Recombinant Lactobacillus plantarum 50S ribosomal protein L21 (rplU)

What is the function of 50S ribosomal protein L21 in Lactobacillus plantarum?

The 50S ribosomal protein L21 (rplU) in Lactobacillus plantarum is a critical component of the large 50S ribosomal subunit, playing an essential role in protein synthesis. It contributes to ribosomal structure stability and participates in the translation process. Similar to L21 proteins in other bacterial species, it likely interacts with rRNA and neighboring proteins to maintain the structural integrity of the ribosome. Research suggests that ribosomal proteins like L21 are highly conserved across bacterial species due to their fundamental role in cellular function, with structural studies indicating its positioning near the peptidyl transferase center, which is essential for peptide bond formation during protein synthesis.

What are the optimal conditions for expressing recombinant L. plantarum 50S ribosomal protein L21?

Based on research with similar ribosomal proteins in L. plantarum, optimal expression conditions typically include:

For heterologous expression in E. coli, standard conditions include induction with 1 mM IPTG and overnight incubation at 25°C in LB medium . The recombinant protein can then be purified using affinity chromatography if a His-tag is incorporated into the design. When expressing in L. plantarum itself, the pSIP expression system has been shown to efficiently express recombinant proteins with yields approaching 1800 Miller Unit equivalents .

How can I optimize codon usage for L. plantarum rplU expression in heterologous systems?

Codon optimization is critical for efficient expression of L. plantarum genes in heterologous systems. Research demonstrates that optimizing codons according to the host's codon usage bias significantly enhances expression efficiency:

• Analyze the codon usage bias of your expression host (e.g., E. coli, L. lactis)

• Optimize the rplU gene sequence according to the host's preferred codons

• Consider GC content adjustment to match the host organism

• Avoid rare codons, especially at the N-terminus of the protein

• Eliminate potential secondary structures in the mRNA that might impede translation

Studies with other L. plantarum recombinant proteins have shown that codon optimization can increase protein yields by several fold . For instance, when expressing the SARS-CoV-2 spike protein in L. plantarum, researchers optimized the codons according to L. plantarum's usage bias, resulting in efficient expression with high antigenicity . The same principles apply to rplU expression.

What purification strategies are most effective for L. plantarum recombinant 50S ribosomal protein L21?

Effective purification of recombinant L. plantarum 50S ribosomal protein L21 can be achieved through several strategies:

For tagged proteins, HisGraviTrap columns have shown good results . For untagged proteins, ion-exchange chromatography using Fractogel EMD DEAE followed by gel filtration on HiLoad Superdex 75 column has been effective for similar ribosomal proteins . After purification, it's recommended to store the protein with 5-50% glycerol at -20°C/-80°C to maintain stability, similar to other recombinant ribosomal proteins .

What are the stability characteristics of recombinant L. plantarum ribosomal proteins?

Research on recombinant L. plantarum proteins has demonstrated remarkable stability properties that are likely applicable to the 50S ribosomal protein L21:

These stability characteristics suggest that recombinant L. plantarum 50S ribosomal protein L21 would likely maintain structural integrity under harsh conditions, making it suitable for various experimental applications. The stability at low pH and in the presence of bile salts is particularly relevant for applications involving gastrointestinal conditions .

How can I assess the activity and functional integrity of purified recombinant L. plantarum 50S ribosomal protein L21?

Assessing the functional integrity of recombinant L. plantarum 50S ribosomal protein L21 requires specialized approaches:

• Structural integrity assessment:

  • Circular Dichroism (CD) spectroscopy to confirm proper folding

  • Size-exclusion chromatography to verify monomeric state

  • Thermal shift assays to assess stability

• Functional assays:

  • In vitro translation assays using reconstituted ribosomes

  • rRNA binding assays to measure interaction with ribosomal RNA

  • Ribosome assembly assays to evaluate incorporation into 50S subunits

• Comparative approaches:

  • Complementation assays in L21-deficient bacterial strains

  • Compare activity with commercially available ribosomal proteins

When establishing activity parameters, it's important to note that active recombinant proteins from L. plantarum have shown similar in vitro activity levels compared to their native counterparts . Activity assessment protocols may need to be adapted from those used for other bacterial ribosomal proteins.

