Recombinant Lactobacillus plantarum Chaperone protein DnaJ (dnaJ)

What is the DnaJ chaperone protein in Lactobacillus plantarum and what is its primary function?

DnaJ is a co-chaperone protein in L. plantarum that functions as part of the molecular chaperone system alongside DnaK. The primary role of DnaJ is to assist in protein quality control, including proper protein folding, prevention of aggregation, and refolding of denatured proteins. DnaJ recognizes and binds to unfolded or misfolded proteins and then recruits DnaK to these substrates, stimulating the ATPase activity of DnaK . This DnaK-DnaJ system is critical for maintaining protein homeostasis in the bacterial cell, especially under stress conditions .

In L. plantarum, the dnaJ gene encodes a 380-amino acid protein with several characteristic domains, including:

• J-domain (involved in DnaK interaction)

• Glycine-phenylalanine rich region

• Zinc finger domain (for substrate binding)

• C-terminal domain

How does the structure of L. plantarum DnaJ differ from other bacterial DnaJ proteins?

L. plantarum DnaJ shares the canonical domain organization with other bacterial DnaJ proteins but exhibits sequence-specific variations that may influence its substrate specificity. The protein contains:

• An N-terminal J-domain (amino acids 1-70) that interacts with DnaK and stimulates its ATPase activity

• A glycine-phenylalanine rich region (approximately amino acids 71-121)

• A zinc finger-like domain (approximately amino acids 122-206)

• A C-terminal domain (approximately amino acids 207-380) involved in substrate binding

The amino acid sequence of L. plantarum DnaJ shows distinctive features in its substrate-binding regions when compared to other lactic acid bacteria, potentially reflecting adaptation to the diverse environmental niches that L. plantarum occupies .

What genomic characteristics of L. plantarum contribute to its versatility as an expression host for recombinant proteins?

L. plantarum possesses several genomic features that make it an excellent host for recombinant protein expression:

• Large and flexible genome (3.3 Mb) containing 3,052 predicted protein-encoding genes, allowing adaptation to various environmental conditions

• Naturally plasmid-free strains available, facilitating transformation and genetic manipulation

• Well-characterized promoter systems such as sppA and constitutive promoter 3a

• Comprehensive pathways for protein quality control, including the DnaK-DnaJ chaperone system

• Relatively high G+C content (44.3%), providing stability for recombinant gene expression

This genomic flexibility contributes to L. plantarum's ability to express and display a variety of heterologous proteins with proper folding and functionality .

What are the most effective expression systems for producing recombinant DnaJ in L. plantarum?

Several expression systems have proven effective for producing recombinant DnaJ in L. plantarum:

• pSIP Expression System: The pSIP inducible expression system, employing the sakacin P promoter (PsppA), offers tight control of expression and high protein yields .

• pWCF Vector Series: These vectors are specifically designed for L. plantarum and provide efficient secretion and surface display of recombinant proteins .

• NICE (NIsin-Controlled gene Expression) System: Though originally developed for L. lactis, this system has been adapted for L. plantarum with good results for chaperone protein expression .

For optimal expression, parameters should be adjusted based on experimental goals:

Codon optimization for L. plantarum can significantly increase expression levels of recombinant DnaJ .

How can recombinant L. plantarum DnaJ be purified with optimal yield and activity?

Purification of recombinant DnaJ from L. plantarum requires careful consideration of protein characteristics to maintain functionality:

• Cell Disruption: Sonication or enzymatic lysis (lysozyme treatment followed by French press) is recommended, as DnaJ is an intracellular protein .

• Tag Selection: His-tag, Avi-tag, or biotinylated versions can be used without significantly affecting protein function .

• Purification Protocol:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged DnaJ

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography for further purification

• Activity Preservation: Include 10% glycerol, 1-5 mM DTT, and avoid freeze-thaw cycles to maintain chaperon activity .

Typical yields of 5-10 mg of purified DnaJ per liter of bacterial culture can be achieved with >85% purity as determined by SDS-PAGE .

What methodological approaches can be used to verify the correct folding and functionality of recombinant DnaJ?

