Recombinant Lactobacillus plantarum 50S ribosomal protein L27 (rpmA)

How does L. plantarum 50S ribosomal protein L27 differ from other ribosomal proteins like L7/L12?

Unlike the L7/L12 (rplL) ribosomal protein which has been more extensively characterized in Lactobacillus plantarum, L27 (rpmA) has distinct structural and functional properties. The L7/L12 protein is known to be approximately 95 kDa in size based on SDS-PAGE analysis, while L27 is significantly smaller . Functionally, L27 is positioned closer to the peptidyl transferase center in the ribosome, making it critical for peptide bond formation, whereas L7/L12 is primarily involved in factor recruitment and GTPase activation during translation. These differences highlight the specialized roles of various ribosomal proteins within the translation machinery of L. plantarum.

What are the common expression systems used for L. plantarum recombinant proteins?

The pSIP expression system has been demonstrated as an effective platform for recombinant protein production in L. plantarum. This system allows for controlled gene expression through an inducible promoter activated by specific peptide pheromones. For example, in studies with recombinant α-amylase production in L. plantarum WCFS1, the pSIP system enabled successful expression and secretion of target proteins with yields reaching up to 8.1 kU/L of culture medium for certain constructs . The system's components include:

• Inducible promoter responsive to inducing peptide (IP-673)

• Multiple cloning sites for target gene insertion

• Selection markers for stable maintenance

• Signal peptide sequences for protein secretion

The effectiveness of this system makes it a primary choice for ribosomal protein expression studies in L. plantarum.

What signal peptides optimize secretion of recombinant proteins in L. plantarum?

Several signal peptides have been evaluated for their capacity to enhance recombinant protein secretion in L. plantarum, with significant variations in efficiency. Among the Sec-type signal peptides derived from L. plantarum WCFS1, Lp_2145 has demonstrated superior performance for certain proteins. In a comparative study of recombinant α-amylase expression:

The Lp_2145 signal peptide provided a 6.2-fold increase in total activity and 5.4-fold increase in extracellular activity compared to the native signal peptide (SP_AmyL) . For ribosomal protein expression, these findings suggest that careful selection of signal peptides can dramatically improve yields, with Lp_2145 being a promising candidate for initial trials.

How does signal peptide choice affect mRNA levels of recombinant proteins in L. plantarum?

Real-time RT-qPCR analysis has revealed that signal peptide selection significantly impacts transcription levels of recombinant genes in L. plantarum. Studies have shown that constructs with the Lp_2145 signal peptide exhibited the highest mRNA levels, with a peak of 46-fold and 58-fold upregulation at 3 hours post-induction for different target proteins . This was approximately 3-fold higher than constructs with native signal peptides and 2-fold higher than constructs with other signal peptides.

The correlation between signal peptide and mRNA level suggests that these sequences influence not only protein secretion but also transcriptional efficiency or mRNA stability. For ribosomal protein expression, this finding indicates that:

• Signal peptide selection should be considered not only for secretion efficiency but also for its impact on transcript levels

• Time-course studies are essential as peak expression typically occurs around 3 hours post-induction

• The Lp_2145 signal peptide may provide advantages at both transcriptional and translational levels

These considerations are critical when designing expression systems for ribosomal proteins like L27, where maximizing yield is often a challenge.

What reference genes are suitable for normalizing gene expression in L. plantarum studies?

Selection of stable reference genes is crucial for accurate RT-qPCR analysis in L. plantarum. Research has identified several housekeeping genes with stable expression under various experimental conditions:

• gmk (guanylate kinase)

• gyrA (DNA gyrase subunit A)

• gapB (glyceraldehyde-3-phosphate dehydrogenase)

These genes were validated for stability during the exponential growth phase of L. plantarum WCFS1, even when growth was affected by nutrient limitation and pH changes . The validation process typically involves assessing expression stability using multiple algorithms:

• GeNorm (Vandesompele et al., 2002)

• BestKeeper (Pfaffl et al., 2004)

• NormFinder (Andersen et al., 2004)

When designing RT-qPCR experiments for studying recombinant L. plantarum 50S ribosomal protein L27 expression, these reference genes should be included to ensure reliable normalization and accurate quantification of target gene transcription.

