantarum expressing surface-displayed frr?

While specific data on frr-expressing L. plantarum is not provided in the search results, we can infer the likely immune responses based on studies with other surface-displayed proteins in L. plantarum:

• Systemic antibody response: Recombinant L. plantarum expressing surface proteins typically triggers specific serum IgG production. For example, L. plantarum expressing pgsA-gp85 showed significant increases in serum IgG titers following oral immunization, with peak levels appearing after the third booster immunization .

• Mucosal immune response: Surface-displayed proteins on L. plantarum effectively stimulate mucosal immunity, particularly secretory IgA (sIgA) production. Studies with recombinant L. plantarum showed elevated sIgA levels in:

  • Bile samples

  • Duodenal-mucosal fluid

  • Other mucosal secretions

This dual immune response makes L. plantarum particularly valuable for applications requiring both systemic and mucosal immunity .

• Time course of response: Based on analogous studies, researchers can expect:

  • Initial antibody detection 1-2 weeks after first immunization

  • Significant increases following booster immunizations

  • Peak levels approximately 35 days after the third booster

For quantitative assessment, ELISA techniques measuring serum IgG and mucosal sIgA would be the recommended approach, with results expressed as sample-to-positive (S/P) values.

How does the metabolic burden of frr expression affect L. plantarum physiology?

The metabolic burden of frr expression on L. plantarum physiology is an important consideration that can influence experimental outcomes:

• Growth characteristics: High-level expression of recombinant proteins like frr can reduce growth rates and final biomass yields. This effect varies based on:

  • Promoter strength (P11 likely creates higher burden than Ptuf promoters)

  • Plasmid copy number (high copy vectors increase burden)

  • Translation efficiency (optimized RBS can increase expression but also metabolic load)

• Fermentation profile changes: L. plantarum normally produces lactic acid, ethanol/acetic acid, and carbon dioxide through fermentation of sugars . Excessive frr expression may alter this profile due to:

  • Redirection of metabolic resources toward protein synthesis

  • Changes in gene expression due to stress responses

  • Altered energy requirements

• Stress response markers: Monitoring stress-related genes and proteins can provide insights into the physiological impact of frr expression. Key markers include:

  • Heat shock proteins (GroEL, DnaK)

  • General stress response sigma factors

  • Metabolic adaptation enzymes

To minimize these effects while maintaining adequate expression, researchers should consider:

• Using inducible rather than constitutive promoters when precise control is needed

• Balancing promoter strength with plasmid copy number

• Optimizing culture conditions (temperature, pH, media composition) to support recombinant protein production

What advanced molecular strategies can enhance the stability and expression of frr in L. plantarum?

Several advanced molecular strategies can enhance the stability and expression of frr in L. plantarum:

• Codon optimization: Analyzing the genome of L. plantarum WCFS1 reveals its codon usage preferences. Adapting the frr gene sequence to match the most frequently used codons in highly expressed L. plantarum genes (those with high codon-adaptation index) can significantly improve translation efficiency .

• Chromosomal integration: While plasmid-based expression provides higher copy numbers, chromosomal integration of frr offers:

  • Greater stability without antibiotic selection

  • Reduced metabolic burden

  • More consistent expression across cell populations
    Integration can be achieved using:

  • Homologous recombination targeting non-essential regions

  • Site-specific recombination systems like Cre-lox

  • CRISPR-Cas9 based approaches for precise integration

• Fusion with stabilizing partners: Frr can be expressed as a fusion with well-expressed L. plantarum proteins (like SlpA) to enhance stability, while incorporating cleavage sites to allow post-translational separation if needed.

• Secretion system optimization: For applications requiring secreted frr:

  • The native Sec pathway can be leveraged by adding appropriate signal peptides

  • Signal peptides from highly secreted L. plantarum proteins are preferable

  • The Sec pathway components can be co-expressed to enhance secretion capacity

• Protective cultivation strategies: Two-stage cultivation processes where biomass is generated prior to induction of frr expression can reduce growth inhibition while maximizing protein yield.

These strategies can be combined and optimized based on specific research objectives and the desired application of the recombinant frr protein.

What are common pitfalls in L. plantarum frr expression systems and how can they be resolved?

Common pitfalls in L. plantarum frr expression systems and their solutions include:

When troubleshooting, a systematic approach is recommended:

• Verify construct integrity by sequencing

• Confirm transcript production by RT-PCR

• Assess protein production by Western blot

• Analyze cellular localization using fractionation techniques

• Test functional activity using targeted assays

How can I develop a standardized protocol for evaluating batch-to-batch consistency in frr expression?

Developing a standardized protocol for evaluating batch-to-batch consistency in frr expression requires rigorous quality control measures across multiple dimensions:

• Growth conditions standardization:

  • Define precise media composition (with lot numbers for complex ingredients)

  • Specify exact temperature, pH, and aeration conditions

  • Standardize inoculum preparation (growth phase, cell density)

  • Document growth curves with specific time points for harvest

• Expression quantification:

  • Protein yield determination via calibrated Western blotting

  • Assessment of surface display efficiency using flow cytometry with defined gating strategies

  • Specific activity measurements using functional assays

  • mRNA expression analysis via RT-qPCR

• Statistical quality control:

  • Establish acceptance criteria (mean ± 2SD) from at least 10 initial production runs

  • Implement control charts for key parameters

  • Define criteria for batch release/rejection

  • Document all deviations with root cause analysis

• Stability assessment:

  • Short-term stability at different temperatures

  • Freeze-thaw stability evaluation

  • Long-term storage behavior

  • Activity retention curves

• Reporting format:

  • Standardized batch record with all critical parameters

  • Certificate of analysis with actual vs. specification values

  • Trend analysis across multiple batches

  • Digital data archiving for retrospective analysis

The BioLector™ system, mentioned in the research literature, can be particularly useful for standardizing growth and expression monitoring across batches as it allows real-time, high-throughput assessment of multiple parameters simultaneously .