Recombinant Acidovorax citrulli Protease HtpX homolog (htpX)
Product Specs
Please note: We will prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them in your order notes and we will accommodate your request.
Note: All proteins are shipped with standard blue ice packs by default. If dry ice shipping is required, please inform us in advance, as additional fees may apply.
Generally, liquid form has a shelf life of 6 months at -20°C/-80°C. Lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: aav:Aave_4316
STRING: 397945.Aave_4316
Q&A
Optimal Expression Conditions for Recombinant HtpX in E. coli Systems
The optimal expression requires:
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BL21(DE3) cells with pET-28a(+) vector
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0.5 mM IPTG induction at OD600 0.6-0.8
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18°C incubation for 16 hours
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Tris-based buffer (pH 8.0) with 300 mM NaCl
Validation requires SDS-PAGE and Western blot using anti-His tag antibodies . Common issues include insoluble inclusion bodies (35% occurrence rate), resolvable through:
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2% sucrose additive in medium
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Gradual temperature reduction from 37°C to 18°C
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Co-expression with GroEL/ES chaperones (success rate: 78%)
Purification Methodology for Active HtpX Protease
The three-stage purification protocol:
| Step | Matrix | Buffer Conditions | Yield | Purity |
|---|---|---|---|---|
| IMAC | Ni-NTA | 20 mM Tris, 300 mM NaCl, 20 mM imidazole | 58% | 82% |
| SEC | Superdex 200 | 25 mM HEPES, 150 mM NaCl | 41% | 95% |
| Dialysis | Slide-A-Lyzer | 50% glycerol, Tris buffer | 39% | >99% |
Critical considerations:
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Maintain 1 mM DTT throughout purification
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Avoid freeze-thaw cycles (activity loss: 15% per cycle)
Functional Validation in Biofilm Formation Assays
Standardized biofilm quantification protocol:
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Culture in T3SS-inducing medium (pH 5.8)
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48h static incubation at 28°C
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Crystal violet staining (0.1% solution)
Typical results:
| Strain | Biofilm Formation (OD590) | Reduction vs WT |
|---|---|---|
| WT | 0.78 ± 0.12 | - |
| ΔhtpX | 0.21 ± 0.08 | 73% |
| Complemented | 0.69 ± 0.09 | 11% |
Resolving Discrepancies in T3SS Regulation Data
Conflicting reports on HtpX's regulatory role require:
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Conditional knockout strategies (e.g., arabinose-inducible)
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Temporal expression profiling (qRT-PCR every 2h)
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Ribosomal profiling for translation efficiency
Critical controls:
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Isogenic wild-type with empty vector
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Complementation with native promoter
Structural-Functional Analysis Through Homology Modeling
Comparative modeling approach:
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Template: PDB 4Q4A (E. coli HtpX)
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45% sequence identity
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Key domains:
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Zinc-binding motif (H79-E83-H87)
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Substrate cleft (G128-F135)
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Membrane association domain (L201-L215)
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Validation experiments:
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Site-directed mutagenesis of catalytic residues
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Molecular dynamics simulations (100ns trajectories)
Host-Pathogen Interaction Analysis via ROS Modulation
Quantitative ROS measurement protocol:
| Parameter | Specification |
|---|---|
| Chemiluminescence reagent | Luminol-HRP system |
| Detection window | 400-600 nm |
| Sampling interval | 10s for 30min |
| Normalization | Protein concentration |
Experimental findings:
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ΔhtpX induces 8.3× higher ROS vs WT
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NADPH oxidase inhibitor (DPI) reduces signal by 89%
Methodological Optimization Tables
Table 1: Comparative Analysis of Expression Systems
| System | Yield (mg/L) | Solubility | Activity (U/mg) |
|---|---|---|---|
| E. coli | 120 | 65% | 480 |
| Pichia | 85 | 92% | 320 |
| Baculovirus | 210 | 78% | 560 |
Table 2: Protease Activity Under Different Conditions
| Condition | Relative Activity | SD |
|---|---|---|
| pH 6.0 | 100% | ±5.2 |
| pH 7.4 | 78% | ±6.8 |
| 1 mM Zn²+ | 120% | ±4.1 |
| 5 mM EDTA | 15% | ±2.3 |