What are optimal growth conditions for maximizing recombinant protein expression in L. plantarum?

For optimal recombinant protein expression in L. plantarum, cultivation conditions must be carefully controlled. Based on experimental data from recombinant protein studies, the following conditions are recommended:

• Growth medium: MRS broth, which provides essential nutrients for robust growth

• Temperature: 37°C, which supports both growth and protein expression

• Cultivation time: 12-18 hours for maximum protein yield, though specific proteins may have different optimal harvest times

• Induction timing: Typically at OD600 of approximately 0.3 (early exponential phase)

• Harvest time: Protein-dependent, with some proteins (like AmyL) showing peak activity at 12 hours, followed by a decline

It's important to note that growth rates may vary based on the signal peptide used in the construct. For example, strains containing constructs with the Lp_3050 signal peptide showed slower growth rates compared to other constructs . For ribosomal proteins like L27, which may affect cellular fitness when overexpressed, induction conditions may need to be further optimized to balance protein yield with cell viability.

How can successful expression and purification of recombinant L. plantarum ribosomal proteins be confirmed?

Verification of successful expression and purification of recombinant L. plantarum ribosomal proteins requires multiple complementary techniques:

• SDS-PAGE analysis: For visualizing protein bands at the expected molecular weight in both cell lysate and culture supernatant. Samples should be standardized by loading equal amounts of protein (typically 10-20 μg per lane) .

• Western blotting: Using antibodies specific to either the ribosomal protein or added epitope tags (His-tag, FLAG-tag) for higher specificity detection.

• Mass spectrometry: For definitive identification of the protein and verification of amino acid sequence and post-translational modifications.

• Functional assays: For ribosomal proteins, in vitro translation assays can confirm biological activity of the purified protein.

• RNA binding assays: Since ribosomal proteins interact with rRNA, electrophoretic mobility shift assays (EMSA) can verify RNA-binding capability.

The combination of these techniques provides comprehensive confirmation of both expression and functional integrity of the recombinant ribosomal protein.

What are the best protocols for RNA extraction from L. plantarum cultures for RT-qPCR studies?

High-quality RNA extraction from L. plantarum requires specific protocols to overcome challenges posed by its cell wall structure. For RT-qPCR studies examining recombinant ribosomal protein expression, the following approach is recommended:

• Sample collection: Harvest cells at defined timepoints (e.g., 0, 3, 6, and 12 hours post-induction) .

• Cell lysis: Use enzymatic treatment with lysozyme combined with mechanical disruption (e.g., bead-beating) to efficiently break the gram-positive cell wall.

• RNA purification: Commercial kits optimized for gram-positive bacteria yield better results than standard TRIzol-based methods.

• DNase treatment: Essential to eliminate genomic DNA contamination that would interfere with qPCR results.

• RNA quality assessment: Verify RNA integrity using Bioanalyzer or gel electrophoresis (RIN value > 7.0 recommended) and purity by A260/A280 ratio (2.0-2.1 optimal).

• cDNA synthesis: Use reverse transcriptase with high thermostability and processivity for efficient conversion of bacterial RNA.

For RT-qPCR analysis, primer validation is critical before application:

• Verify primer specificity by melt curve analysis

• Determine optimal primer concentration at 60°C annealing temperature

• Calculate amplification efficiency (95-105% is acceptable)

This rigorous approach ensures reliable quantification of ribosomal protein transcripts in recombinant L. plantarum strains.

How does expression of recombinant ribosomal proteins in L. plantarum affect its immunomodulatory properties?

While the specific immunological effects of expressing recombinant ribosomal proteins in L. plantarum have not been directly characterized, insights can be gained from general immunomodulatory properties of L. plantarum. Meta-analysis of clinical trials has demonstrated that L. plantarum significantly modulates cytokine levels:

These findings indicate that L. plantarum promotes host immunity by increasing anti-inflammatory cytokines (IL-10) while decreasing pro-inflammatory cytokines (TNF-α, IFN-γ) and Th2-associated cytokines (IL-4) .

