Recombinant Lactobacillus johnsonii 50S ribosomal protein L36 (rpmJ)

What is Recombinant Lactobacillus johnsonii 50S ribosomal protein L36 (rpmJ)?

Recombinant Lactobacillus johnsonii 50S ribosomal protein L36 (rpmJ) is a small ribosomal protein that forms part of the large (50S) ribosomal subunit. When produced recombinantly, the protein typically has the amino acid sequence MKVRPSVKPM CEHCKIIKRQ GRVMVICSAN PKHKQRQG and spans the full expression region of 1-38 amino acids. The protein has the Uniprot identifier Q74L67 and is commonly expressed in E. coli expression systems for research purposes . The protein plays a significant role in ribosomal assembly and function, with its absence leading to alterations in translation fidelity and ribosomal activity.

What are the optimal storage conditions for recombinant rpmJ protein?

The shelf life of recombinant rpmJ protein depends on several factors including storage state, buffer ingredients, and storage temperature. For optimal stability:

• Liquid form: Store at -20°C/-80°C for up to 6 months

• Lyophilized form: Store at -20°C/-80°C for up to 12 months

• Avoid repeated freeze-thaw cycles

• For working aliquots, store at 4°C for up to one week

• Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of 5-50% glycerol (with 50% as the default concentration) is recommended for long-term storage

How can researchers verify the purity and identity of recombinant rpmJ protein?

Researchers should implement a multi-verification approach:

• SDS-PAGE analysis: Confirm a single band at approximately 4.3 kDa with purity >85%

• Western blotting: Use anti-His tag antibodies if the protein contains a histidine tag

• Mass spectrometry: Validate the molecular weight and sequence coverage

• Functional assays: Test ribosome assembly capacity in cell-free systems

• Sequence verification: Compare with the reference sequence: MKVRPSVKPM CEHCKIIKRQ GRVMVICSAN PKHKQRQG

What are the recommended protocols for engineering recombinant Lactobacillus johnsonii to express rpmJ?

Engineering recombinant L. johnsonii to express rpmJ follows similar methodologies to those established for other recombinant protein expression in this species:

• Plasmid construction:

  • Insert the rpmJ gene into a suitable expression vector (e.g., pPG-612)

  • Use appropriate restriction sites (e.g., EcoRI and EcoRV)

  • Verify the recombinant plasmid by restriction enzyme digestion and sequencing

• Transformation:

  • Prepare L. johnsonii competent cells in an ice-water slurry for 10 minutes

  • Mix 5 μL of the recombinant plasmid with 100 μL of competent cells

  • After 5 minutes of incubation on ice, transfer to a pre-chilled electroporation cuvette

  • Apply a single electric pulse of 2.1 kV for 3 ms

  • Incubate on ice for 10 minutes post-electroporation

• Selection and culture:

  • Transfer cells to MRS broth containing 15% sucrose

  • Incubate anaerobically at 37°C for 2 hours

  • Centrifuge at 3,000 rpm for 10 minutes and resuspend in 200 μL MRS broth

  • Plate on MRS agar containing appropriate antibiotic (e.g., 10 μg/mL chloramphenicol)

  • Incubate anaerobically at 37°C overnight

• Verification:

  • Confirm transformation by PCR

  • Verify protein expression via Western blotting

  • Test stability over multiple generations (at least 40) to ensure inheritance

How can researchers optimize the expression of recombinant rpmJ in Lactobacillus johnsonii?

Optimization of rpmJ expression in L. johnsonii can be achieved through Design of Experiments (DoE) methodology:

• Variable identification using Plackett-Burman design:

  • Test carbon sources (glucose/lactose concentration)

  • Nitrogen sources (ammonium citrate concentration)

  • Physical parameters (pH, temperature, incubation time)

• Optimization using central composite design (CCD) of response surface methodology (RSM):

  • Create a full experimental design with identified variables

  • Conduct submerged fermentation experiments

  • Quantify protein production

  • Analyze using ANOVA to generate a polynomial equation

  • Determine optimal conditions

• Example optimization parameters for L. johnsonii:

  • Carbon source: glucose 10 g/L or lactose 10.07 g/L

  • Nitrogen source: ammonium citrate 2.49 g/L

  • Incubation time: 48-94 hours (strain-dependent)

  • pH: 5.4-7.6 (strain-dependent)

What is the role of rpmJ in zinc resistance in bacteria?

