Recombinant Lactobacillus plantarum 30S ribosomal protein S11 (rpsK)

What is the 30S ribosomal protein S11 and why express it recombinantly in L. plantarum?

The 30S ribosomal protein S11 is a critical component of the small ribosomal subunit involved in protein synthesis. In bacterial systems like E. coli, it's encoded by the rpsK gene and plays essential roles in translation, making it significant for both basic and applied research . Expressing S11 recombinantly in L. plantarum offers several advantages over conventional expression systems. L. plantarum provides a food-grade, generally recognized as safe (GRAS) expression host that can survive harsh gastrointestinal conditions, making it suitable for both research applications and potential therapeutic protein delivery. Unlike E. coli-based systems, L. plantarum-based expression eliminates endotoxin concerns and can be used for surface display of target proteins, offering greater versatility for structural and functional studies of ribosomal proteins .

How does recombinant S11 from L. plantarum differ from native S11 protein?

Recombinant S11 protein expression in L. plantarum typically involves the addition of affinity tags (such as His-tags) and potentially signal peptides that facilitate protein targeting and purification. These modifications can affect protein structure and function compared to the native form. Like other recombinant proteins expressed in L. plantarum, recombinant S11 may undergo specific post-translational modifications that differ from those in the native system. While the amino acid sequence of S11 is highly conserved among bacterial species, expression in heterologous systems like L. plantarum may result in protein folding variations that require validation through structural and functional assays. Researchers should verify that the recombinant protein maintains its expected ribosomal assembly capabilities through appropriate biochemical characterization methods .

What are the fundamental differences between expressing ribosomal proteins in E. coli versus L. plantarum?

The expression of ribosomal proteins presents distinct challenges in different bacterial hosts. E. coli systems typically provide higher protein yields but may form inclusion bodies requiring complex refolding procedures. In contrast, L. plantarum offers advantages in protein solubility and proper folding for certain ribosomal proteins. Selection marker systems also differ significantly - while E. coli typically relies on antibiotic resistance markers, L. plantarum can utilize food-grade markers like the alanine racemase gene (alr), which shows considerable potential for production of ingredients for the food industry .

The cellular machinery in L. plantarum may process recombinant proteins differently, affecting factors such as codon usage optimization, plasmid stability, and expression efficiency. For instance, expression yields in L. plantarum using the alanine racemase-based pSIP609 system tend to be slightly higher than with erythromycin-dependent expression systems (pSIP409), likely due to avoiding antibiotic detoxification processes and subsequent reduced plasmid loss .

Which expression vectors are most effective for ribosomal protein expression in L. plantarum?

For ribosomal protein expression in L. plantarum, the pSIP series of vectors has demonstrated high efficiency and versatility. The pSIP409 vector (erythromycin resistance-based) and pSIP609 vector (alanine racemase-based) are particularly suitable for recombinant protein expression. The pSIP609 system offers an advantage as a food-grade, antibiotic-independent expression system that can yield higher expression levels compared to antibiotic-dependent systems . These vectors utilize inducible promoters that allow controlled expression of the target protein.

For surface display applications, vectors incorporating anchoring domains like pgsA are effective. Based on successful expression of other recombinant proteins, researchers can construct plasmids such as pSIP409-pgsA-rpsK to anchor the S11 protein to the bacterial surface, similar to approaches used for other recombinant proteins in L. plantarum NC8 . The choice between secreted expression and surface display depends on the specific research objectives and downstream applications.

How can codon optimization improve S11 protein expression in L. plantarum?

Codon optimization is crucial for efficient expression of heterologous proteins in L. plantarum. When expressing genes from other organisms, researchers should optimize the coding sequence according to the codon usage bias of L. plantarum to enhance translation efficiency and protein yield. This process typically involves:

• Analyzing the codon usage bias of L. plantarum strains (such as Lp18)

• Modifying the target gene sequence to use preferred codons without altering the amino acid sequence

• Potentially incorporating endogenous signal peptides (like ALX04_001320) at the 5' terminus to enhance secretion or surface display

For example, this approach has been successfully applied when expressing viral proteins in L. plantarum, where genes were synthesized with optimized codons followed by linking endogenous signal peptide sequences to enhance expression efficiency . Similar strategies would be applicable for optimizing S11 ribosomal protein expression.

What induction conditions yield optimal S11 protein expression in L. plantarum?

Optimal induction conditions for protein expression in L. plantarum vary based on the specific expression system used. For the pSIP-based expression systems, the following parameters should be considered:

The highest protein yields are typically obtained with induction using 50 ng/mL SppIP at 30-37°C for 6-10 hours, as demonstrated with other recombinant proteins in L. plantarum . Researchers should conduct optimization experiments for their specific construct, as expression efficiency can vary significantly between different proteins and expression systems.