Verification of proper folding and functionality of recombinant DnaJ requires multiple complementary approaches:

• Structural Verification:

  • Circular dichroism spectroscopy to assess secondary structure

  • Limited proteolysis to assess domain integrity

  • Thermal shift assays to measure protein stability

• Functional Assays:

  • ATPase stimulation assay: DnaJ should stimulate the ATPase activity of DnaK in vitro

  • Protein aggregation prevention assay: Measurement of the ability to prevent thermal aggregation of model substrates like luciferase

  • Protein refolding assay: Assessment of the ability to assist in refolding of denatured proteins

• Interaction Studies:

  • Co-immunoprecipitation with DnaK

  • Surface plasmon resonance to measure binding kinetics

  • Size-exclusion chromatography to detect complex formation with substrate proteins

To distinguish between inactive and active forms, researchers should include appropriate positive controls (e.g., commercially available E. coli DnaJ) and negative controls (e.g., heat-inactivated DnaJ) .

How does recombinant L. plantarum expressing DnaJ influence host immune responses?

Recombinant L. plantarum expressing DnaJ has been found to modulate host immune responses in several ways:

• Dendritic Cell (DC) Activation: DnaJ-expressing L. plantarum significantly stimulates the differentiation of bone marrow-derived dendritic cells and increases secretion of interleukin (IL)-6, as demonstrated by flow cytometry, ELISA, and qRT-PCR analyses .

• T Helper Cell Modulation: In vivo studies show that DnaJ-expressing L. plantarum promotes differentiation of IL-4+ and IL-17A+ T helper (Th) cells in the spleen, leading to increased serum levels of IL-4 and IL-17A .

• B Cell Responses: Recombinant L. plantarum expressing DnaJ increases the production of B220+ B cells in mesenteric lymph nodes and Peyer's patches, enhancing antibody production .

• Mucosal Immunity: L. plantarum expressing DnaJ alongside antigenic proteins can significantly enhance mucosal IgA responses, as demonstrated in studies examining intestinal and lung IgA expression .

These immunomodulatory effects suggest potential applications in vaccine development and immunotherapeutic approaches .

What are the advantages of using L. plantarum as a host for recombinant DnaJ expression compared to other expression systems?

L. plantarum offers several distinct advantages as a host for recombinant DnaJ expression:

• Safety Profile: L. plantarum has Generally Recognized As Safe (GRAS) status, making it suitable for in vivo applications and potential clinical use .

• Mucosal Delivery: As a lactic acid bacterium, L. plantarum can survive gastric passage and interact with intestinal mucosal surfaces, facilitating immune response induction .

• Adjuvant Properties: L. plantarum itself has inherent adjuvant properties that can enhance immune responses to co-expressed antigens .

• Protein Folding Capability: The endogenous protein quality control system of L. plantarum supports proper folding of complex proteins like DnaJ .

• Genetic Tractability: Recent advances in genetic manipulation techniques, including CRISPR/Cas9-assisted genome editing, allow precise modification of L. plantarum .

Comparative analysis with other expression systems:

How can recombinant L. plantarum DnaJ be engineered to enhance its chaperone activity?

Several engineering approaches have been developed to enhance the chaperone activity of recombinant L. plantarum DnaJ:

• Site-Directed Mutagenesis:

  • Modification of the HPD motif in the J-domain to optimize interaction with DnaK

  • Engineering the zinc finger domain to alter substrate binding specificity

  • Introduction of additional substrate binding sites

• Domain Swapping:

  • Creation of chimeric DnaJ proteins by combining domains from different species to enhance specific functions

  • Fusion of the L. plantarum DnaJ J-domain with alternative substrate binding domains

• Co-expression Strategies:

  • Co-expression with compatible chaperones like DnaK and GrpE for enhanced folding capacity

  • Development of polycistronic expression constructs for balanced chaperone production

• Structural Stabilization:

  • Introduction of disulfide bridges to enhance thermal stability

  • Surface charge engineering to improve solubility

  • Rational design based on homology modeling and molecular dynamics simulations

These approaches can be combined and optimized based on the specific application requirements .

