Recombinant Lactobacillus plantarum Putative Holliday junction resolvase (lp_2274)

What is the putative Holliday junction resolvase (lp_2274) in Lactobacillus plantarum?

The putative Holliday junction resolvase (lp_2274) in Lactobacillus plantarum is an enzyme involved in DNA recombination processes. It functions by resolving Holliday junctions, which are four-stranded DNA structures that form during genetic recombination and DNA repair. As a resolvase, this enzyme catalyzes the cleavage of these junctions, allowing for the separation of recombinant DNA molecules. The lp_2274 designation refers to the specific gene locus in the L. plantarum genome encoding this protein. While this protein shares sequence homology with other known resolvases, its specific structural and functional characteristics in L. plantarum make it a subject of interest for researchers studying DNA repair mechanisms and genetic recombination in probiotic bacteria.

Which expression systems are most suitable for recombinant L. plantarum proteins?

Several expression systems have been developed for L. plantarum, with the pSIP system being particularly effective for recombinant protein production. The pSIP expression system is based on the promoters and regulatory genes involved in the production of the class-II bacteriocin sakacin P in Latilactobacillus sakei . This system offers tight regulation of gene expression, as the promoter upstream of the target gene is strictly controlled by the two-component system sppKR, which depends on the external addition of the peptide pheromone SppIP for induction .

The choice of expression system depends on several factors:

The pSIP system has demonstrated effectiveness for expressing various recombinant proteins in L. plantarum, including membrane proteins like RseP , making it a promising choice for expressing putative Holliday junction resolvase.

What are the advantages of using L. plantarum as an expression host compared to E. coli?

L. plantarum offers several distinct advantages as an expression host compared to traditional E. coli systems, particularly for certain types of proteins:

• Membrane protein expression: L. plantarum has shown superior capability for expressing integral membrane proteins. In a comparative study of RseP expression, detection of soluble protein failed in two of three E. coli strains tested, while L. plantarum successfully expressed the protein . Additionally, purification of RseP expressed in E. coli C43(DE3) resulted in a protein with lower purity compared to the same protein expressed in L. plantarum .

• Simplified cell envelope: As a Gram-positive bacterium, L. plantarum possesses a monoderm cell envelope that can facilitate easier protein extraction and purification, especially for membrane-associated proteins .

• Limited proteolytic activity: L. plantarum has a relatively small genome with limited proteolytic activity compared to E. coli, potentially resulting in higher yields of intact recombinant proteins .

• Growth without aeration: L. plantarum can reach high cell densities without requiring aeration, allowing for expression at industrial scale using simplified bioreactors .

• GRAS status: L. plantarum has Generally Recognized As Safe (GRAS) status, making it suitable for applications in vaccine development and therapeutic protein production.

How can I confirm the successful expression of recombinant proteins in L. plantarum?

Confirming successful expression of recombinant proteins in L. plantarum requires multiple analytical approaches:

• Western blot analysis: This is a standard method for confirming protein expression. Add a detection tag (such as 6xHis-tag) to your construct to facilitate identification. Western blotting has been successfully used to confirm the production of various recombinant proteins in L. plantarum, including different RseP orthologs .

• Flow cytometry: For surface-expressed proteins, flow cytometry using specific antibodies can be employed. This technique was used to detect the expression of influenza virus antigen HA1 on recombinant L. plantarum .

• Immunofluorescence microscopy: This technique allows visualization of the expressed protein. For example, recombinant L. plantarum expressing DCpep were identified using a DCpep-specific polyclonal antibody followed by a FITC-labeled secondary antibody and observed under a fluorescence microscope .

• Functional assays: For enzymes like Holliday junction resolvase, activity assays using synthetic Holliday junctions can confirm not only expression but also proper folding and functionality of the protein.

• Immunoblotting: Bacterial lysates can be analyzed by immunoblotting to detect the target protein. This method was used to detect fusion antigens in recombinant L. plantarum after sonication or freeze-thaw cycles .

What are the optimal conditions for expressing recombinant proteins in L. plantarum using the pSIP system?

