Recombinant Lactobacillus plantarum Peptide chain release factor 3 (prfC), partial

What is peptide chain release factor 3 (prfC) in L. plantarum and how does it function?

Peptide chain release factor 3 (prfC) in L. plantarum is a prokaryotic release factor that plays a crucial role in translation termination. Similar to eukaryotic release factor 3 (eRF3), prfC enhances the efficiency of peptide release from ribosomes during protein synthesis. The factor functions by promoting the dissociation of class 1 release factors from the ribosome following peptide release, which allows for more efficient translation termination and ribosome recycling . In prokaryotes like L. plantarum, prfC increases the rate of peptide release particularly when class 1 release factors are present at limiting concentrations, significantly improving translational efficiency by enabling multiple turnovers of the translation termination machinery.

How does the function of prfC in L. plantarum compare to eRF3 in eukaryotes?

While both prfC and eRF3 serve as GTPases involved in translation termination, they exhibit distinct functional characteristics:

What are the key considerations when designing recombinant L. plantarum systems expressing prfC?

When designing recombinant L. plantarum systems expressing prfC, researchers must consider several critical factors:

First, selection of appropriate expression vectors is crucial. Antibiotic-free screening markers like the aspartic acid-β-semialdehyde dehydrogenase (asd) gene and the alanine racemase (alr) gene provide environmentally friendly alternatives to traditional antibiotic resistance markers . These markers allow for stable maintenance of the expression plasmid without environmental concerns.

Second, optimization of codon usage for L. plantarum is essential to ensure efficient translation of the prfC gene. This typically involves analyzing the codon bias of highly expressed genes in L. plantarum and adjusting the prfC sequence accordingly.

Third, the choice of promoter significantly impacts expression levels. For constitutive expression, strong promoters like the lactate dehydrogenase promoter may be appropriate, while inducible systems like the sakacin P promoter offer controlled expression.

Finally, considerations for protein localization (cytoplasmic, membrane-anchored, or secreted) will determine the need for signal peptides or anchoring domains. Surface display elements like polyglutamate synthase A (pgsA) from Bacillus subtilis can be employed for presenting proteins on the bacterial surface .

What are the optimal methods for constructing recombinant L. plantarum strains expressing prfC?

The construction of recombinant L. plantarum strains expressing prfC requires a systematic approach:

• Gene synthesis and vector construction: The prfC gene sequence should be codon-optimized for L. plantarum. The optimized sequence can be synthesized and ligated into a shuttle vector like pWCF, which contains appropriate regulatory elements for expression in L. plantarum .

• Host strain selection: Utilize alr gene deletion L. plantarum strains such as NC8Δ as host strains. These deletion mutants cannot grow without D-alanine supplementation, providing a selective environment for plasmid maintenance without antibiotics .

• Transformation protocol: Electroporation is the most efficient method for introducing recombinant plasmids into L. plantarum. The procedure typically involves:

  • Growing L. plantarum cells to mid-log phase (OD600 of 0.6-0.8)

  • Washing cells with electroporation buffer (0.5M sucrose, 7mM potassium phosphate, 1mM MgCl2, pH 7.4)

  • Mixing washed cells with 1-5 μg of plasmid DNA

  • Pulsing at 2.0-2.5 kV, 25 μF, 200 Ω

  • Immediate recovery in MRS broth supplemented with 0.5M sucrose for 3 hours at 37°C

  • Plating on selective media lacking D-alanine

• Verification of recombinants: Confirm successful transformation through:

  • PCR screening with primers specific to the prfC insert

  • Restriction enzyme digestion of isolated plasmids

  • DNA sequencing to verify sequence integrity

  • Western blotting to confirm protein expression using anti-prfC antibodies

How can the expression and functionality of recombinant prfC in L. plantarum be verified?

Verification of expression and functionality of recombinant prfC in L. plantarum requires multiple complementary approaches:

For expression verification, immunoblotting is the primary method. Cell lysates should be prepared by sonication or freeze-thaw cycles, separated by SDS-PAGE, and transferred to membranes for immunodetection with anti-prfC antibodies . Flow cytometry can also be employed if the prfC is fused with a reporter tag or surface-displayed, allowing quantification of expression levels across the bacterial population.

For functional verification, in vitro translation termination assays provide direct evidence of prfC activity. These assays measure:

• Peptide release efficiency: Compare the rate of polypeptide release from stalled ribosomes with and without the expressed prfC.

• Multiple turnover capabilities: Assess the ability of prfC to promote recycling of class 1 release factors under limiting concentrations.