How does the DnaJ-DnaK chaperone system in L. plantarum differ functionally from homologous systems in other bacterial species?

The DnaJ-DnaK chaperone system in L. plantarum exhibits several distinctive functional characteristics compared to homologous systems in other bacteria:

• Substrate Specificity: L. plantarum DnaJ shows broader substrate specificity when transferred to heterologous hosts compared to human Hsp40 and Hsp70, suggesting it may target a wider range of misfolded protein substrates .

• Thermal Stability: The DnaJ-DnaK system in L. plantarum operates efficiently at lower temperatures (25-37°C) compared to thermophilic bacteria, reflecting its adaptation to mesophilic environments .

• Co-chaperone Interactions: While E. coli DnaJ interacts with multiple co-chaperones including ClpB, FtsH, and Lon proteases, the L. plantarum DnaJ-DnaK system appears to have fewer documented interactions with proteolytic systems .

• Functional Activities: Comparative analysis of DnaJ activities across species reveals interesting distinctions:

These functional differences may reflect the adaptation of L. plantarum to diverse environmental niches and its role as a commensal organism .

What is the role of recombinant L. plantarum DnaJ in enhancing heterologous protein expression in bacterial and insect cell systems?

Recombinant L. plantarum DnaJ has shown significant potential for enhancing heterologous protein expression across different host systems:

• Bacterial Expression Systems:

  • Co-expression of L. plantarum DnaJ with target proteins in E. coli can improve soluble protein yield by preventing aggregation

  • The unique substrate binding properties of L. plantarum DnaJ make it complementary to endogenous E. coli chaperones

• Insect Cell Expression:

  • Co-expression of bacterial DnaJ and DnaK in insect cells dramatically enhances protein yield and solubility

  • L. plantarum DnaJ has been shown to improve the proteolytic stability of recombinant proteins in insect cells

  • Fluorescence studies with model proteins like GFP show more homogeneous distribution of properly folded protein when DnaJ is co-expressed

• Mechanism of Enhancement:

  • L. plantarum DnaJ appears to separate the beneficial foldase activity from undesirable proteolytic enhancement effects observed with E. coli DnaJ

  • This suggests that "chaperone rehosting" (using chaperones from one species in a different host) can be an effective strategy for high-quality recombinant protein production

Quantitative studies show that co-expression of DnaJ can increase soluble protein yield by 2-10 fold depending on the target protein and expression system .

How can genome-wide analysis techniques be applied to optimize recombinant DnaJ expression in L. plantarum strains?

Advanced genome-wide analysis techniques offer powerful approaches to optimize recombinant DnaJ expression in L. plantarum:

• Whole Genome Sequencing and Comparative Genomics:

  • Comparison of multiple L. plantarum strains (such as WCFS1, ATCC BAA-793, and NCIMB 8826) to identify optimal genetic backgrounds for DnaJ expression

  • Identification of strain-specific SNPs that could influence protein production capacity

• Transcriptomics and Proteomics Integration:

  • RNA-seq analysis to identify genes co-regulated with dnaJ under various stress conditions

  • Proteomic profiling to understand the impact of DnaJ overexpression on the global protein landscape

  • Integration of these datasets to identify potential bottlenecks in expression

• CRISPR/Cas9-Assisted Genome Editing:

  • Precise modification of the L. plantarum genome to optimize dnaJ expression

  • Examples include:

    • Promoter engineering for fine-tuned expression

    • Deletion of competing chaperone systems

    • Integration of additional gene copies at favorable genomic locations

• Metabolomics-Guided Optimization:

  • Analysis of metabolic shifts during recombinant protein production

  • Identification of limiting metabolites or stress indicators

  • Rational modification of culture conditions based on metabolomic data

Practical implementation involves iterative cycles of genomic modification, expression testing, and multiomics analysis to achieve optimal expression levels .

What experimental controls are essential when studying the effects of recombinant L. plantarum DnaJ on host immune responses?