The knockout of rpmJ has been demonstrated to lead to zinc resistance in bacteria, revealing important insights into metal homeostasis mechanisms:

• Observed phenotype:

  • Deletion of rpmJ (L36) in E. coli results in zinc resistance

  • The rpmJ mutant shows decreased intracellular zinc concentration under excess zinc conditions

  • The zinc-resistant phenotype depends on ZntA, a zinc efflux pump

• Mechanisms:

  • The rpmJ mutant exhibits ribosomal dysfunction evidenced by:

    • Increased sensitivity to protein synthesis inhibitors (chloramphenicol, erythromycin, clarithromycin, tetracycline)

    • Altered translation fidelity measured by dual luciferase assay

    • Higher UGA stop codon readthrough

• Gene expression changes:

  • RNA sequencing analysis revealed 195 upregulated and 275 downregulated genes in the rpmJ mutant

  • No direct alterations in zinc uptake or efflux genes

  • Decreased expression of 6 genes encoding iron-sulfur cluster synthases, which may contribute to zinc resistance as iron-sulfur clusters are toxic targets of zinc

  • Changes in expression of respiratory genes, metabolic genes, and stress response genes

How does rpmJ affect translation fidelity and what are the implications for protein synthesis?

The rpmJ protein significantly impacts translation accuracy and efficiency:

• Translation accuracy metrics:

  • In rpmJ knockout mutants, UGA stop codon readthrough increases, indicating decreased translation fidelity

  • This suggests the protein plays a role in maintaining accurate termination during protein synthesis

• Sensitivity to antibiotics:

  • The rpmJ mutant shows increased sensitivity to:

    • Chloramphenicol (binds to the 50S ribosomal subunit)

    • Erythromycin and clarithromycin (macrolides targeting the 50S subunit)

    • Tetracycline (binds to the 30S ribosomal subunit)

  • This heightened sensitivity confirms alteration of ribosomal function

• Implications for recombinant protein production:

  • When using L. johnsonii as an expression system, researchers should consider how manipulation of rpmJ might affect:

    • Translation accuracy of the recombinant protein

    • Potential readthrough events leading to extended protein variants

    • Antibiotic selection strategies during strain maintenance

How can recombinant L. johnsonii expressing modified rpmJ be used in probiotic applications?

L. johnsonii has proven probiotic properties that could be enhanced by engineering its ribosomal proteins:

• Acid resistance enhancement:

  • L. johnsonii 456 naturally exhibits higher viability at lower pH conditions compared to other probiotic strains

  • Engineering rpmJ could potentially modify translation of acid stress response proteins

  • This could further enhance survival through the gastric environment, a critical feature for probiotic delivery

• Pathogen inhibition capabilities:

  • L. johnsonii has demonstrated contact-dependent and independent inhibition of pathogen growth

  • Engineered rpmJ could alter translation of antimicrobial peptides or modify metabolic pathways to enhance this inhibitory effect

  • Testing protocols involve co-culture experiments with pathogens such as Enterotoxigenic E. coli, Salmonella, and E. faecalis

• Mutant development protocols:

  • Create rpmJ variants via site-directed mutagenesis

  • Transform into L. johnsonii using established electroporation protocols

  • Screen for enhanced probiotic properties:

    • Acid survival assays (pH 2.0-4.0 for 1-3 hours)

    • Bile resistance tests (0.1-0.5% bile salts)

    • Pathogen inhibition zones on agar plates

    • Co-culture growth curves with pathogens

What methods are available for detecting contradictory data when studying rpmJ function across different experimental conditions?

When analyzing potentially contradictory data regarding rpmJ function, researchers should employ systematic approaches:

How can researchers systematically design experiments to understand the structure-function relationship of rpmJ in L. johnsonii?

A comprehensive structure-function investigation requires:

• Mutagenesis approach:

  • Site-directed mutagenesis targeting conserved residues in the sequence: MKVRPSVKPM CEHCKIIKRQ GRVMVICSAN PKHKQRQG

  • Focus on cysteine residues (C, positions 12 and 15) that may form zinc-binding motifs

  • Create systematic alanine scanning mutants to identify critical residues

  • Design chimeric proteins with rpmJ from different bacterial species to identify species-specific functional domains

• Functional assessment:

  • Translation fidelity assays using dual-luciferase reporters with programmed errors

  • Ribosome assembly analysis using sucrose gradient centrifugation

  • In vitro translation efficiency using cell-free systems

  • Metal binding capacity through isothermal titration calorimetry

  • Growth assays under various stress conditions (zinc excess, antibiotics, acid stress)

• Structural analysis:

  • X-ray crystallography of purified rpmJ protein

  • Cryo-EM analysis of ribosomes containing wild-type versus mutant rpmJ

  • In silico molecular dynamics simulations to predict conformational changes

  • Hydrogen-deuterium exchange mass spectrometry to assess protein dynamics and binding interfaces

What are the most effective methods for resolving expression and purification issues with recombinant rpmJ?