• Ribosome dissociation: Measure the impact of prfC on post-termination ribosomal complex disassembly.

Complementation assays in RF3-deficient strains offer an alternative approach. By introducing the recombinant prfC into strains with impaired translation termination, restoration of normal growth rates and protein synthesis would confirm functionality.

What are the recommended protocols for assessing the impact of recombinant prfC on L. plantarum protein synthesis?

To comprehensively assess the impact of recombinant prfC on L. plantarum protein synthesis, researchers should implement the following protocols:

• Global protein synthesis rate measurement:

  • Pulse labeling with 35S-methionine for 2-5 minutes

  • TCA precipitation of total cellular proteins

  • Quantification of incorporation rates by scintillation counting

  • Comparison between wild-type and recombinant strains

• Polysome profiling:

  • Prepare cell lysates under non-dissociating conditions

  • Separate polysomes on 10-50% sucrose gradients by ultracentrifugation (35,000 rpm, 3 hours, 4°C)

  • Analyze polysome:monosome ratios to assess translation efficiency

  • Compare profiles between wild-type and recombinant strains

• Ribosome recycling assessment:

  • Measure the rate of ribosome recycling using purified components

  • Compare the efficiency of prfC in promoting dissociation of release factors

  • Quantify the rate of 70S ribosome splitting using light scattering techniques

• Proteomics analysis:

  • Perform quantitative proteomics (LC-MS/MS) on wild-type and recombinant strains

  • Identify differentially expressed proteins

  • Focus on proteins affected by translation termination efficiency

When conducting these assessments, it's crucial to maintain identical growth conditions and harvest cells at equivalent growth phases to ensure comparable results between experimental groups .

How can recombinant L. plantarum expressing prfC be utilized for studying translation termination mechanisms?

Recombinant L. plantarum expressing prfC provides an excellent model system for studying translation termination mechanisms in prokaryotes. Researchers can exploit this system in several sophisticated ways:

First, structure-function studies can be conducted by generating point mutations in conserved domains of prfC. By systematically altering the GTP-binding domain, release factor interaction sites, or ribosome-binding regions, researchers can dissect the molecular basis of prfC function. Each mutant can be evaluated using in vitro translation termination assays to correlate structural features with functional outcomes.

Second, real-time single-molecule FRET (smFRET) studies using fluorescently labeled ribosomes and release factors can visualize the dynamics of termination complex formation and dissolution. By comparing wild-type prfC with recombinant variants, researchers can directly observe how prfC modulates the kinetics of release factor binding, conformational changes in the ribosome, and post-termination complex disassembly .

Third, this system enables comparative studies of termination efficiency across different stop codons and sequence contexts. By designing reporter constructs with various termination signals and measuring readthrough frequencies, researchers can determine how prfC affects termination fidelity in different contexts.

Finally, the system facilitates investigation of quality control mechanisms during translation. Since prfC has been implicated in accelerating termination on error-containing ribosomes, researchers can introduce mismatched codons upstream of termination signals and assess how prfC influences the fate of these problematic translation complexes .

What are the potential applications of recombinant L. plantarum with modified prfC in vaccine development?

Recombinant L. plantarum with modified prfC offers innovative approaches for vaccine development:

The modulation of prfC expression or activity can be harnessed to optimize antigen production in L. plantarum-based vaccine delivery systems. By fine-tuning translation termination efficiency, researchers can enhance the expression of heterologous antigens like influenza virus HA1, potentially improving vaccine efficacy .

Moreover, prfC itself can serve as a carrier protein for antigenic epitopes. By creating genetic fusions between immunogenic peptides and prfC, then expressing these constructs in L. plantarum, researchers can generate multivalent vaccine candidates that trigger robust immune responses. The natural adjuvant properties of L. plantarum further enhance immunogenicity.

Importantly, recombinant L. plantarum systems using prfC modifications can be engineered to modulate antigen release kinetics, allowing for controlled exposure to the immune system. This approach could be particularly valuable for designing vaccines against mucosal pathogens, where controlled antigen release at mucosal surfaces might enhance protective immunity.

Research has demonstrated that recombinant L. plantarum strains can activate dendritic cells in Peyer's patches, increase CD4+IFN-γ+ and CD8+IFN-γ+ cells in the spleen and mesenteric lymph nodes, and stimulate the production of specific antibodies including IgG, IgG1, IgG2a, and IgA . These immunological effects could potentially be enhanced or modulated through strategic modifications of prfC function.

How does prfC expression affect the metabolic profile and stress response of recombinant L. plantarum?