Optimizing expression conditions for recombinant proteins in L. plantarum using the pSIP system requires careful consideration of multiple parameters:

Induction parameters:

• Inducer concentration: The optimal concentration of peptide pheromone SppIP typically ranges from 25-100 ng/ml, but this should be titrated for each specific protein.

• Induction timing: Induction at mid-log phase (OD600 ~0.6-0.8) often yields best results, but this can vary depending on protein toxicity.

• Post-induction growth time: For most proteins, 3-5 hours is optimal, but expression of complex proteins may benefit from overnight induction at lower temperatures.

Culture conditions:

• Growth temperature: Standard growth at 30°C works well for most proteins, but lowering to 25°C during induction can improve folding of complex proteins.

• Media composition: MRS broth supplemented with 2% glucose is standard, but protein-specific optimization may be necessary.

• pH: Maintaining pH at 6.0-6.5 during growth and induction phases can improve protein yield.

Strain selection:

• Screening different L. plantarum strains can significantly impact expression levels. In an ortholog screening approach for RseP, expression levels varied considerably among different constructs .

• For expressing Holliday junction resolvase specifically, considering codon optimization for L. plantarum can improve translation efficiency.

Vector design considerations:

• Including a C-terminal 6xHis-tag facilitates both purification and detection of the recombinant protein .

• Optimizing ribosome binding sites and codon usage for L. plantarum can significantly enhance expression levels.

What purification strategies are most effective for recombinant proteins expressed in L. plantarum?

Purification of recombinant proteins from L. plantarum requires a strategic approach, particularly for proteins like Holliday junction resolvase that may have DNA-binding properties:

Cell lysis and initial extraction:

• Mechanical disruption: French press or sonication methods are effective for L. plantarum cell lysis. For membrane-associated proteins, multiple sonication cycles may be necessary.

• Enzymatic lysis: Lysozyme treatment (1-5 mg/ml) in combination with mutanolysin can improve cell wall disruption prior to mechanical lysis.

• Buffer optimization: For DNA-binding proteins like resolvases, high-salt buffers (300-500 mM NaCl) help prevent non-specific binding to nucleic acids during extraction.

Chromatography methods:

Optimizing purification conditions:

• Detergent selection: For membrane-associated proteins, screening different detergents (DDM, LDAO, etc.) is crucial.

• Buffer composition: Including stabilizing agents like glycerol (10-20%) can improve protein stability during purification.

• Temperature considerations: Maintaining purification steps at 4°C helps preserve protein activity.

A typical purification workflow for a His-tagged Holliday junction resolvase from L. plantarum would involve cell lysis, IMAC purification, and SEC, potentially yielding protein of sufficient purity for structural and functional studies.

How can I assess the structural integrity and activity of purified recombinant Holliday junction resolvase?

Assessing both structural integrity and enzymatic activity of purified recombinant Holliday junction resolvase requires a multi-technique approach:

Structural integrity assessment:

• Circular Dichroism (CD) spectroscopy: CD can confirm the secondary structure content and proper folding of the purified protein. This technique was successfully used to confirm the structural integrity of RseP purified from L. plantarum .

• Thermal shift assays: These can assess protein stability and help optimize buffer conditions for maximum stability.

• Size-exclusion chromatography with multi-angle light scattering (SEC-MALS): This technique provides information about the oligomeric state and homogeneity of the purified protein.

• Limited proteolysis: This can probe the compactness of the protein structure and identify flexible regions.

Activity assays for Holliday junction resolvase:

• Synthetic Holliday junction cleavage: Using synthetic four-way junctions labeled with fluorophores to monitor cleavage activity.

  • Prepare synthetic Holliday junctions using oligonucleotides with one strand fluorescently labeled

  • Incubate with purified resolvase under varying conditions (pH, salt, metal ions)

  • Analyze products by denaturing PAGE to quantify cleavage efficiency

• DNA binding assays: Using electrophoretic mobility shift assays (EMSA) to assess binding to Holliday junction structures.

  • Incubate labeled Holliday junctions with increasing concentrations of purified resolvase

  • Analyze complex formation by native PAGE

  • Determine binding affinity from titration curves

• Branch migration assays: To assess if the resolvase also possesses branch migration activity.

