Recombinant Bovine Tetraspanin-18 (TSPAN18)
Description
Physical and Chemical Properties
The recombinant bovine TSPAN18 protein has several distinct physical and chemical properties that are important for its handling and application in research settings. These properties are summarized in the following table:
| Property | Description |
|---|---|
| Molecular Weight | ~27 kDa (with His-tag) |
| Form | Lyophilized powder |
| Purity | >90% as determined by SDS-PAGE |
| Storage Buffer | Tris/PBS-based buffer, 6% Trehalose, pH 8.0 |
| Stability | Sensitive to freeze-thaw cycles; stable at -20°C/-80°C |
| Solubility | Requires reconstitution in deionized sterile water |
| Expression System | E. coli |
| Tag | N-terminal His tag |
These properties are crucial considerations for researchers working with this protein, as they affect storage conditions, experimental design, and potential applications .
Expression Systems
The expression construct typically includes the full-length TSPAN18 coding sequence (amino acids 1-249) fused to an N-terminal histidine tag, which facilitates downstream purification processes . This approach allows for the production of sufficient quantities of protein for structural and functional studies.
Purification Strategies
The purification of recombinant bovine TSPAN18 typically involves a multi-step process that leverages the presence of the His-tag. The general purification workflow includes:
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Cell lysis to release the expressed protein
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Immobilized metal affinity chromatography (IMAC) using the His-tag
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Additional chromatographic steps to achieve high purity
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Final quality control assessment by SDS-PAGE
The purified protein is generally formulated in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0, which enhances stability during lyophilization and storage . The final product is typically provided as a lyophilized powder with purity greater than 90% as determined by SDS-PAGE analysis.
Calcium Channel Regulation
One of the most significant functions of TSPAN18 is its role in calcium signaling through regulation of the Orai1 calcium channel. Studies have shown that TSPAN18 directly interacts with Orai1, a store-operated calcium channel that mediates calcium influx into cells .
In endothelial cells, TSPAN18 knockdown results in a 55-70% decrease in calcium mobilization upon stimulation with inflammatory mediators such as thrombin or histamine, similar to the effects observed with Orai1 knockdown . This suggests that TSPAN18 is essential for proper Orai1 function. Further analysis has revealed that TSPAN18 influences Orai1 cell surface localization, with a 70% reduction in Orai1 surface expression observed in TSPAN18-knockdown endothelial cells .
The functional relationship between TSPAN18 and calcium signaling extends beyond endothelial cells. Overexpression of TSPAN18 in lymphocyte model cell lines induces a 20-fold activation of calcium-responsive nuclear factor of activated T cell (NFAT) signaling in an Orai1-dependent manner . This finding indicates a conserved role for TSPAN18 in calcium regulation across different cell types.
Endothelial Cell Function
TSPAN18 is highly expressed in endothelial cells at levels several-fold higher than in most other cell types analyzed, suggesting a specialized role in vascular function . The protein plays a crucial role in endothelial responses to inflammatory stimuli, particularly through its effects on calcium signaling and subsequent von Willebrand factor (vWF) release.
Studies using TSPAN18-knockdown human umbilical vein endothelial cells have demonstrated that TSPAN18 is required for normal calcium mobilization in response to thrombin or histamine stimulation . This calcium signaling is essential for various endothelial cell functions, including barrier regulation, vasomotor tone control, and thromboinflammatory responses.
Von Willebrand Factor Release and Hemostasis
TSPAN18 plays a critical role in the regulated release of von Willebrand factor (vWF) from endothelial cells, a process essential for proper hemostasis. In vitro studies have shown that histamine- or thrombin-induced vWF release is reduced by approximately 90% following TSPAN18 knockdown in endothelial cells .
This effect on vWF release has significant physiological consequences. TSPAN18-knockout mice exhibit impaired hemostasis, losing on average 6-fold more blood in tail-bleed assays compared to wild-type controls . Through studies with chimeric mice, this bleeding phenotype has been attributed to TSPAN18 deficiency in non-hematopoietic cells (specifically endothelial cells) rather than in blood cells .
The role of TSPAN18 in vWF regulation is further evidenced by in vivo studies showing that histamine-induced increases in plasma vWF are reduced by approximately 45% in TSPAN18-knockout mice compared to wild-type controls . This confirms that TSPAN18's function in regulating stimulated vWF release is physiologically relevant.
