Recombinant Torpedo marmorata 14 kDa transmembrane protein
Description
Definition and Overview
The Recombinant Torpedo marmorata 14 kDa transmembrane protein is a synthetically produced, full-length protein derived from the marbled electric ray (Torpedo marmorata). It is classified under the Dispanin subfamily A member 2b (DSPA2b) and is expressed in Escherichia coli with an N-terminal hexahistidine (His) tag for purification . This protein, encoded by UniProt ID Q91499, spans 107 amino acids and is characterized by its hydrophobic transmembrane domains, making it integral to membrane-associated studies .
Key Features:
The protein’s sequence includes conserved hydrophobic regions critical for membrane integration, as predicted by algorithms like TopPred II and PHDTopography .
Expression System:
Purification Protocol:
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Lysis: Cells are lysed in Tris/PBS-based buffer.
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Chromatography: Ni-NTA affinity chromatography isolates the His-tagged protein.
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Formulation: Lyophilized in a buffer containing 6% trehalose (pH 8.0) for long-term stability .
Predicted Roles:
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Membrane Insertion: Structural homology with Dispanin family proteins suggests involvement in membrane organization or lipid interactions .
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Choline Transport: Indirect evidence links homologs (e.g., CTL1) to choline uptake, though direct functional validation for this protein remains limited .
Experimental Findings:
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Yeast Complementation: Torpedo marmorata CTL1 (a homolog) restored choline transport in deficient yeast, hinting at potential functional overlap .
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Mast Cell Studies: Transient expression in mammalian cells showed marginal increases in choline uptake, though results were inconclusive .
Primary Uses:
Product Specs
Note: We will prioritize shipping the format that we have in stock. However, if you have any specific format requirements, please indicate them when placing your order, and we will prepare the product accordingly.
Note: All of our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please contact us in advance, as additional fees will apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Target Background
Q&A
What expression systems are suitable for producing functional Recombinant Torpedo marmorata 14 kDa transmembrane protein?
The protein is optimally expressed in Escherichia coli systems with N-terminal His-tag fusion for purification efficiency . Key parameters include:
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Codon optimization: Critical for expressing marine organism-derived sequences in prokaryotic systems.
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Induction conditions: Use of IPTG at 0.5 mM for 16–20 hours at 18°C to enhance soluble fraction yield .
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Post-lysis handling: Inclusion body resolubilization requires Tris/PBS-based buffers with 6% trehalose to maintain structural stability .
Table 1: Production Parameters
| Parameter | Specification | Source |
|---|---|---|
| Host organism | E. coli BL21(DE3) | |
| Tag system | N-terminal His-tag | |
| Purity | >90% (SDS-PAGE verified) | |
| Storage stability | -80°C in 50% glycerol |
How to validate the structural integrity of the recombinant protein?
Combine circular dichroism (CD) spectroscopy and immunoblotting for multi-level verification:
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CD spectroscopy: Confirm α-helical content matching predicted transmembrane domains (residues 30–90) .
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Immunoblotting: Use anti-His tag antibodies to verify molecular weight (~14 kDa) and epitope accessibility .
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N-terminal sequencing: Validate the intactness of the MEYRTD... N-terminal sequence via Edman degradation .
Critical consideration: Lyophilization alters secondary structure; reconstitute in Tris buffer with 0.1% DDM for functional assays .
What methods are recommended for studying membrane integration dynamics?
Employ protease protection assays and fluorescence quenching:
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Trypsin digestion: Expose intact liposomes containing reconstituted protein to 0.1 mg/mL trypsin for 10 min. Protected fragments indicate transmembrane topology .
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Acrylamide quenching: Monitor Trp fluorescence (ex 280 nm/em 340 nm) to assess solvent accessibility of residue W52 in the putative membrane-spanning region .
How to resolve conflicting data regarding the protein’s role in acetylcholine receptor clustering?
The 14 kDa protein’s relationship to synaptic components requires comparative co-purification analysis:
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Co-immunoprecipitation: Use anti-Q91499 antibodies with Torpedo postsynaptic membrane extracts .
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Quantitative mass spectrometry: Compare protein abundance in synaptic vs. non-synaptic membrane fractions .
Key finding: While the 43 kDa postsynaptic protein shows direct acetylcholine receptor interaction , the 14 kDa protein exhibits <5% co-purification in alkaline-extracted membranes, suggesting auxiliary roles .
What strategies mitigate hydrophobicity-driven aggregation during in vitro studies?
Implement multi-step solubilization protocols:
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Primary solubilization: 1% (w/v) n-dodecyl-β-D-maltoside (DDM) in 20 mM Tris-HCl (pH 8.0) .
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Size-exclusion chromatography: Use Superdex 200 Increase column with 0.03% DDM to isolate monodisperse particles .
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Lipid reconstitution: Incorporate into asolectin liposomes at 1:100 (protein:lipid ratio) for functional assays .
How to model the protein’s interaction with dystrophin-associated complexes?
Apply surface plasmon resonance (SPR) and cryo-EM:
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SPR analysis: Immobilize recombinant β-dystroglycan on CM5 chips; measure binding kinetics at 25°C in HBS-EP buffer .
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Cryo-EM grid preparation: Embed protein-lipid nanodiscs in 2% trehalose for vitrification .
Table 2: Interaction Partners in Torpedo Muscle Membranes
| Protein Complex | Observed Interaction | Method Used | Reference |
|---|---|---|---|
| Dystrophin-glycoprotein | Negative | Co-IP/Western | |
| Acetylcholine receptor | Weak | Sucrose gradient |
Addressing discrepancies in post-translational modification analyses
The protein’s lack of glycosylation sites (predicted via NetNGlyc 1.0) contrasts with observed 15–16 kDa bands in some Western blots . Resolution strategies:
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Phos-tag SDS-PAGE: Detect potential phosphorylation at S27/S34 using 50 μM Phos-tag acrylamide .
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EndoH treatment: Confirm absence of N-linked glycans by comparing electrophoretic mobility shifts .
Optimizing electrophysiological characterization protocols
For ion channel interaction studies:
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Planar lipid bilayer setup: Incorporate protein into DPhPC bilayers under 200 mV holding potential .
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Single-channel recording: Use 2 kHz low-pass filtering and 50 μs sampling intervals to detect <5 pA currents .
Note: No intrinsic channel activity detected to date, suggesting non-conducting regulatory roles .
Reconciling conflicting subcellular localization reports
Combine immunoelectron microscopy and differential centrifugation:
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Immunogold labeling: 10 nm gold particles with anti-Q91499 antibodies show 82% PM localization vs. 18% ER .
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Sucrose gradient centrifugation: 14 kDa protein co-sediments with light membrane fractions (20–25% sucrose) .
Validating evolutionary conservation through cross-species studies
Comparative immunoblotting protocol:
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Extract membrane proteins from rat diaphragm (1% Triton X-114).
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Transfer to PVDF membranes using semi-dry apparatus (1.5 mA/cm², 1 hr).
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Probe with anti-Torpedo 14 kDa antibody (1:1000 dilution).
Outcome: No cross-reactivity observed, indicating unique evolutionary adaptations in elasmobranchs .