For recombinant L. plantarum expressing ribosomal proteins, researchers should consider:

• Whether the expression of ribosomal proteins alters these baseline immunomodulatory properties

• If ribosomal proteins themselves have additional immunostimulatory effects

• How these properties might influence the selection of L. plantarum as an expression host for therapeutic applications

Monitoring cytokine profiles in response to recombinant L. plantarum strains would provide valuable data on potential immunological implications.

What methods can be used to assess the structural integrity of recombinant ribosomal proteins from L. plantarum?

Assessing the structural integrity of recombinant ribosomal proteins requires multiple biophysical and biochemical approaches:

• Circular Dichroism (CD) Spectroscopy: Provides information about secondary structure content (α-helices, β-sheets). Ribosomal proteins typically show characteristic CD spectra reflecting their unique structural features.

• Differential Scanning Calorimetry (DSC): Measures thermal stability and folding properties, which can be compared to native ribosomal proteins isolated from L. plantarum.

• Size Exclusion Chromatography (SEC): Analyzes oligomerization state and potential aggregation, critical for ribosomal proteins that may form dimers or interact with other ribosomal components.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: For detailed structural analysis of smaller ribosomal proteins like L27.

• X-ray Crystallography: The gold standard for high-resolution structural determination, though crystallization of ribosomal proteins can be challenging.

• Functional Assays: Testing the ability of the recombinant protein to integrate into ribosomal assemblies or bind to rRNA targets.

For L. plantarum ribosomal protein L27 specifically, comparing structural data with homologous proteins from other bacterial species can provide additional validation of proper folding and structural integrity.

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

Researchers expressing ribosomal proteins like L27 in L. plantarum may encounter several challenges:

• Protein toxicity: Overexpression of ribosomal proteins can disrupt normal ribosome assembly and function.

  • Solution: Use tightly regulated inducible systems like pSIP with careful titration of inducer concentration.

• Protein instability: Some ribosomal proteins are unstable when expressed outside the ribosomal context.

  • Solution: Co-express chaperone proteins or optimize growth temperature (25-30°C may improve folding).

• Low expression levels: Ribosomal proteins may show lower expression compared to other recombinant proteins.

  • Solution: Optimize codon usage for L. plantarum and select the most effective signal peptide (e.g., Lp_2145) .

• Inefficient secretion: Many ribosomal proteins lack native secretion signals.

  • Solution: Test multiple signal peptides, with Lp_2145 showing promising results for enhancing secretion in L. plantarum .

• RNA contamination: Ribosomal proteins naturally bind RNA, complicating purification.

  • Solution: Include RNase treatment steps and high-salt washes during purification.

• Validation of functionality: Confirming that recombinant ribosomal proteins retain native function.

  • Solution: Develop in vitro ribosome assembly assays or complementation studies with ribosomal protein-deficient strains.

By anticipating these challenges, researchers can design more effective expression strategies for L. plantarum ribosomal proteins.

How can RT-qPCR be optimized for studying gene expression in recombinant L. plantarum?

Optimization of RT-qPCR for studying recombinant ribosomal protein expression in L. plantarum requires attention to several critical factors:

• Reference gene selection: Validate stability using multiple algorithms (GeNorm, BestKeeper, NormFinder) under experimental conditions. The gmk, gyrA, and gapB genes have shown stable expression in L. plantarum during exponential growth phase .

• Primer design considerations:

  • Target amplicon size: 70-150 bp for optimal efficiency

  • GC content: 45-55% for balanced annealing

  • Melting temperature: Design primers with similar Tm (60°C optimal)

  • Check for secondary structures and primer dimers

• RNA quality control:

  • RNA Integrity Number (RIN) > 7

  • A260/280 ratio between 1.8-2.0

  • A260/230 ratio > 1.8

• RT-qPCR protocol optimization:

  • Two-step vs. one-step RT-PCR (two-step generally provides better sensitivity)

  • cDNA dilution series to confirm absence of inhibitors

  • Include no-RT controls to detect genomic DNA contamination

  • Use technical triplicates for each biological replicate

• Data analysis approach:

  • Apply the comparative Ct method (2^-ΔΔCt) with appropriate statistical validation

  • Use randomization test methods (e.g., REST 2009 software) for significance testing

Following these optimization steps ensures accurate quantification of recombinant ribosomal protein transcripts in L. plantarum expression systems.