When investigating immunomodulatory effects of recombinant L. plantarum DnaJ, several critical controls must be included:

• Vector Controls:

  • L. plantarum containing empty expression vector (e.g., pWCF with no insert)

  • This controls for effects of the bacterial backbone and expression system

• Protein Specificity Controls:

  • L. plantarum expressing an unrelated protein of similar size

  • L. plantarum expressing a mutant DnaJ lacking key functional domains

  • These controls help attribute observed effects specifically to DnaJ functionality

• Dose-Response Controls:

  • Administration of different bacterial cell concentrations (typically 10^8-10^10 CFU/ml)

  • Multiple time points for analysis (e.g., 24h, 48h, 72h post-administration)

  • These establish the dose-dependency and kinetics of immune responses

• Immune Cell Population Controls:

  • Inclusion of isotype antibody controls for flow cytometry

  • Positive stimulants (e.g., LPS for dendritic cells, PMA/ionomycin for T cells)

  • Controls for cell viability to distinguish activation from cytotoxicity

• In vivo Controls:

  • Vehicle-only administration group

  • Non-recombinant wild-type L. plantarum administration

  • Comparison with established adjuvants or immune modulators

What are the most sensitive molecular techniques for detecting and quantifying L. plantarum DnaJ expression levels?

Several molecular techniques offer varying degrees of sensitivity and specificity for detecting and quantifying L. plantarum DnaJ expression:

• Quantitative Real-Time PCR (qRT-PCR):

  • Highly sensitive for measuring dnaJ transcript levels

  • Requires careful primer design to distinguish recombinant from endogenous dnaJ

  • Can detect as few as 10-100 copies of target sequence

  • For accurate quantification, normalization with multiple reference genes is essential

• Digital Droplet PCR (ddPCR):

  • Offers absolute quantification without standard curves

  • Higher precision than traditional qPCR for low-abundance templates

  • Less susceptible to PCR inhibitors in complex samples

• Protein Quantification Methods:

  • Western blotting with specific anti-DnaJ antibodies (detection limit ~0.1 ng)

  • ELISA for quantitative analysis (detection limit ~10 pg/ml)

  • Flow cytometry for cell surface-displayed DnaJ (detection limit ~1000 molecules/cell)

• Mass Spectrometry-Based Approaches:

  • Targeted proteomics using multiple reaction monitoring (MRM)

  • Label-free quantification using high-resolution MS

  • Can detect DnaJ in complex mixtures with detection limits in femtomole range

Comparative sensitivity analysis:

For optimal results, combining transcript and protein-level quantification is recommended .

What strategies can be employed to address stability issues when working with recombinant L. plantarum DnaJ proteins?

Several strategies have been developed to address stability challenges with recombinant L. plantarum DnaJ proteins:

• Genetic Stabilization Approaches:

  • Codon optimization for L. plantarum (increasing GC content at wobble positions)

  • Introduction of stabilizing mutations based on consensus sequence analysis

  • Removal of protease recognition sites through silent mutations

  • Construction of fusion proteins with stabilizing partners

• Expression Optimization:

  • Tuning expression levels to prevent aggregation (using titratable promoters)

  • Co-expression with compatible chaperones (DnaK, GrpE)

  • Low-temperature induction protocols (16-25°C) to slow folding and prevent inclusion body formation

• Protein Engineering Solutions:

  • Addition of solubility tags (MBP, SUMO, Thioredoxin)

  • Surface charge engineering to improve solubility

  • Disulfide bridge introduction for structural stabilization

• Formulation and Storage Considerations:

  • Inclusion of stabilizing agents:

    • 10-20% glycerol to prevent freeze-thaw damage

    • 1-5 mM DTT or β-mercaptoethanol to maintain reduced cysteines

    • Protease inhibitors to prevent degradation

  • Optimal pH maintenance (typically pH 7.0-7.5)

  • Storage as aliquots at -80°C to minimize freeze-thaw cycles

These strategies can be combined based on the specific application requirements and properties of the recombinant DnaJ protein .

How might new genome editing technologies be applied to enhance recombinant DnaJ expression and functionality in L. plantarum?