What are the challenges in expressing L. plantarum ribosomal proteins in heterologous systems?

Researchers face several challenges when expressing L. plantarum ribosomal proteins in heterologous systems:

• Protein solubility issues:

  • Ribosomal proteins often aggregate when expressed outside their native context

  • Solution: Optimize expression conditions (temperature, induction timing) or use solubility-enhancing tags

• Toxicity to host cells:

  • Overexpression of ribosomal proteins can disrupt host cell translation

  • Solution: Use tightly regulated expression systems or lower expression temperatures

• Proper folding:

  • Studies show that inefficient heterologous folding can be a significant barrier

  • Solution: Co-express with chaperones or use ancestral sequence reconstruction to identify stabilizing mutations

• Post-translational modifications:

  • Modifications present in the native host may be absent in heterologous systems

  • Solution: Select expression hosts with similar modification machinery

Research has shown that combining ancestral reconstruction with folding-landscape analysis can significantly improve heterologous expression efficiency . For example, identifying problematic regions (like the 1-22 fragment or 70-77 loop in other proteins) and replacing them with ancestral sequences has improved solubility dramatically in some cases.

How does the expression of recombinant L. plantarum ribosomal proteins affect host cell physiology?

The expression of recombinant L. plantarum ribosomal proteins can significantly impact host cell physiology:

• Metabolic burden:

  • Diverts cellular resources from normal growth to heterologous protein production

  • Can result in reduced growth rates and biomass yield

• Stress responses:

  • Induces heat shock response due to accumulation of misfolded proteins

  • Upregulates chaperones and proteases

• Ribosomal interference:

  • May compete with host ribosomal proteins for assembly into ribosomes

  • Can disrupt normal translation processes

• Transcriptional changes:

  • Proteomics studies of bacteria expressing recombinant proteins show upregulation of stress-related genes

  • For example, in E. coli, expression of foreign proteins has been shown to upregulate proteins like ParE, RimO, and several 50S ribosomal proteins

These physiological changes should be considered when optimizing expression conditions. For instance, the induction time and concentration of inducer can be adjusted to balance protein yield with host cell viability, as demonstrated in studies with L. plantarum expression systems where the highest protein yields were obtained with specific induction conditions (50 ng/mL SppIP for 8h at 30°C) .

What is the role of L. plantarum 50S ribosomal protein L21 in chloroplast ribosome biogenesis in plants?

Although L. plantarum and plant chloroplasts have distinct ribosomal systems, research on chloroplast ribosomal protein L21 provides insights relevant to bacterial L21 proteins:

• Functional conservation:

  • The chloroplast ribosomal protein L21 gene is essential for plastid development and embryogenesis in Arabidopsis

  • The mutated asd phenotype in Arabidopsis demonstrates that L21 is indispensable for chloroplast biogenesis

• Expression patterns:

  • In plants, RPL21C exhibits higher expression in leaves and flowers compared to roots and developing seeds

  • This suggests tissue-specific roles for ribosomal proteins that may have parallels in bacterial expression

• Subcellular localization:

  • In plant cells, the RPL21C-GFP fusion protein localizes to chloroplasts

  • This confirms its role in plastid ribosome assembly

This research on plant L21 proteins highlights the fundamental importance of this ribosomal component across different kingdoms of life and suggests that bacterial L21, including from L. plantarum, likely plays a similarly critical role in ribosome assembly and function.

How can recombinant L. plantarum 50S ribosomal protein L21 be used in immunological studies?

Recombinant L. plantarum 50S ribosomal protein L21 can be valuable for immunological studies in several ways:

• As an antigen in vaccine development:

  • Ribosomal proteins are conserved bacterial antigens that can elicit immune responses

  • Can be used to develop vaccines against pathogenic bacteria with homologous L21 proteins

• For studying host-microbe interactions:

  • Can help elucidate how commensal bacteria like L. plantarum interact with the host immune system

  • Research shows L. plantarum can induce specific immune responses, including antibody production and T-cell proliferation

• As a dendritic cell stimulant:

  • L. plantarum components have been shown to mature dendritic cells and upregulate co-stimulatory molecules

  • Studies indicate DCs incubated with L. plantarum upregulate expression of co-stimulatory molecules and secrete cytokines like IL-12 and TNF-α

• Experimental protocols:

  • Mice can be immunized orally with L. plantarum expressing the protein of interest

  • Immune responses can be assessed by measuring serum IgG, IgG1, IgG2a, and mucosal IgA in intestinal lavages

  • T-cell responses can be evaluated through splenocyte proliferation assays

What are the evolutionary implications of studying L. plantarum 50S ribosomal protein L21?