Quantitative enzymatic parameters to determine:

• Optimal reaction conditions (pH, temperature, salt concentration)

• Metal ion requirements (typically Mg2+ or Mn2+)

• Sequence specificity of junction resolution

• Kinetic parameters (KM, kcat, catalytic efficiency)

What strategies can improve expression yields of recombinant Holliday junction resolvase in L. plantarum?

Improving expression yields of recombinant Holliday junction resolvase in L. plantarum requires a multi-faceted optimization approach:

Genetic optimization strategies:

• Codon optimization: Adapting the coding sequence to the codon usage bias of L. plantarum can significantly improve translation efficiency.

• Promoter selection: Testing different promoter strengths within the pSIP system can help balance expression levels with protein folding capacity.

• Fusion partners: Adding solubility-enhancing fusion partners such as thioredoxin or SUMO may increase soluble protein yields.

• Signal sequence optimization: For secreted versions, testing different signal peptides can improve translocation efficiency.

Expression condition optimization:

• Ortholog screening approach: Testing Holliday junction resolvases from different bacterial species may identify variants with superior expression characteristics in L. plantarum. This approach proved successful for RseP, where orthologs from different species showed varied expression levels .

• Temperature reduction during induction: Lowering growth temperature to 20-25°C after induction can improve protein folding.

• Inducer concentration titration: Systematically vary the concentration of inducing peptide to find the optimal balance between expression level and protein quality.

• Growth media supplementation: Adding specific amino acids or cofactors relevant to the protein of interest.

Host strain engineering:

• Knockout of specific proteases: Generating protease-deficient strains can reduce degradation of the recombinant protein.

• Overexpression of chaperones: Co-expressing molecular chaperones can assist proper protein folding.

• Adaptation of host strains: Subjecting L. plantarum to directed evolution under protein expression stress can select for strains with improved expression capacity.

Scale-up considerations:

• Bioreactor parameters: Optimizing oxygen transfer, pH control, and feeding strategies for high-density cultures.

• Fed-batch cultivation: Implementing feeding strategies to maintain optimal growth while minimizing stress responses.

How can I investigate the role of Holliday junction resolvase in L. plantarum DNA recombination and repair mechanisms?

Investigating the biological role of Holliday junction resolvase (lp_2274) in L. plantarum requires multiple complementary approaches:

Genetic manipulation strategies:

• Gene knockout studies: Generate a clean deletion of the lp_2274 gene to assess its essentiality and phenotypic consequences.

  • Use CRISPR-Cas9 or homologous recombination methods optimized for L. plantarum

  • Evaluate growth under standard and stress conditions (DNA damaging agents)

  • Measure recombination frequencies in the knockout strain

• Complementation experiments: Reintroduce wild-type or mutant versions of the gene to confirm phenotype specificity.

  • Express wild-type gene from an inducible promoter in the knockout background

  • Introduce specific mutations in catalytic residues to create separation-of-function alleles

• Protein localization studies: Track the subcellular localization of the resolvase during normal growth and under DNA damage conditions.

  • Create fluorescent protein fusions while ensuring protein functionality is maintained

  • Use immunofluorescence microscopy to visualize protein localization, similar to techniques used for detecting recombinant proteins in L. plantarum

Functional analyses:

• DNA damage sensitivity assays: Evaluate sensitivity of the knockout strain to various DNA-damaging agents.

  • Test sensitivity to UV radiation, mitomycin C, methyl methanesulfonate

  • Quantify survival curves and recovery rates compared to wild-type

• Recombination frequency measurements: Develop systems to measure homologous recombination rates in vivo.

  • Use selectable marker systems for quantifying recombination events

  • Measure conjugation efficiency as a proxy for recombination proficiency

• Chromatin immunoprecipitation (ChIP): Identify genomic binding sites of the resolvase under different conditions.

  • Develop protocols for ChIP in L. plantarum using tagged versions of resolvase

  • Map binding sites during normal growth versus DNA damage response

Protein interaction studies:

• Co-immunoprecipitation: Identify protein interaction partners that may regulate resolvase activity.

• Bacterial two-hybrid assays: Screen for specific protein-protein interactions with other DNA repair components.

• In vitro reconstitution: Reconstitute minimal recombination systems using purified components including the recombinant resolvase.