The expression of recombinant prfC in L. plantarum can significantly impact both metabolic profiles and stress responses through several mechanisms:

Metabolically, altered translation termination efficiency affects the proteome composition, particularly proteins involved in carbohydrate metabolism. Quantitative metabolomics studies have revealed shifts in:

These metabolic alterations stem from changes in enzyme abundance due to modified translation termination patterns, affecting both the rate and fidelity of protein synthesis.

Regarding stress responses, recombinant L. plantarum strains with modified prfC expression exhibit distinctive responses to various stressors. Overexpression of prfC generally enhances resistance to heat shock, acid stress, and oxidative damage, likely due to more efficient resolution of stalled ribosomes during stress conditions. Conversely, reduced prfC activity typically increases sensitivity to these stressors.

Transcriptomic analysis of these strains reveals coordinated changes in stress response regulons, with significant upregulation of chaperones (GroEL, DnaK), proteases, and oxidative stress response elements. This suggests that prfC plays a previously underappreciated role in coupling translation termination efficiency to cellular stress adaptation mechanisms in L. plantarum .

What are common challenges in expressing functional prfC in L. plantarum and how can they be addressed?

Researchers frequently encounter several challenges when expressing functional prfC in L. plantarum:

Protein misfolding and insolubility often occur due to overexpression or improper folding environments. This can be addressed by:

• Optimizing growth temperatures (typically reducing to 25-30°C during induction)

• Co-expressing molecular chaperones (GroEL/GroES system)

• Using fusion partners like thioredoxin or NusA to enhance solubility

• Employing inducible promoters for tighter expression control

Plasmid instability presents another common obstacle, especially with large inserts like prfC. Solutions include:

• Utilizing balanced growth selection systems instead of antibiotic selection

• Implementing chromosomal integration strategies for stable expression

• Optimizing copy number through appropriate replicon selection

• Monitoring plasmid retention through regular PCR screening of colonies

Toxicity from inappropriate prfC activity levels can disrupt normal translation, impacting bacterial viability. Researchers can mitigate this by:

• Employing tightly regulated inducible promoters like nisin-controlled systems

• Creating attenuated variants with reduced GTPase activity

• Developing degron-tagged versions for controlled protein turnover

• Careful titration of inducer concentrations to find optimal expression windows

Incorrect post-translational modifications may impact prfC functionality. Approaches to address this include:

• Characterizing native modifications in L. plantarum prfC

• Engineering constructs to include necessary modification sites

• Co-expressing relevant modification enzymes if required

• Validating modification status through mass spectrometry

How should researchers interpret conflicting data regarding prfC function in translation termination studies?

When faced with conflicting data regarding prfC function in translation termination studies, researchers should implement a systematic analytical approach:

First, evaluate methodological differences between studies, particularly focusing on:

• The specific strain backgrounds used (wild-type vs. knockout complementation)

• The nature of the reporter systems (different stop codons, sequence contexts)

• The experimental conditions (in vitro vs. in vivo, buffer compositions)

• The concentrations of factors used (stoichiometric vs. catalytic amounts)

Second, consider the multifunctional nature of prfC, which could explain apparently contradictory results. Research indicates that prfC impacts different aspects of translation termination depending on experimental conditions. While some studies emphasize its role in peptide release rate enhancement, others highlight its function in release factor recycling . Both observations may be correct but represent different facets of prfC activity.

Finally, use computational modeling to reconcile conflicting datasets. Kinetic models incorporating all aspects of termination (binding, GTP hydrolysis, peptide release, factor dissociation, and ribosome recycling) can often explain seemingly contradictory experimental results by revealing how different rate-limiting steps dominate under different conditions.

What statistical approaches are most appropriate for analyzing the effects of recombinant prfC on L. plantarum physiology?

For growth rate and metabolic studies comparing wild-type and recombinant strains, repeated measures ANOVA or mixed-effects models are recommended. These approaches account for the temporal correlation in measurements and can incorporate both fixed effects (strain differences) and random effects (batch-to-batch variation).

For proteomics data, which typically involve thousands of measurements with complex correlation structures, researchers should employ:

• False discovery rate (FDR) control methods like Benjamini-Hochberg procedure

• LIMMA (Linear Models for Microarray Data) approaches adapted for proteomics

• Pathway enrichment analysis using tools like GSEA or DAVID

• Multivariate techniques such as principal component analysis or partial least squares discriminant analysis

For ribosome profiling or translation efficiency studies, specific statistical frameworks include:

• DESeq2 or edgeR for differential ribosome occupancy analysis

• Specialized metrics like translation efficiency (TE) ratios with appropriate variance modeling

• Bayesian approaches for modeling ribosome pause sites and termination efficiency

Power analysis is crucial when designing experiments, particularly when subtle physiological effects are expected. For typical studies measuring translation effects, sample sizes should be calculated to detect changes of 15-20% with 80% power at α=0.05, which often requires 6-8 biological replicates per condition .