Studying L. plantarum 50S ribosomal protein L21 offers valuable evolutionary insights:

• Conservation across species:

  • Ribosomal proteins are among the most conserved proteins in all living organisms

  • Comparative analysis of L21 sequences can reveal evolutionary relationships between different bacterial lineages

• Ancestral sequence reconstruction:

  • Research indicates that ancestral sequence reconstruction can improve heterologous expression and folding properties

  • This approach has been successful with other proteins and could be applied to L21

• Horizontal gene transfer assessment:

  • Analysis of ribosomal protein sequences can help identify potential horizontal gene transfer events

  • Such events contribute to bacterial evolution and adaptation

• Molecular clock applications:

  • Due to their high conservation, ribosomal proteins can serve as molecular clocks for dating evolutionary events

  • Divergence in ribosomal protein sequences correlates with evolutionary distance

• Host-microbe co-evolution:

  • L. plantarum is a commensal organism with applications in probiotics and food fermentation

  • Understanding its ribosomal proteins can provide insights into how it has co-evolved with various hosts

This evolutionary perspective not only enhances our fundamental understanding of bacterial evolution but also has practical applications in taxonomy, ecological studies, and comparative genomics.

How does phosphate concentration affect the expression and function of ribosomal proteins in L. plantarum?

Research indicates that phosphate concentration significantly impacts ribosomal protein expression and function in Lactobacillus species:

• Physiological adaptations:

  • Phosphate concentration in culture media affects bacterial fitness during stationary phase

  • Modifying inorganic phosphate (Pi) concentration leads to physiological adaptations correlated with variations in intracellular polyphosphate levels

• Protein expression changes:

  • Differential Pi conditions can trigger proteomic changes, including alterations in ribosomal protein expression

  • Stress-related chaperones and cell surface modification enzymes are upregulated in phosphate-limited media

• Growth phase considerations:

  • Pi effects are most pronounced during stationary phase

  • This has implications for recombinant protein production, which often extends into stationary phase

• Practical applications:

  • Adjusting environmental Pi concentration constitutes a simple strategy to improve cellular fitness of Lactobacillus species

  • This approach could benefit L. plantarum as a microbial cell factory, including for ribosomal protein production

These findings suggest that researchers should consider phosphate concentration as a critical parameter when optimizing expression conditions for L. plantarum ribosomal proteins, including L21.

What are the latest advances in using recombinant L. plantarum for vaccine delivery and how might ribosomal proteins contribute?

Recent advances in using recombinant L. plantarum for vaccine delivery offer potential applications for ribosomal proteins:

• Food-grade oral vaccine development:

  • L. plantarum has been successfully used to express and deliver viral antigens like SARS-CoV-2 spike protein

  • Similar approaches could be applied to express ribosomal proteins as antigens

• Immune response induction:

  • Recombinant L. plantarum can induce both systemic (serum IgG) and mucosal (intestinal IgA) antibody responses

  • It can also stimulate T-cell responses as measured by splenocyte proliferation

• Gut microbiota modulation:

  • Recombinant L. plantarum can influence gut microbial diversity and structure

  • This has implications for immune regulation and potential vaccine efficacy

• Enhanced stability:

  • L. plantarum-expressed proteins show remarkable stability under harsh conditions (50°C, pH 1.5, high salt)

  • This makes them suitable for oral delivery through the gastrointestinal tract

• Adjuvant properties:

  • L. plantarum itself has inherent adjuvanticity, enhancing immune responses to the expressed antigen

  • L. plantarum components mature dendritic cells and upregulate co-stimulatory molecules

Ribosomal proteins like L21 could serve as conserved bacterial antigens in such vaccine approaches, potentially providing cross-protection against multiple bacterial species with homologous ribosomal proteins.