What are the most effective purification strategies for recombinant S11 protein from L. plantarum?

Purification of recombinant S11 protein from L. plantarum typically employs affinity chromatography techniques, leveraging fusion tags incorporated into the expression construct. The most common approaches include:

• His-tag based purification: For constructs with polyhistidine tags, immobilized metal affinity chromatography (IMAC) provides efficient single-step purification. Cell lysis can be achieved through repeated freeze-thaw cycles or sonication in an appropriate buffer supplemented with protease inhibitors .

• Surface-displayed protein recovery: For S11 anchored to the cell surface (using systems like pgsA), mild detergent treatment or enzymatic cleavage may be required to release the protein while maintaining its structural integrity.

The choice of buffer systems is crucial for maintaining protein stability. Based on observations with other recombinant proteins expressed in L. plantarum, Tris-based buffers with 50% glycerol can provide stability during storage . For co-expressed proteins containing the same affinity tag, sequential elution with an imidazole gradient may successfully separate different proteins, as demonstrated with other recombinant proteins from L. plantarum .

How can researchers verify the structural integrity of purified recombinant S11 protein?

Verifying the structural integrity and functionality of purified recombinant S11 protein requires multiple complementary approaches:

• SDS-PAGE and Western blotting: Primary verification using antibodies against incorporated tags (such as His-tag) or against the S11 protein itself can confirm expression and approximate molecular weight (typically showing bands matching the expected size with fusion tags) .

• Mass spectrometry: For precise molecular characterization, peptide mass fingerprinting and intact protein analysis provide accurate molecular weight determination and sequence verification.

• Circular dichroism (CD) spectroscopy: This technique assesses secondary structure elements and can be used to compare recombinant S11 with native protein standards.

• Functional assays: Since S11 is involved in ribosomal assembly, in vitro ribosome reconstitution assays can verify whether the recombinant protein retains the ability to associate with other ribosomal components.

• Quaternary structure analysis: Techniques like size exclusion chromatography and analytical ultracentrifugation can verify whether the recombinant S11 maintains expected oligomeric states, which may differ between bacterial species (dimeric, tetrameric, etc.) .

What analytical methods best characterize protein-protein interactions involving recombinant S11?

Characterizing protein-protein interactions involving recombinant S11 requires specialized techniques to understand its role in ribosome assembly and function:

• Surface plasmon resonance (SPR): Provides real-time kinetic data on binding interactions between S11 and other ribosomal proteins or rRNA molecules.

• Microscale thermophoresis (MST): Allows detection of interactions in solution with minimal sample consumption and without immobilization requirements.

• Isothermal titration calorimetry (ITC): Delivers thermodynamic parameters of binding interactions, including binding constants, enthalpy changes, and stoichiometry.

• Cryo-electron microscopy: For structural studies, this technique can visualize S11 within the context of ribosomal assemblies, providing insights into conformational changes upon binding.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Maps interaction interfaces by measuring changes in hydrogen exchange rates upon complex formation.

These methods complement each other and provide comprehensive characterization of how recombinant S11 interacts with ribosomal components and potentially with other cellular factors. For surface-displayed variants, additional techniques like indirect immunofluorescence assay (IFA) and flow cytometry can quantify surface expression levels and accessibility, similar to approaches used for other surface-displayed proteins in L. plantarum .

How can recombinant L. plantarum expressing S11 be used for structural biology studies?

Recombinant L. plantarum expressing the 30S ribosomal protein S11 offers unique advantages for structural biology studies:

• Surface display systems: Using anchoring domains like pgsA allows presentation of S11 on the bacterial surface, facilitating structural studies of the protein in a native-like membrane environment . This approach enables accessibility studies using antibodies or ligands without the need for protein purification.

• Production of labeled protein: L. plantarum can be grown in media containing isotope-labeled amino acids (13C, 15N) for NMR spectroscopy studies, providing insights into protein dynamics and interactions.

• Crystallization screening: Purified recombinant S11 can be used for crystallization trials to determine high-resolution structures, complementing existing structural information derived from other bacterial species.

• Cryo-EM studies: The recombinant protein can be incorporated into ribosomal assembly assays for cryo-electron microscopy, allowing visualization of S11's position and interactions within the ribosomal complex.

• Comparative structural biology: Expression of S11 from different bacterial species in the standardized L. plantarum system allows direct structural comparisons to identify conserved and variable features.