Deep Vein Thrombosis Models
The functional importance of TSPAN18 extends to pathological thrombotic conditions. In a deep vein thrombosis model, which is dependent on endothelial vWF, TSPAN18-knockout mice developed thrombi that were approximately 60% smaller in both length and weight compared to wild-type littermate controls . Moreover, 44% of TSPAN18-knockout mice (4 out of 9) failed to develop any thrombus, whereas thrombus formation occurred in 100% of wild-type mice .
These findings suggest that TSPAN18 plays a significant role in thrombo-inflammatory processes, likely through its regulation of endothelial vWF release in response to inflammatory stimuli. The macroscopic appearance of thrombi that did form in TSPAN18-knockout mice was similar to those in wild-type mice, suggesting that TSPAN18 influences the initiation or growth of thrombi rather than their composition .
Myocardial Ischemia-Reperfusion Injury
TSPAN18 also contributes to platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. In a vWF-dependent model of this condition, platelet deposition and aggregate size in the microcirculation were reduced by approximately 50% in TSPAN18-knockout mice . This finding further supports the role of TSPAN18 in thrombo-inflammatory responses through its regulation of endothelial vWF release.
The reduced severity observed in these thrombo-inflammatory models is consistent with TSPAN18's role in facilitating endothelial vWF release in response to inflammatory mediators. This suggests that the protein may represent a potential therapeutic target for conditions characterized by excessive thrombo-inflammatory responses.
Tetraspanin Clustering Patterns
While the search results do not provide specific information about bovine TSPAN18 clustering, general research on tetraspanins provides insight into their molecular organization in cell membranes. Tetraspanins, including TSPAN18, are known to form clusters or "tetraspanin-enriched microdomains" (TEMs) in the plasma membrane .
Super-resolution microscopy studies of other tetraspanin family members have revealed that these proteins form clusters with a typical size of around 120 nm in the plasma membrane . These clusters appear to be relatively small, with estimates suggesting that some tetraspanin clusters may contain fewer than 10 molecules per domain .
Interactions with Other Membrane Proteins
Tetraspanins interact with specific partner proteins and regulate their trafficking and clustering . In the case of TSPAN18, a key interaction partner is the Orai1 calcium channel. This interaction is functionally significant, as it affects Orai1 surface localization and consequently calcium influx into cells .
Interestingly, studies on other tetraspanins have shown that different tetraspanin family members may form distinct clusters rather than mixing together in the same microdomains . This suggests a potential specialization of function among different tetraspanins, with each potentially regulating distinct sets of partner proteins.
Current Research Applications
Recombinant bovine TSPAN18 serves as a valuable tool for various research applications:
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Structural studies to understand the molecular architecture of tetraspanins
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Interaction studies to identify and characterize binding partners
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Functional assays to investigate calcium signaling mechanisms
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Development of antibodies and other detection reagents
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Comparative studies across species to identify conserved features
The availability of purified recombinant protein facilitates these investigations by providing a consistent and well-characterized reagent for experimental use.
Potential Therapeutic Implications
The functional roles of TSPAN18 in calcium signaling, vWF release, and thrombo-inflammatory processes suggest several potential therapeutic applications:
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Targeting TSPAN18-Orai1 interactions might modulate calcium signaling in endothelial cells, with implications for vascular disorders.
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Modulating TSPAN18 function could potentially control vWF release in conditions characterized by excessive thrombosis.
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TSPAN18 could serve as a therapeutic target in thrombo-inflammatory conditions such as deep vein thrombosis.
Technical Challenges and Future Directions
Several technical challenges remain in the study of recombinant bovine TSPAN18:
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Ensuring proper folding and membrane insertion when expressed in recombinant systems
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Developing improved methods for structural characterization of membrane proteins
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Elucidating the complete interactome of TSPAN18 in different cell types
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Understanding species-specific differences in TSPAN18 function
Future research directions might include detailed structural studies using techniques such as cryo-electron microscopy, comprehensive interactome mapping, and development of specific modulators of TSPAN18 function for both research and potential therapeutic applications.
Product Specs
Please note: We will prioritize shipping the format currently in stock. However, if you have a specific format preference, please indicate it in your order notes. We will accommodate your request whenever possible.