When encountering challenges with rpmJ expression and purification:

• Low expression levels:

  • Optimize codon usage for the host organism

  • Test different promoter systems (constitutive vs. inducible)

  • Adjust induction parameters (inducer concentration, temperature, duration)

  • Co-express with chaperones to assist proper folding

  • Use fusion tags that enhance solubility (MBP, SUMO, TrxA)

• Protein degradation:

  • Add protease inhibitors during cell lysis

  • Reduce processing time and keep samples cold

  • Test different buffer compositions to enhance stability

  • Consider expressing a more stable fusion protein

  • Purify under denaturing conditions if necessary

• Purification challenges:

  Challenge	Solution	Implementation
  Poor binding to affinity resin	Adjust imidazole concentration in binding buffer	Test 5-20 mM imidazole to reduce non-specific binding
  Co-purification of contaminants	Increase washing stringency	Use step gradients with increasing imidazole (50, 100, 250 mM)
  Protein aggregation	Add stabilizing agents	Include 5-10% glycerol, 0.1-0.5 M NaCl, or 0.05% Tween-20
  Low yield from gel filtration	Optimize sample concentration	Concentrate sample to 5-10 mg/mL before gel filtration
  Precipitation after buffer exchange	Screen buffer conditions	Test various pH values (6.0-8.0) and salt concentrations (0-500 mM)

How can researchers overcome challenges in analyzing the role of rpmJ in zinc homeostasis?

To effectively study zinc homeostasis related to rpmJ:

• Zinc concentration measurement techniques:

  • Inductively coupled plasma mass spectrometry (ICP-MS) for precise total zinc quantification

  • Fluorescent zinc probes (FluoZin-3, Zinpyr-1) for live-cell imaging of free zinc

  • Genetically encoded FRET sensors for subcellular zinc monitoring

  • X-ray fluorescence microscopy for high-resolution mapping of zinc distribution

• Genetic background considerations:

  • Create double knockouts with zinc transporters (ZntA, ZnuABC) to understand interactions

  • Use regulatable promoters to control rpmJ expression levels

  • Complement mutants with wild-type and modified rpmJ to confirm phenotype specificity

  • Consider strain-specific differences in zinc homeostasis pathways

• Controlling for experimental variables:

  • Standardize media composition, particularly trace metal content

  • Use metal-defined media prepared with ultrapure water and chemicals

  • Employ chelators (EDTA, TPEN) as controls to confirm zinc-specific effects

  • Include multiple time points to capture dynamic responses to zinc stress

  • Normalize measurements to cell number or protein content

What are promising avenues for leveraging rpmJ modification in developing next-generation Lactobacillus-based therapeutics?

Emerging research directions include:

• Engineered delivery systems:

  • Create L. johnsonii strains with modified rpmJ to serve as live delivery vectors for therapeutic molecules

  • Design strains that can co-express rpmJ variants along with bioactive peptides or immunomodulatory proteins

  • Develop conditional expression systems that activate in specific gut environments

  • Engineer strains with enhanced mucosal adhesion and colonization properties

• Immunomodulatory applications:

  • Explore the impact of rpmJ modifications on L. johnsonii's anti-inflammatory properties

  • Investigate how alterations in ribosomal function might affect the production of immunomodulatory metabolites

  • Develop L. johnsonii strains with modified rpmJ that can express immunomodulatory molecules like GM-CSF

  • Evaluate these strains in models of inflammatory bowel disease and other inflammatory conditions

• Synthetic biology approaches:

  • Design minimal synthetic ribosomes with engineered rpmJ to create L. johnsonii strains with orthogonal translation systems

  • Develop ribosome-targeting antibiotics specifically against pathogenic bacteria while sparing beneficial Lactobacillus species

  • Create biosensor strains where rpmJ-dependent translation is linked to reporter gene expression for tracking intestinal conditions

How might advances in structural biology and computational methods enhance our understanding of rpmJ function in L. johnsonii?

Advanced methodologies will drive future research:

• Cryo-EM and structural studies:

  • High-resolution structures of L. johnsonii ribosomes with and without rpmJ

  • Visualization of zinc binding sites and coordination geometry

  • Structural comparison across different growth conditions and zinc concentrations

  • Identification of interaction partners and conformational changes during translation

• Molecular dynamics simulations:

  • Predict the impact of mutations on rpmJ structure and zinc binding

  • Model interactions between rpmJ and other ribosomal components

  • Simulate the dynamics of zinc binding and release

  • Identify potential allosteric effects that influence ribosome function

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics) to map the impact of rpmJ on cellular physiology

  • Network analysis to identify regulatory hubs influenced by rpmJ-dependent translation

  • Machine learning models to predict phenotypic outcomes of rpmJ modifications

  • Whole-cell computational models incorporating translation dynamics to predict growth and stress responses