Recombinant Lactobacillus fermentum Large-conductance mechanosensitive channel (mscL)
Description
Introduction to Recombinant Lactobacillus fermentum Large-conductance Mechanosensitive Channel (mscL)
The Recombinant Lactobacillus fermentum Large-conductance mechanosensitive channel (mscL) is a protein expressed in the bacterium Lactobacillus fermentum. Mechanosensitive channels are crucial for bacterial survival, allowing them to maintain cellular integrity by responding to changes in osmotic pressure. The mscL channel is particularly significant due to its large conductance, which enables rapid efflux of ions and water from the cell, thereby preventing cell lysis under osmotic stress conditions.
Structure and Function of mscL
The mscL channel is a pentameric structure composed of five identical subunits. Each subunit contains two transmembrane helices, with the channel's pore being formed by the second helix of each subunit. The channel is gated by mechanical stress, opening in response to membrane tension caused by osmotic downshock. This allows ions and water to flow out of the cell, reducing the internal pressure and preventing cell rupture.
| Feature | Description |
|---|---|
| Structure | Pentameric structure with five identical subunits, each containing two transmembrane helices. |
| Function | Responds to mechanical stress by opening to allow rapid efflux of ions and water, maintaining cellular integrity under osmotic stress. |
| Conductance | Large conductance, facilitating rapid ion and water flow. |
Recombinant Expression in Lactobacillus fermentum
Recombinant expression of the mscL channel in Lactobacillus fermentum involves cloning the mscL gene into a suitable plasmid and transforming it into L. fermentum cells. This process allows for the production of large quantities of the mscL protein for research and potential applications. The recombinant protein is typically purified and characterized using various biochemical and biophysical techniques.
| Recombinant Expression Details | Description |
|---|---|
| Host Organism | Lactobacillus fermentum |
| Gene | mscL |
| Plasmid | Custom-designed plasmid for expression in L. fermentum |
| Purification | Techniques such as affinity chromatography or gel filtration |
References:
- GeneBioSystems. Recombinant Lactobacillus fermentum Large-conductance mechanosensitive channel (mscL).
- Expression of catalase in Lactobacillus fermentum and evaluation of its survival under oxidative stress.
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General literature on mechanosensitive channels and their role in bacteria.
Product Specs
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Target Background
KEGG: lfe:LAF_1837
Q&A
What is the structural configuration of recombinant L. fermentum mscL, and how does it compare to native homologs?
The recombinant L. fermentum mscL is a 125-amino-acid protein with a His-tag fused to its N-terminus for purification. Structural analysis reveals a conserved mechanosensitive channel architecture, including transmembrane domains (TMD1 and TMD2) critical for pore formation and gating mechanisms . Key residues involved in tension sensing (e.g., Gly-22, Ala-23) align with E. coli MscL homologs, but unique features like a truncated C-terminal domain differentiate it from other bacterial mechanosensitive channels . Researchers should verify structural integrity using circular dichroism (for secondary structure) and cryo-EM (for tertiary conformation), comparing results against native L. fermentum mscL controls.
Table 1: Structural Features of Recombinant vs. Native mscL
What experimental methodologies confirm successful cloning and expression of mscL in heterologous systems?
Three-tier validation is required:
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Genetic confirmation: Amplify the mscL gene (372 bp) using colony PCR with primers targeting the His-tag insertion site . For Lactobacillus systems, use species-specific promoters (e.g., ldhL in ) and codon-optimize sequences to match host preferences (e.g., increase GC content to 45-50% for L. fermentum) .
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Expression verification: Perform SDS-PAGE with Coomassie staining to detect the ~14 kDa band . For low-expression systems, use Western blotting with anti-His antibodies .
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Functional assays: Conduct patch-clamp electrophysiology to confirm mechanosensitivity, applying 20-40 mmHg pressure to observe channel activation .
How do researchers standardize dosage for in vitro studies of recombinant mscL?
Dosage depends on the expression system:
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E. coli-expressed protein: Use 0.1–1.0 mg/mL in Tris/PBS buffer with 6% trehalose .
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Lactobacillus-delivered mscL: Calculate colony-forming units (CFU) based on optical density (OD600 = 1.0 ≈ 1 × 10^9 CFU/mL) . For co-culture experiments with intestinal epithelial cells (e.g., NCM460), maintain a 10:1 bacteria-to-cell ratio . Always include vector-only controls (e.g., empty pLC13.9 plasmid ) to isolate mscL-specific effects.
How should researchers address discrepancies between in vitro and in vivo activity data for recombinant mscL?
Contradictions often arise from:
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Post-translational modifications: L. fermentum may glycosylate mscL absent in E. coli systems . Validate using PNGase F treatment followed by Western blot.
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Microenvironmental factors: Gut microbiota in murine models degrade 30–40% of recombinant proteins within 2 hours . Use protease inhibitors (e.g., 1 mM PMSF) in ex vivo assays.
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Channel oligomerization: Native mscL forms pentamers, while recombinant versions may misfold. Perform blue native PAGE to confirm oligomeric state .
Table 2: Troubleshooting Data Discrepancies
| Issue | In Vitro Result | In Vivo Result | Resolution Strategy |
|---|---|---|---|
| Low activity | 90% channel opening | 45% opening | Check for bile salt inhibition |
| Protein degradation | Stable for 72 hrs | Undetectable after 6 hrs | Use enteric-coated bacterial vectors |
| Immune response | None observed | IgG elevation | Test for His-tag immunogenicity |
What strategies optimize heterologous expression of mscL in Lactobacillus systems?
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Codon optimization: Redesign the mscL gene using L. fermentum-preferred codons (e.g., TTT for Phe instead of TTC) . The OPTIMIZER tool (http://genomes.urv.es/OPTIMIZER) increases expression by 2.5-fold .
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Promoter selection: Constitutive promoters like ldhL yield 3× higher expression than inducible systems in Lactobacillus .
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Electroporation parameters: For L. fermentum RCEID01, use 2.5 kV/cm, 25 μF, and 200 Ω in 0.2 cm cuvettes with 1 mM MgCl₂ in electroporation buffer .
How do researchers evaluate mscL’s role in disease models like colorectal inflammation?
Adopt a three-phase approach:
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Mechanistic studies: Co-culture recombinant L. fermentum with NCM460 colonocytes under MC-LR exposure (200 μM) . Measure CSF1R pathway activation via qPCR (primers for CSF1R, Rap1b) and Western blot (phospho-CSF1R antibodies) .
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Microbiome modulation: Use 16S rRNA sequencing to track Lactobacillus abundance (expect ≥50% increase in treated groups) .
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In vivo validation: Administer 10^8 CFU/day of recombinant L. fermentum to MC-LR-exposed mice for 28 days. Collect colorectal tissues for histopathology (HE staining) and cytokine profiling (ELISA for IL-6, TNF-α) .
Problem: Low protein yield in L. fermentum expression systems
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Cause: Poor plasmid stability in Lactobacillus.
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Solution: Use high-copy plasmids (e.g., pLEM415) with repA origin and erythromycin resistance . Supplement media with 1% glycine to weaken cell walls pre-electroporation .
