Recombinant Lactobacillus acidophilus 50S ribosomal protein L29 (rpmC)

What is the structural and functional role of 50S ribosomal protein L29 in Lactobacillus acidophilus?

The 50S ribosomal protein L29 (rpmC) functions as a component of the large ribosomal subunit in L. acidophilus, participating in the assembly and stability of the ribosome. This protein belongs to the conserved ribosomal protein set found in bacterial species. In the bacterial ribosome architecture, L29 is positioned near the peptide exit tunnel and may play a role in ribosome-nascent chain interactions . Unlike many ribosomal proteins, some studies have suggested L29 may sometimes be non-essential in certain bacterial species, though this varies across organisms . Within L. acidophilus NCFM (a commercially important probiotic strain), the genome contains 1,864 predicted ORFs, including ribosomal proteins that contribute to the organism's protein synthesis machinery .

How does growth phase affect expression of 50S ribosomal proteins in L. acidophilus?

Growth phase significantly impacts the expression of ribosomal proteins in L. acidophilus. Research using TMT (tandem mass tag) labeling combined with triple-stage mass spectrometry (MS3) has demonstrated that the L. acidophilus surface-associated proteome undergoes significant alterations between logarithmic and early stationary growth phases .
Specifically, many highly abundant proteins including several ribosomal proteins show differential expression between growth phases. During the transition from logarithmic to stationary phase, the 50S ribosomal protein L29 expression pattern may shift along with other ribosomal components. One study identified a 50S ribosomal protein (LBA0292) that was upregulated to exceptionally high abundance in stationary phase . This indicates that ribosomal proteins in L. acidophilus are not simply constitutive but respond to growth conditions, potentially serving additional functions beyond translation.

What expression systems are most effective for producing recombinant L. acidophilus 50S ribosomal protein L29?

Multiple expression systems can be employed for producing recombinant L. acidophilus 50S ribosomal protein L29, each with specific advantages:

What purification strategies yield the highest purity of recombinant L. acidophilus 50S ribosomal protein L29?

For optimal purification of recombinant L. acidophilus 50S ribosomal protein L29, a multi-step purification strategy is recommended:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using a histidine tag is effective for initial purification if the recombinant protein includes a His-tag.

• Intermediate Purification: Ion exchange chromatography based on the protein's theoretical isoelectric point.

• Polishing Step: Size exclusion chromatography to remove aggregates and achieve high purity.
  For L. acidophilus surface-associated proteins, including potential ribosomal proteins that may associate with the cell surface, lithium chloride extraction protocols have proven effective for releasing proteins non-covalently bound to the L. acidophilus S-layer . This approach could be considered if studying potential moonlighting functions of L29 at the cell surface.

What methods are most effective for characterizing the structure and stability of recombinant L. acidophilus 50S ribosomal protein L29?

For comprehensive characterization of recombinant L. acidophilus 50S ribosomal protein L29, the following complementary approaches are recommended:

• Circular Dichroism (CD) Spectroscopy: To analyze secondary structure content and thermal stability.

• Differential Scanning Calorimetry (DSC): For detailed thermodynamic analysis of protein unfolding.

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): To determine oligomeric state and homogeneity.

• Mass Spectrometry: For accurate mass determination and identification of post-translational modifications.

• X-ray Crystallography or Cryo-EM: For high-resolution structural characterization.
  Studies with other proteins from L. acidophilus have employed molecular dynamics (MD) to identify highly flexible regions in protein structures, as evaluated by root mean square fluctuation (RMSF) values . Similar approaches could be applied to L29 to understand its structural dynamics and stability.

How can computational methods be used to predict and improve the stability of L. acidophilus 50S ribosomal protein L29?

Computational approaches can significantly contribute to understanding and enhancing the stability of L. acidophilus 50S ribosomal protein L29:

• Molecular Dynamics (MD) Simulations: To identify highly flexible regions that may impact protein stability. Specific regions with high RMSF values would be candidates for stabilization through mutagenesis.

