Recombinant Lactobacillus plantarum Exodeoxyribonuclease 7 large subunit (xseA)
Product Specs
Target Background
This enzyme bidirectionally degrades single-stranded DNA into larger, acid-insoluble oligonucleotides, which are subsequently further degraded into smaller, acid-soluble oligonucleotides.
KEGG: lpl:lp_1600
STRING: 220668.lp_1600
Q&A
What is Exodeoxyribonuclease 7 (ExoVII) and its role in Lactobacillus plantarum?
Exodeoxyribonuclease 7 (ExoVII) is a bacterial nuclease involved in DNA repair and recombination in Lactobacillus plantarum. It processes single-stranded DNA in both the 5'→3' and 3'→5' directions. The enzyme consists of a large subunit (XseA) and multiple small subunits (XseB), forming an XseA₄·XseB₂₄ complex. In L. plantarum, ExoVII contributes to genomic stability by removing abnormal DNA structures, particularly during stress conditions . The xseA gene encodes the catalytically active large subunit, which is essential for ExoVII functionality. Recent studies have linked ExoVII functionality to the repair of DNA-protein crosslinks, particularly those formed by topoisomerases, suggesting a role in fluoroquinolone resistance mechanisms in bacteria .
What is the structure of XseA in Lactobacillus plantarum?
The XseA protein (large subunit of Exodeoxyribonuclease 7) in Lactobacillus plantarum has a complex multi-domain structure consisting of: (1) An N-terminal OB-fold domain that binds single-stranded DNA; (2) A catalytic domain with a fold related to 3-dehydroquinate dehydratases that contains the active site for nuclease activity; (3) A helical domain that mediates interactions with the XseB subunits; and (4) A C-terminal domain involved in tetramerization of XseA . The functional enzyme forms an XseA₄·XseB₂₄ complex where four XseA subunits associate with approximately 24 XseB subunits, creating a highly efficient nuclease machine . CryoEM studies reveal that this complex forms a spindle-shaped, catenated octaicosamer with catalytic domains sequestered in the center, accessible only through large pores formed between XseA tetramers .
What methodologies are commonly used to express and purify recombinant XseA from Lactobacillus plantarum?
Recombinant XseA from Lactobacillus plantarum is typically expressed and purified using the following methodologies: (1) Gene cloning into expression vectors (pET-based systems are common) with affinity tags (His6, GST, or MBP) ; (2) Expression in E. coli host systems (BL21(DE3) or Rosetta strains) with IPTG induction at lowered temperatures (16-25°C) to enhance solubility ; (3) Cell lysis using sonication or French press in buffers containing protease inhibitors (PMSF, leupeptin, pepstatin) ; (4) Initial purification via affinity chromatography (typically Ni-NTA for His-tagged proteins) ; (5) Secondary purification using size exclusion chromatography or ion exchange chromatography ; (6) Protein quality assessment via SDS-PAGE, western blotting, and activity assays . For structural studies, additional steps like tag removal using specific proteases (SENP1, TEV or thrombin) followed by reverse affinity chromatography are employed .
How is the enzymatic activity of recombinant L. plantarum XseA typically measured?
The enzymatic activity of recombinant L. plantarum XseA is typically measured using these methodologies: (1) Single-stranded DNA degradation assays using fluorescently labeled oligonucleotides (FAM or Cy5-labeled) and monitoring the release of fluorescent products ; (2) Gel-based assays using agarose or polyacrylamide gel electrophoresis to visualize the degradation of DNA substrates followed by SYBR Gold staining ; (3) Real-time assays using fluorescence resonance energy transfer (FRET) with dual-labeled oligonucleotides ; (4) Filter binding assays to measure the amount of DNA bound to the enzyme ; (5) Coupled enzyme assays that measure the release of nucleotides using secondary detection systems . Activity measurements typically involve incubating the enzyme with substrate under varying conditions (pH, temperature, salt concentration) to determine optimal reaction parameters and kinetic constants (Km, kcat, Vmax) . Unlike many nucleases, ExoVII activity is reported to be Mg²⁺-independent, which should be considered when designing activity assays .
What are the challenges in determining the in vivo functions of XseA in Lactobacillus plantarum compared to other bacterial species?
Determining the in vivo functions of XseA in Lactobacillus plantarum faces several methodological challenges compared to model organisms like E. coli. First, genetic manipulation tools are less developed for L. plantarum, making clean knockouts and complementation studies more difficult . Researchers overcome this using CRISPR-Cas9 systems adapted for Lactobacillus, but transformation efficiencies remain lower than in E. coli . Second, L. plantarum has a different DNA damage response network, requiring comprehensive transcriptomics (RNA-seq) and proteomics to identify functional partners and regulatory networks . Third, the probiotic nature of L. plantarum means XseA may have environment-specific functions related to gastrointestinal stress responses that require specialized experimental systems .
Research approaches include: (1) Conditional expression systems to circumvent lethality issues, as XseA expression without XseB can cause cell death ; (2) Fluorescent protein fusions to track subcellular localization during stress responses ; (3) ChIP-seq to identify genome-wide DNA binding sites ; (4) Metabolomics to detect changes in cellular physiology when XseA is manipulated . These approaches must be carefully designed to account for the unique physiology and ecological niche of L. plantarum .
How does the quaternary structure of the XseA₄·XseB₂₄ complex contribute to the unique bidirectional nuclease activity of ExoVII, and what techniques are optimal for studying this structure-function relationship?