These approaches would provide comprehensive insights into the biological function of Holliday junction resolvase in L. plantarum and its contribution to DNA recombination mechanisms in this probiotic bacterium.

How do I troubleshoot low expression levels of recombinant proteins in L. plantarum?

When encountering low expression levels of recombinant proteins like Holliday junction resolvase in L. plantarum, a systematic troubleshooting approach is necessary:

Construct design issues:

• Check sequence integrity: Verify the construct sequence to ensure no mutations have been introduced during cloning.

• Examine regulatory elements: Confirm that promoter, ribosome binding site, and terminator sequences are correct and properly positioned.

• Codon optimization: If initial expression is low, consider codon optimization for L. plantarum. Different RseP orthologs showed varying expression levels in L. plantarum despite similar functions .

• Fusion tags: Test alternative positions (N- vs C-terminal) or different types of affinity tags that may improve expression or detection.

Expression conditions:

• Induction parameters: Systematically vary inducer concentration, induction timing, and post-induction incubation periods.

• Growth temperature: Test expression at different temperatures (25°C, 30°C, 37°C) as lower temperatures often favor proper folding.

• Media composition: Try different media formulations or supplements that may enhance expression.

• Cell density at induction: Test induction at different growth phases (early, mid, or late logarithmic phase).

Protein stability issues:

• Proteolytic degradation: Include protease inhibitors during extraction or consider using protease-deficient host strains.

• Protein toxicity: If the protein is toxic to the host, use tighter promoter control and shorter induction times.

• Inclusion body formation: Modify solubilization conditions or add solubility-enhancing tags.

Detection methods:

• Extraction efficiency: Ensure your extraction method is appropriate for the predicted cellular localization of the protein.

• Detection sensitivity: Use more sensitive detection methods like enhanced chemiluminescence or fluorescence-based western blot.

• Antibody specificity: If using antibodies for detection, verify their specificity and consider using anti-tag antibodies which are often more reliable.

What are the key considerations for designing experiments to measure Holliday junction resolvase activity?

Designing robust experiments to measure Holliday junction resolvase activity requires careful attention to multiple parameters:

Substrate design and preparation:

• Synthetic Holliday junction construction: Design oligonucleotides that form stable four-way junctions when annealed.

  • Include fluorescent or radioactive labels for sensitive detection

  • Consider incorporating sequence elements that may influence specificity

  • Verify junction formation by native PAGE before use

• Substrate quality control: Ensure homogeneity of substrate preparation.

  • Purify annealed junctions by gel electrophoresis if necessary

  • Quantify substrate concentration accurately for kinetic measurements

  • Verify stability under assay conditions

Reaction conditions optimization:

• Buffer composition: Systematically test:

  • pH range (typically 6.5-8.5)

  • Salt concentration (50-200 mM)

  • Divalent cation requirements (Mg2+, Mn2+, Ca2+)

  • Reducing agents if the protein contains cysteines

• Enzyme concentration titration: Determine the linear range of enzyme activity.

• Time course analysis: Establish initial velocity conditions for kinetic measurements.

Controls and specificity tests:

• Essential controls:

  • Negative control (no enzyme)

  • Heat-inactivated enzyme control

  • Control with catalytically inactive mutant

  • Non-junction DNA substrate control

• Specificity determination:

  • Test activity on different junction sequences

  • Compare with other DNA structures (3-way junctions, nicked duplexes)

  • Competition assays with unlabeled substrates

Analysis methods:

• Product analysis by denaturing PAGE to identify specific cleavage sites.

• Quantification approaches:

  • Phosphorimager analysis for radioactive substrates

  • Fluorescence quantification for fluorescently labeled substrates

• Kinetic parameter determination:

  • Initial velocity measurements at varying substrate concentrations

  • Fitting to appropriate enzyme kinetic models

This methodical approach will enable reliable measurement of Holliday junction resolvase activity from recombinantly expressed protein, providing insights into its biochemical properties and potential roles in L. plantarum DNA metabolism.

What are emerging applications for recombinant proteins expressed in L. plantarum?