• Computational Protein Design (e.g., Rosetta): To predict mutations that could increase rigidity and favorable interactions in highly flexible regions. The Rosetta platform has been successfully applied to other L. acidophilus proteins .

• ΔΔG Calculations: To assess the energetic impact of potential mutations on protein stability. Mutations with ΔΔG < 0 REU typically indicate stabilizing modifications.

• Hydrogen Bond Network Analysis: To identify possible sites for introducing additional hydrogen bonds to enhance structural integrity.
  A similar workflow has been successfully applied to L. acidophilus α-L-rhamnosidase, where MD simulations identified five highly flexible regions, and computational design predicted stabilizing mutations that increased hydrogen bond interactions . Applying this approach to L29 could yield stabilized variants with enhanced thermal tolerance for research applications.

How can recombinant L. acidophilus 50S ribosomal protein L29 be employed in surface display systems?

Recombinant L. acidophilus 50S ribosomal protein L29 could be integrated into surface display systems using established anchoring strategies for L. acidophilus. Based on research with other surface-displayed proteins, two primary anchoring approaches could be employed:

• Non-covalent Cell Wall Association: Fusion of L29 to the C-terminal region of a cell envelope proteinase (PrtP), resulting in attachment to the cell wall via electrostatic bonds .

• Covalent Cell Wall Attachment: Conjugation of L29 to the anchor region of mucus binding protein (Mub) containing an LPXTG motif, which would allow covalent association with the cell wall through sortase activity .
  Research has shown that these two different anchoring methods for surface-displayed proteins in L. acidophilus can result in dissimilar maturation and cytokine production patterns in human myeloid dendritic cells . When considering L29 for surface display, researchers should anticipate that the method of attachment may significantly impact the immune response to the displayed protein.

What is the potential role of L. acidophilus 50S ribosomal protein L29 in moonlighting functions?

While the primary function of L29 is as a component of the ribosome, several studies suggest that ribosomal proteins may serve moonlighting functions, particularly at the cell surface:

• Surface Association: Research on the L. acidophilus surface proteome has identified various traditionally cytoplasmic proteins associated with the cell surface, including several ribosomal proteins . A 50S ribosomal protein (LBA0292) was found to be significantly upregulated in stationary phase and present on the cell surface .

• Potential Functions: Based on studies of other ribosomal proteins found on bacterial surfaces, L29 might potentially participate in:

  • Adhesion to host tissues

  • Immune modulation

  • Extracellular matrix interactions
    The presence of ribosomal proteins on the exterior of Enterococcus faecalis and Staphylococcus aureus has been documented, with evidence suggesting roles beyond translation . In L. acidophilus, many surface-exposed cytoplasmic proteins show growth phase-dependent expression, indicating possible regulatory mechanisms for these moonlighting functions .

How does simulated microgravity affect the expression of 50S ribosomal proteins in L. acidophilus?

Simulated microgravity (SMG) conditions have been shown to impact the growth and gene expression of various bacteria, including L. acidophilus. Research indicates:

How does recombinant L. acidophilus 50S ribosomal protein L29 interact with the immune system?

The interactions between recombinant L. acidophilus 50S ribosomal protein L29 and the immune system can be understood through several perspectives:

• Dendritic Cell Responses: When displayed on the L. acidophilus surface, recombinant proteins can induce maturation of human myeloid dendritic cells (DCs). Studies with other surface-displayed proteins have shown upregulation of CD40, CD80, CD83, and CD86 markers, with particular emphasis on CD83 and CD40 expression .

• Cytokine Production: Surface-displayed proteins on L. acidophilus can trigger the secretion of cytokines from DCs, including IL-1β, IL-6, IL-10, IL-12 (p70), and TNF-α . The specific pattern of cytokine production may vary depending on how L29 is anchored to the cell surface.