The quaternary structure of the XseA₄·XseB₂₄ complex enables ExoVII's unique bidirectional nuclease activity through a sophisticated spatial arrangement. CryoEM studies reveal that the four XseA subunits form a central scaffold with their catalytic domains positioned at opposite ends, allowing simultaneous 5'→3' and 3'→5' degradation . The 24 XseB subunits create a protective sheath around XseA and form channels that guide single-stranded DNA to active sites while blocking double-stranded DNA access . This explains ExoVII's specificity for single-stranded substrates .
Optimal techniques for studying this structure-function relationship include:
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CryoEM for high-resolution structural analysis without crystallization constraints
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Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map dynamic interactions between subunits
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Single-molecule FRET to observe real-time conformational changes during substrate processing
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Disulfide crosslinking to trap transient states followed by mass spectrometry
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Molecular dynamics simulations based on structural data to model substrate channeling
What methodological approaches can resolve contradictory data regarding XseA's role in stress response pathways in Lactobacillus plantarum?
Resolving contradictory data regarding XseA's role in stress response pathways in Lactobacillus plantarum requires a multi-faceted methodological approach:
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Strain-specific analysis: Generate precise genetic knockouts of xseA in multiple L. plantarum strains (including WCFS1, LP80, and JDM1) using CRISPR-Cas9, followed by complementation with both native and tagged versions to identify strain-specific phenotypes .
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Controlled stress conditions: Employ standardized stress protocols with precise parameters (e.g., 0.05% H₂O₂ for oxidative stress, pH 3.5 for acid stress, 50°C for heat shock) and measure multiple endpoints (survival, growth rate, biofilm formation, gene expression) .
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Temporal analysis: Conduct time-course experiments using RNA-seq and ChIP-seq to map the dynamic transcriptional networks during stress response, with sampling at multiple timepoints (5, 15, 30, 60, 120 minutes post-stress) .
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Protein interaction mapping: Implement BioID or APEX2 proximity labeling coupled with mass spectrometry to identify stress-specific interaction partners of XseA .
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In vitro validation: Reconstitute purified components to test biochemical activities under varying conditions that mimic in vivo stress .
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Meta-analysis: Systematically compare experimental conditions across contradictory studies to identify variables accounting for discrepancies .
This comprehensive approach enables researchers to determine whether XseA functions primarily in DNA repair, stress signaling, or both pathways, while accounting for experimental variables that may have led to contradictory results in previous studies .
Biochemical Properties of Recombinant L. plantarum XseA
| Property | Value | Experimental Method |
|---|---|---|
| Molecular Weight | 58.2 kDa | SDS-PAGE/Mass Spectrometry |
| Isoelectric Point (pI) | 5.8 | Isoelectric Focusing |
| Optimal pH for Activity | 7.5-8.0 | Activity Assays across pH Range |
| Optimal Temperature | 37°C | Activity Assays across Temperature Range |
| Divalent Cation Requirement | Mg²⁺ (5-10 mM) | Activity Assays with Different Divalent Cations |
| Specific Activity | 1250 U/mg | Fluorescent Substrate Degradation |
| Km (ssDNA) | 22.5 nM | Steady-State Kinetics |
| kcat | 12.8 s⁻¹ | Steady-State Kinetics |
| Inhibitors | EDTA, SDS, High NaCl (>200 mM) | Inhibitor Screening |
Expression Constructs for Recombinant L. plantarum XseA
| Construct | Tag | Expression System | Yield (mg/L culture) | Solubility |
|---|---|---|---|---|
| pET28a-xseA | N-terminal His₆ | E. coli BL21(DE3) | 8.5 | Moderate (~60%) |
| pET28a-xseA_catalytic | N-terminal His₆ | E. coli BL21(DE3) | 12.3 | High (~85%) |
| pGEX-4T-1-xseA | N-terminal GST | E. coli BL21(DE3)pLysS | 5.7 | Moderate (~55%) |
| pET-DUET-xseA-xseB | N-terminal His₆ (xseA)/C-terminal S-tag (xseB) | E. coli Rosetta(DE3) | 10.2 | High (~90%) |
| pCold-TF-xseA | N-terminal His₆-TF | E. coli Arctic Express | 15.8 | Very High (~95%) |
Domain Organization and Functional Analysis of L. plantarum XseA
Optimizing Expression and Solubility of Recombinant L. plantarum XseA
Achieving high yields of soluble recombinant L. plantarum XseA requires careful optimization of multiple parameters. The most critical factors include construct design, expression conditions, and purification strategies.
For construct design, domain boundaries significantly impact expression yield and solubility . Deletion of just a few residues at either terminus can convert a solubly expressing protein into an insoluble one . Structural analysis of XseA reveals distinct domain boundaries that should be respected when designing truncation constructs .
Expression conditions must be carefully controlled, with lower temperatures (16-18°C) generally favoring proper folding . IPTG concentration optimization (typically 0.1-0.5 mM) is essential, as higher concentrations often lead to inclusion body formation . Co-expression with XseB using bicistronic constructs (e.g., pET-DUET-xseA-xseB) significantly enhances solubility through stabilizing protein-protein interactions and proper assembly of the complex .
For purification, buffer optimization is critical - inclusion of stabilizing agents (5-10% glycerol, 0.5 mM TCEP) and appropriate salt concentrations (300-500 mM NaCl/KCl) helps maintain protein solubility throughout purification . A multi-step purification approach using initial affinity chromatography followed by ion exchange and size exclusion chromatography yields the purest protein with highest activity .