What are promising avenues for engineering prfC to enhance recombinant protein production in L. plantarum?

Several innovative approaches for engineering prfC to enhance recombinant protein production in L. plantarum show significant promise:

Development of context-specific prfC variants represents another promising direction. By engineering prfC to interact differently with specific termination signals, researchers could create expression systems with enhanced efficiency for particular recombinant proteins. This approach could be particularly valuable for proteins with challenging C-terminal sequences or those containing multiple rare codons near the termination site.

Fusion of prfC with other translation factors offers an intriguing strategy. Chimeric constructs combining domains from prfC and elongation factors could create bifunctional proteins that improve both elongation and termination, potentially resolving bottlenecks in the translation cycle. Initial experiments with prfC-EF-G fusions have shown up to 40% increases in recombinant protein yields in laboratory strains.

Implementation of feedback-regulated prfC expression systems represents a sophisticated approach for maintaining optimal translation termination conditions. By linking prfC expression to sensors of translation stress, researchers could develop adaptive systems that automatically adjust termination efficiency in response to the burden of recombinant protein production .

How might advances in understanding prfC function contribute to developing L. plantarum as a therapeutic delivery vehicle?

Advances in understanding prfC function can significantly enhance the development of L. plantarum as a therapeutic delivery vehicle through several mechanisms:

Optimization of heterologous protein expression is the most direct application. By fine-tuning prfC activity, researchers can enhance the production of therapeutic proteins, peptides, or antigens within L. plantarum. This optimization could increase the potency of live bacterial therapeutics by ensuring higher delivery levels of the therapeutic molecule to target tissues.

Engineering of controlled release systems represents a sophisticated application of prfC knowledge. By creating conditionally regulated prfC variants, researchers could develop L. plantarum strains that release therapeutics in response to specific environmental triggers found in disease sites. For example, intestinal inflammation markers could trigger enhanced termination of secretion signal-containing therapeutic proteins.

Development of immunomodulatory capabilities is another promising direction. Research has shown that recombinant L. plantarum can activate dendritic cells and stimulate both cellular and humoral immune responses . By engineering prfC to optimize the expression of immunomodulatory molecules, researchers could create bacterial therapeutics with precisely controlled effects on host immunity.

Creation of environmental sensing and responsive systems represents an advanced application of prfC engineering. By linking prfC activity to biosensors that detect disease biomarkers, researchers could develop L. plantarum therapeutics that dynamically adjust their protein expression profiles in response to changing conditions in the disease environment, potentially providing personalized therapeutic responses .

What are the key unresolved questions regarding prfC function in L. plantarum that warrant further investigation?

Despite significant progress in understanding prfC function, several critical questions remain unresolved and merit further investigation:

The molecular mechanism of prfC's role in translation quality control remains poorly understood. While research in other organisms suggests that prfC accelerates termination on ribosomes that have experienced recent errors, the specific recognition mechanisms and signal transduction pathways involved in L. plantarum are unknown. Structural and biochemical studies examining how prfC interacts with ribosomes containing errors could provide valuable insights with implications for recombinant protein quality.

The potential regulatory roles of prfC beyond canonical translation termination represent an intriguing frontier. Preliminary evidence suggests that prfC may participate in ribosome hibernation during stress, influence mRNA decay pathways, or interact with other cellular systems. Comprehensive interactome studies combined with conditional knockdown approaches could reveal unexpected functions that impact L. plantarum physiology.

The evolutionary adaptation of prfC across different Lactobacillus species presents an interesting comparative question. Different species show varying levels of prfC conservation and potentially different termination preferences. Systematic comparative genomics coupled with biochemical characterization could reveal how prfC has evolved to optimize translation in different ecological niches, potentially identifying specialized variants with unique properties for biotechnological applications.

The structure-function relationships in L. plantarum prfC, particularly how specific domains contribute to different aspects of its activity, remain incompletely characterized. While general features of bacterial release factors are known, species-specific differences in domain organization and interaction could significantly impact function. Cryogenic electron microscopy studies of L. plantarum termination complexes would provide crucial structural insights to guide future engineering efforts .