The stability of recombinant proteins expressed in L. plantarum under various conditions (demonstrated temperature stability up to 50°C and pH stability at 1.5) provides additional advantages for structural studies requiring various buffer conditions .

What are the potential applications of S11-expressing L. plantarum in immunological research?

The expression of ribosomal protein S11 in L. plantarum creates opportunities for innovative immunological research:

• Antigen delivery system: Surface-displayed S11 on L. plantarum can function as an antigen delivery vehicle, potentially eliciting immune responses against this bacterial protein. The ability of L. plantarum to survive gastrointestinal conditions makes it suitable for oral immunization studies .

• Adjuvant development: The immunomodulatory properties of L. plantarum combined with surface-displayed S11 could enhance immune responses to co-administered antigens, serving as a potential adjuvant platform.

• Epitope mapping: By expressing different segments of S11 on the bacterial surface, researchers can identify immunodominant epitopes recognized by the immune system. This approach has been successful with other recombinant proteins expressed in L. plantarum .

• Autoimmune disease models: Since ribosomal proteins can sometimes be targets of autoantibodies, S11-expressing L. plantarum could be used to study or potentially modulate autoimmune responses in research models.

• Cross-reactivity studies: The recombinant system allows investigation of antigenic cross-reactivity between S11 proteins from different bacterial species, providing insights into evolutionary conservation of immune epitopes.

The recombinant L. plantarum platform offers advantages over traditional protein delivery systems due to its stability under gastrointestinal conditions and potential food-grade status, eliminating concerns associated with other expression systems .

How does the study of recombinant S11 contribute to understanding ribosome assembly?

Recombinant expression of S11 in L. plantarum provides valuable tools for investigating the complex process of ribosome assembly:

• Assembly intermediate analysis: Purified recombinant S11 can be used in in vitro reconstitution assays to study the sequential assembly of the 30S ribosomal subunit, helping identify critical assembly intermediates.

• Interaction network mapping: Modified versions of S11 (with point mutations or domain deletions) can reveal the contribution of specific regions to interactions with other ribosomal proteins and rRNA.

• Species-specific assembly differences: By comparing the incorporation of S11 from different bacterial species into partial ribosomal assemblies, researchers can identify species-specific assembly pathways.

• Co-expression studies: The L. plantarum expression system allows co-expression of multiple ribosomal proteins, enabling studies of cooperative assembly processes. This approach has been demonstrated with other protein pairs in L. plantarum, with proteins containing the same affinity tag being successfully separated during purification .

• Chaperone involvement: The system can be used to investigate whether specific chaperones facilitate S11 incorporation into the ribosome, enhancing our understanding of assisted ribosome assembly.

These applications contribute to fundamental knowledge about ribosome biogenesis, which has implications for antibiotic development and understanding bacterial adaptation mechanisms.

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

Researchers working with recombinant S11 expression in L. plantarum frequently encounter several challenges:

• Plasmid stability issues: The expression plasmid can be lost during prolonged cultivation, particularly with antibiotic-based selection systems. This can be mitigated by using food-grade selection markers like alanine racemase (alr), which show less plasmid loss compared to antibiotic resistance markers . Regular verification of plasmid retention through colony PCR is recommended.

• Low expression levels: Sub-optimal codon usage often leads to poor translation efficiency. Researchers should conduct thorough codon optimization based on L. plantarum's codon preference and consider incorporating strong ribosome binding sites. The expression level can be monitored by Western blot analysis, with detection using anti-tag antibodies .

• Protein misfolding: Ribosomal proteins typically interact with rRNA and other proteins, which can lead to misfolding when expressed in isolation. Optimizing growth and induction conditions (37°C, 6-10 hours induction with appropriate inducer concentration) can improve proper folding .

• Proteolytic degradation: Host proteases may target recombinant proteins. This can be addressed by incorporating protease inhibitors during extraction and optimizing harvest timing to avoid the stationary phase when protease activity increases.

• Induction variability: Expression levels can vary between bacterial passages due to differences in induction states. Maintaining consistent inoculation amounts and induction conditions can minimize this variability .

How can researchers troubleshoot insufficient surface display of S11 protein?

Surface display of S11 protein on L. plantarum may present specific challenges that require targeted troubleshooting approaches:

• Anchoring domain optimization: If surface display efficiency is low, researchers should consider testing alternative anchoring domains beyond pgsA. Each protein may interact differently with various anchoring systems.

• Signal peptide selection: The efficiency of protein translocation depends heavily on the signal peptide used. Testing multiple endogenous signal peptides from L. plantarum (such as ALX04_001320) can identify optimal combinations for S11 surface display .