L. plantarum is increasingly recognized as a versatile platform for recombinant protein expression with several emerging applications:

Vaccine development and delivery:

• Mucosal vaccine delivery: L. plantarum expressing antigens can induce both systemic and mucosal immune responses. Recombinant L. plantarum expressing influenza virus antigen HA1 demonstrated the ability to induce strong immune responses, including T cell activation in mesenteric lymph nodes and spleen, as well as specific antibody production .

• Adjuvant co-expression: Co-expressing immunomodulatory molecules with antigens can enhance vaccine efficacy. For example, L. plantarum expressing HA1 with dendritic cell-targeting peptide (DCpep) showed enhanced immunogenicity compared to HA1 alone . The HA1-DCpep group exhibited significantly increased CD4+IFN-γ+ cells compared to control groups .

• Multi-valent vaccine platforms: Expressing multiple antigens simultaneously to create combination vaccines.

Therapeutic protein production:

• Enzyme replacement therapies: Production of therapeutic enzymes for oral delivery to treat gastrointestinal disorders.

• Antimicrobial protein production: Expression of bacteriocins or antimicrobial peptides for targeted pathogen control. The successful expression of RseP, which serves as a receptor for bacteriocins of the LsbB family, demonstrates the potential for engineering bacteriocin-producing strains .

• Metabolic health modulators: Enzymes or signaling molecules that can influence host metabolism when delivered to the gut.

Research tool development:

• Structure-function studies: L. plantarum has shown promise for production of membrane proteins for structural studies. The successful purification of the membrane protein RseP with retained structural integrity (confirmed by circular dichroism) demonstrates this potential .

• Protein-protein interaction studies: Developing bacterial surface display systems to study protein interactions.

• Holliday junction resolvases: These could serve as tools for studying DNA recombination mechanisms and potentially as components of gene editing systems.

Biotechnology applications:

• Biocatalyst development: Expression of industrial enzymes for food processing or biocatalysis applications.

• Biosensor development: Engineered L. plantarum expressing reporter proteins in response to specific stimuli.

• Bioremediation approaches: Expression of enzymes capable of degrading environmental contaminants.

The versatility of L. plantarum as an expression host continues to expand as genetic tools and expression systems become more sophisticated.

How might structural analysis of Holliday junction resolvase contribute to understanding DNA recombination in probiotic bacteria?

Structural analysis of Holliday junction resolvase from L. plantarum could provide crucial insights into DNA recombination mechanisms in probiotic bacteria:

Evolutionary insights:

• Comparative structural biology: Comparing the structure of L. plantarum resolvase with those from pathogenic bacteria could reveal adaptations specific to probiotic lifestyles.

• Functional diversification: Structural analysis may reveal how resolvases in probiotic bacteria have evolved specialized functions related to their ecological niche.

• Horizontal gene transfer mechanisms: Understanding the structural basis of Holliday junction resolution could illuminate mechanisms facilitating genetic exchange in gut microbiota communities.

Mechanistic understanding:

• Catalytic mechanism: Structural studies would clarify how the resolvase recognizes, binds, and cleaves Holliday junctions, potentially revealing unique features compared to other bacterial resolvases.

• DNA sequence specificity: Crystal structures with bound DNA substrates could reveal the molecular basis for sequence preferences in junction resolution.

• Conformational changes: Structures in different states (apo vs. DNA-bound) would elucidate the dynamics of the resolution process.

Applications in biotechnology:

• Engineered recombinases: Structural insights could guide the development of engineered resolvases with altered specificity or activity for biotechnological applications.

• Novel antimicrobial targets: If the structure reveals unique features compared to human resolvases, this could inform the development of narrow-spectrum antimicrobials.

• Tools for genome engineering: Understanding the structural basis of resolution specificity could enable the development of new site-specific recombination tools.

Experimental approaches needed:

• X-ray crystallography: This would require high-yield expression and purification of resolvase from L. plantarum, which the pSIP system has proven capable of achieving for other membrane proteins .

• Cryo-electron microscopy: For visualization of larger complexes with DNA substrates.

• NMR spectroscopy: For studying dynamics of protein-DNA interactions.

• Molecular dynamics simulations: To complement experimental structures with insights into conformational changes during catalysis.