Recombinant Burkholderia thailandensis Large-conductance mechanosensitive channel (mscL)
Description
Functional Role
MscL acts as an osmotic emergency release valve, discharging cytoplasmic osmolytes during rapid osmotic shifts to prevent cell lysis. Its conductance reaches ~3 nS in the open state .
Mechanosensitive Gating Mechanisms
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Key Residues:
| Mutation | Effect |
|---|---|
| N103C (MTSSL) | Stabilizes expanded pore conformation; reduces lipid access to TM pockets |
| L89W | Destabilizes closed state; enhances channel activation under tension |
Modulation by Agonists and Inhibitors
MscL is activated by small molecules targeting the S1-TM2 interface:
| Compound | Mechanism | Effect |
|---|---|---|
| SCH-79797 | Binds near TM2, disrupting lipid interactions | Activates channel; bactericidal |
| Curcumin | Indirect activation; enhances membrane permeabilization | Inhibits bacterial growth |
| Gadolinium | Blocks pore in closed state | Inhibits channel activity |
These compounds highlight MscL’s potential as an antimicrobial target .
Expression and Reconstitution
MscL is expressed in E. coli as a GST fusion protein, purified via glutathione affinity chromatography, and cleaved with thrombin to yield the native protein . Functional reconstitution into liposomes confirms:
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your preferred format in order notes for customized preparation.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires advance notification and incurs additional charges.
The tag type is determined during production. If you require a specific tag, please inform us; we will prioritize its development.
Target Background
KEGG: bte:BTH_I2079
Q&A
Basic Research Questions
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What is the Burkholderia thailandensis MscL protein and what is its physiological role?
The Large-conductance mechanosensitive channel (MscL) in B. thailandensis is a pore-forming membrane protein that responds to mechanical stress in the cell membrane. It functions as a stretch-activated osmotic release valve, opening in response to membrane tension to prevent cell lysis during osmotic shock . The channel forms a homopentamer with each subunit containing two transmembrane regions. When open, MscL has a large conductance (approximately 3 nS), making it permeable to ions, water, and small proteins . In B. thailandensis, as in other bacteria, MscL is upregulated during stationary phase and osmotic shock conditions to protect cellular integrity .
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What expression systems are commonly used for recombinant B. thailandensis MscL production?
E. coli is the predominant expression system for recombinant production of B. thailandensis MscL. The protein is typically expressed with an N-terminal His-tag to facilitate purification . The expression construct generally includes the full-length MscL protein (amino acids 1-143) cloned into an appropriate expression vector. After expression, the protein can be purified using affinity chromatography and is often supplied as a lyophilized powder that requires reconstitution in an appropriate buffer system, typically containing 6% trehalose at pH 8.0 . Storage recommendations include keeping aliquots at -20°C to -80°C and avoiding repeated freeze-thaw cycles.
Experimental Data and Resources
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What characterized properties should researchers expect when working with recombinant B. thailandensis MscL?
When working with recombinant B. thailandensis MscL, researchers should expect the following properties:
Property Characteristic Notes Protein length 143 amino acids Full-length protein Molecular weight ~15-16 kDa per monomer ~75-80 kDa for pentameric complex Oligomeric state Pentamer Forms homopentameric complex Conductance ~3 nS One of the largest conductances among ion channels Gating threshold ~10-12 mN/m Membrane tension required for opening Storage stability Stable at -20°C/-80°C Avoid repeated freeze-thaw cycles Buffer compatibility Tris/PBS-based, pH 8.0 Contains 6% trehalose as stabilizer Reconstitution 0.1-1.0 mg/mL In deionized sterile water Purification tag N-terminal His-tag Facilitates purification by metal affinity chromatography For electrophysiological studies, researchers typically incorporate purified MscL into liposomes or planar lipid bilayers. The channel exhibits voltage-independent gating and is primarily activated by membrane tension, with a characteristic large conductance that allows passage of ions and small molecules up to ~30 Å in diameter when fully open .
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What genetic resources and tools are available for studying MscL in B. thailandensis?
Researchers have access to various genetic resources and tools for studying MscL in B. thailandensis:
Resource Type Examples Applications Genome sequence B. thailandensis E264 complete genome Gene identification, primer design Expression vectors pUC18miniTn7T-based vectors Complementation, overexpression Selectable markers sacB, pheS, gat, dhfR Selection for recombinants Promoter systems P<sub>s12</sub>, P<sub>lac</sub> Constitutive or inducible expression Recombineering systems λ Red proteins Targeted mutagenesis Natural transformation PCR transformation protocols Gene knockout and replacement Transposon systems Mini-Tn7, Tn5 derivatives Site-specific or random mutagenesis Fluorescent tagging BONCAT Protein labeling and visualization The availability of these tools enables comprehensive genetic manipulation of B. thailandensis, including targeted gene knockouts, complementation studies, and protein expression analyses. Particularly useful are the natural competence-based methods and λ Red recombineering systems that allow for efficient genetic modifications with relatively small PCR products, reducing the number of primers and amplification steps required .