• Expression verification techniques: Flow cytometry analysis can quantify the percentage of bacteria successfully displaying the target protein. Positive rates of 30-40% might be expected based on similar recombinant proteins, compared to background levels of 2-3% in parental strains .

• Immunofluorescence microscopy: This technique provides visual confirmation of surface display and can identify potential aggregation or uneven distribution issues. Indirect immunofluorescence assay (IFA) using appropriate antibodies can reveal high-efficiency reactivity patterns .

• Transmission electron microscopy (TEM): For detailed visualization, TEM can confirm the presence of recombinant proteins on the bacterial surface without affecting bacterial morphology .

What strategies can improve stability and yield of purified recombinant S11 protein?

Maximizing stability and yield of purified recombinant S11 requires attention to multiple factors throughout the expression and purification process:

• Buffer optimization: Tris-based buffers containing 50% glycerol have been shown to enhance stability of recombinant proteins from bacterial systems . For S11, testing buffer components that mimic the ribosomal environment may improve stability.

• Temperature and pH considerations: Recombinant proteins expressed in L. plantarum can demonstrate remarkable stability at elevated temperatures (up to 50°C) and acidic conditions (pH 1.5), suggesting that stability assessment across various conditions is valuable for optimizing purification and storage protocols .

• Salt concentration effects: High salt concentrations may affect protein stability differently depending on the specific protein. Testing the effect of various salt concentrations during purification can identify optimal conditions for S11 .

• Co-expression strategies: Co-expressing S11 with natural binding partners (other ribosomal proteins or RNA fragments) may improve stability by satisfying interaction interfaces that might otherwise lead to aggregation.

• Chromatography optimization: Sequential chromatography steps (affinity followed by size exclusion) can improve purity while maintaining structural integrity. The elution strategy should be optimized specifically for S11, with appropriate imidazole gradients if using His-tag purification.

For long-term storage, flash-freezing aliquots in liquid nitrogen and storing at -80°C with cryoprotectants can maintain activity. Testing protein stability under various storage conditions is recommended for developing optimal preservation protocols.

How might recombinant S11 expression in L. plantarum contribute to antibiotic development?

The expression of ribosomal protein S11 in L. plantarum opens several avenues for antibiotic research:

• Target validation studies: Since the bacterial ribosome is a major antibiotic target, recombinant S11 can be used to validate its potential as a specific target for new antimicrobials through binding and inhibition assays.

• Screening platforms: Surface-displayed S11 on L. plantarum could serve as a screening platform for compounds that specifically bind to this ribosomal protein, potentially identifying novel antibiotic candidates.

• Resistance mechanism studies: By expressing S11 variants containing mutations associated with antibiotic resistance, researchers can investigate the structural and functional basis of resistance mechanisms.

• Species-selective targeting: Comparative studies of S11 from different bacterial species could reveal structural differences that enable development of species-selective antibiotics, addressing the challenge of broad-spectrum antibiotic resistance.

• Structure-based drug design: High-resolution structural data from purified recombinant S11 can inform computational and medicinal chemistry approaches to design molecules that specifically interfere with S11 function or its interactions within the ribosome.

These approaches may contribute to addressing the urgent need for new antibiotics against multidrug-resistant pathogens by targeting essential components of the bacterial translation machinery.

What emerging technologies might enhance recombinant S11 research in L. plantarum?

Several cutting-edge technologies show promise for advancing recombinant S11 research in L. plantarum:

• CRISPR-Cas genome editing: Precise genomic integration of S11 variants could provide more stable expression compared to plasmid-based systems and allow for targeted manipulation of host factors affecting recombinant protein production.

• Synthetic biology approaches: Designer expression systems with orthogonal translation machinery could allow incorporation of non-canonical amino acids into S11, enabling novel studies of ribosome function with minimal interference from endogenous ribosomes.

• Microfluidics and high-throughput screening: These technologies can accelerate the optimization of expression conditions and facilitate rapid assessment of S11 variants for specific properties or interactions.

• Artificial intelligence for protein engineering: Machine learning approaches can predict optimal sequences for improved expression, stability, and functionality of recombinant S11 in the L. plantarum host.

• Advanced imaging techniques: Super-resolution microscopy and correlative light and electron microscopy (CLEM) could provide unprecedented insights into the localization and dynamics of S11 within L. plantarum cells or in reconstituted ribosomal assemblies.

Integration of these technologies with established expression systems will likely enhance our ability to produce and study recombinant ribosomal proteins with increased efficiency and experimental possibilities.