• TLR Signaling: As a bacterial protein, L29 might potentially interact with pattern recognition receptors like TLRs. Research with other Lactobacillus proteins has shown activation of TLR pathways, leading to NF-κB activation .
  In therapeutic contexts, understanding these immunological interactions is crucial for predicting how recombinant L29 might modulate immune responses when delivered by L. acidophilus.

What strategies can protect recombinant surface-displayed L. acidophilus 50S ribosomal protein L29 from degradation in the gastrointestinal tract?

For therapeutic applications involving orally administered recombinant L. acidophilus displaying 50S ribosomal protein L29, protection from gastrointestinal degradation is critical. Research suggests the following approaches:

• Buffer Supplementation: Addition of bicarbonate buffer can protect surface-displayed proteins from proteolytic enzymes during gastric challenge in vitro .

• Enzyme Inhibitors: Supplementation with soybean trypsin inhibitor has been shown to protect cell surface antigens from degradation .

• Combined Approach: Using both bicarbonate buffer and soybean trypsin inhibitor provides dual protection, not only for the surface-displayed proteins but also increasing the viability of the L. acidophilus cells themselves upon challenge with simulated digestive juices .
  Experimental data has demonstrated that surface-associated proteins on L. acidophilus are highly sensitive to simulated gastric and small intestinal juices, with rapid degradation observed even after a 100× dilution of proteolytic enzymes . Both covalently and non-covalently bound surface proteins showed similar sensitivities to enzymatic proteolysis, underscoring the importance of protective measures regardless of the anchoring strategy employed.

What quantitative proteomics approaches are most effective for studying L. acidophilus 50S ribosomal protein L29 expression?

For accurate quantification of L. acidophilus 50S ribosomal protein L29 expression under different conditions, advanced proteomics approaches are recommended:

• TMT Labeling with MS3 Technology: Tandem mass tag (TMT) labeling combined with synchronous precursor selection (SPS)-based MS3 technology has demonstrated high sensitivity and reproducibility in quantifying the L. acidophilus surface-associated proteome . This approach allows multiplexed analysis of samples from different conditions while avoiding ratio compression effects.

• iTRAQ Labeling: For comparing protein expression across multiple conditions, iTRAQ (isobaric tags for relative and absolute quantification) has been successfully employed for stress response studies in bacterial systems .

• Sample Preparation Considerations: For comprehensive analysis, extraction protocols should be optimized based on the subcellular localization of interest:

  • For cytoplasmic fraction: Standard cell lysis and protein extraction

  • For surface-associated proteins: Lithium chloride (LiCl) isolation protocol

  • For membrane-embedded proteins: Specialized detergent-based extraction
    MS3 technology has yielded almost a tenfold increase in protein identification compared to previous L. acidophilus exoproteome studies, making it particularly valuable for detecting and quantifying lower-abundance proteins like L29 under various experimental conditions .

How can researchers verify the functionality of recombinant L. acidophilus 50S ribosomal protein L29?

Verifying the functionality of recombinant L. acidophilus 50S ribosomal protein L29 requires assays that assess both its primary ribosomal role and potential moonlighting functions:

• Ribosomal Assembly Assays:

  • In vitro reconstitution of partial ribosomal subunits

  • Complementation assays in conditional L29 mutants

• Binding Assays:

  • RNA binding assays to confirm interaction with ribosomal RNA

  • Protein-protein interaction studies with other ribosomal components

• Surface Functionality (if studying moonlighting roles):

  • Adhesion assays to human intestinal epithelial cells

  • Immunomodulatory effects using human dendritic cell models

  • Fluorescent labeling to track surface localization

• Structural Integrity Validation:

  • Circular dichroism to confirm proper folding

  • Thermal stability assays to assess structural robustness For functional ribosomal studies, it's important to note that the 50S ribosomal protein L29 works in concert with many other ribosomal proteins, and its functionality may be best assessed in the context of the assembled ribosome or ribosomal subunit rather than as an isolated protein.