Emerging genome editing technologies offer exciting possibilities for enhancing DnaJ expression and functionality in L. plantarum:

• CRISPR/Cas9-Assisted Seamless Genome Editing:

  • Direct integration of optimized dnaJ genes into the L. plantarum genome

  • Modification of native gene regulatory elements to enhance expression

  • Deletion of competing chaperone systems or negative regulators

  • Introduction of point mutations to enhance functionality without the need for plasmid-based expression

• Base Editing and Prime Editing:

  • Introduction of specific nucleotide changes without double-strand breaks

  • Fine-tuning of the dnaJ gene sequence and regulatory elements

  • Correction of deleterious mutations in industrial strains

• Multiplexed Genome Engineering:

  • Simultaneous modification of multiple genes involved in the chaperone network

  • Creation of synthetic chaperone operons with optimized expression ratios

  • Integration of heterologous chaperone systems from different organisms

• Systems Biology-Guided Genome Editing:

  • Identification of optimal integration sites based on genomic accessibility mapping

  • Modification of global regulators affecting chaperone expression

  • Engineering of metabolism to support high-level protein production

These advanced genome editing approaches offer the potential to create stable, highly productive L. plantarum strains with enhanced DnaJ functionality for various biotechnological applications .

What potential therapeutic applications could emerge from further research on recombinant L. plantarum DnaJ proteins?

Several promising therapeutic applications are emerging from research on recombinant L. plantarum DnaJ proteins:

• Vaccine Delivery Platforms:

  • Co-expression of DnaJ with pathogen antigens to enhance immunogenicity

  • The adjuvant effect of DnaJ could potentiate mucosal and systemic immune responses

  • This approach has shown promise in models of influenza vaccination

• Protein Misfolding Disorders:

  • The chaperone activity of DnaJ could be harnessed to address protein misfolding in neurodegenerative diseases

  • Studies with DnaJ chaperones in Huntington's disease models have shown rescue of protein aggregation phenotypes

• Inflammatory Bowel Disease Therapy:

  • The immunomodulatory properties of L. plantarum expressing DnaJ could help regulate intestinal inflammation

  • Specific immune cell populations targeted by DnaJ (dendritic cells, Th17 cells) play key roles in IBD pathogenesis

• Allergy Desensitization:

  • Recombinant L. plantarum expressing allergen-DnaJ fusion proteins could modulate allergic responses

  • The ability to skew T cell responses toward Th1/Treg phenotypes may help rebalance allergic Th2 responses

• Microbiome Engineering:

  • Integration of optimized dnaJ genes into probiotic L. plantarum strains could enhance their stress resistance and colonization potential

  • This could improve therapeutic efficacy in various microbiome-related disorders

These applications are supported by growing evidence of the immunomodulatory and chaperone functions of recombinant DnaJ proteins, with clinical trials beginning to explore their therapeutic potential .

How might systems biology approaches contribute to understanding the complex interactions of DnaJ in the L. plantarum chaperone network?

Systems biology approaches offer powerful frameworks for unraveling the complex interactions of DnaJ within the L. plantarum chaperone network:

• Interactomics:

  • Protein-protein interaction mapping using techniques like affinity purification-mass spectrometry (AP-MS)

  • Identification of the complete DnaJ interactome beyond known partners like DnaK

  • Construction of chaperone interaction networks in different stress conditions

• Multi-omics Integration:

  • Combined analysis of transcriptomics, proteomics, and metabolomics data

  • Correlation of DnaJ expression with global cellular responses

  • Identification of regulatory networks controlling chaperone expression

• Computational Modeling:

  • Development of mathematical models of the chaperone network dynamics

  • Prediction of system behavior under different stress conditions

  • In silico testing of genetic modifications before experimental validation

• Single-Cell Analysis:

  • Examination of cell-to-cell variability in DnaJ expression and function

  • Correlation with cellular phenotypes like stress resistance

  • Identification of subpopulations with distinct chaperone activities

• Evolutionary Systems Biology:

  • Comparative analysis of chaperone networks across Lactobacillus species

  • Identification of conserved and divergent features reflecting adaptation to different niches

  • Reconstruction of the evolutionary history of the DnaJ-DnaK system

These approaches would provide a comprehensive understanding of how DnaJ functions within the broader context of cellular physiology, potentially leading to novel applications in synthetic biology and biotechnology .