Note: All protein shipments are standardly packaged with blue ice packs. If you require dry ice packaging, please inform us beforehand. Additional fees will apply.
Generally, the shelf life of liquid protein is 6 months at -20°C/-80°C. Lyophilized protein typically has a shelf life of 12 months at -20°C/-80°C.
Target Background
Q&A
What is the molecular structure of TSPAN18 and how does it compare to other tetraspanins?
TSPAN18 belongs to the tetraspanin superfamily characterized by four transmembrane domains. Like other tetraspanins, it contains a small extracellular loop (EC1), a large extracellular loop (EC2), and short intracellular N- and C-terminal tails. The EC2 domain is particularly important for protein-protein interactions and contains conserved cysteine residues that form disulfide bonds critical for structural stability .
Unlike some other tetraspanins that are broadly expressed, TSPAN18 shows relatively selective expression in endothelial cells at several-fold higher levels than most other cell types analyzed . This suggests a specialized function in the vasculature compared to more ubiquitously expressed tetraspanins like CD9, CD63, and CD81.
What are the primary functional roles of TSPAN18 in cellular processes?
TSPAN18 functions primarily as a regulator of calcium signaling in endothelial cells through its interaction with the calcium channel Orai1. Research has demonstrated that:
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TSPAN18 interacts directly with Orai1 and regulates its cell surface localization, with Orai1 surface expression reduced by approximately 70% in TSPAN18-knockdown endothelial cells .
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TSPAN18-knockdown primary human umbilical vein endothelial cells show 55-70% decreased Ca²⁺ mobilization upon stimulation with inflammatory mediators like thrombin or histamine, similar to effects seen with Orai1-knockdown .
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TSPAN18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca²⁺-responsive NFAT signaling in an Orai1-dependent manner .
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TSPAN18 plays a critical role in von Willebrand factor release from endothelial cells in response to inflammatory stimuli, with histamine- or thrombin-induced release reduced by 90% following TSPAN18-knockdown .
How do TSPAN18 knockout models inform our understanding of its physiological function?
TSPAN18 knockout mice studies have revealed significant phenotypes related to vascular function:
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TSPAN18-knockout mice are viable but lose on average 6-fold more blood in tail-bleed assays compared to wild-type controls .
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The bleeding phenotype is attributable to TSPAN18 deficiency in non-hematopoietic cells, as demonstrated through chimeric mouse studies .
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TSPAN18-knockout mice show 60% reduced thrombus size in deep vein thrombosis models and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury .
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Histamine-induced increase of plasma von Willebrand factor is reduced by 45% in TSPAN18-knockout mice .
This constellation of phenotypes indicates TSPAN18's physiological importance in hemostasis and thrombosis through regulation of endothelial cell function.
What expression systems are most effective for producing recombinant bovine TSPAN18?
| Expression System | Advantages | Disadvantages | Typical Yield | Best For |
|---|---|---|---|---|
| Mammalian (HEK293, CHO) | Native-like post-translational modifications; proper folding | Higher cost; longer production time | Moderate (1-5 mg/L) | Functional studies; protein-protein interactions |
| Insect cells (Sf9, Hi5) | Higher yield than mammalian; some post-translational modifications | May lack mammalian-specific glycosylation | Higher (5-20 mg/L) | Structural studies; antibody production |
| E. coli | Lowest cost; highest potential yield | Lacks post-translational modifications; refolding often required | Highest (potential >50 mg/L) | Domain studies; peptide generation |
For functional studies of recombinant bovine TSPAN18, mammalian expression systems are generally preferred due to their ability to properly fold multi-pass membrane proteins and provide appropriate post-translational modifications. When studying tetraspanin interactions, it's critical to maintain the native conformation of the extracellular domains, particularly the large extracellular loop (EC2) which mediates many protein-protein interactions .
Researchers have successfully used recombinant EC2 domains of tetraspanins to study their functions, suggesting that expression of the EC2 domain of TSPAN18 alone may be sufficient for certain applications .
What are effective methods for studying TSPAN18-mediated calcium signaling in experimental systems?
Studying TSPAN18's role in calcium signaling requires specialized techniques:
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Calcium imaging: Fluorescent calcium indicators (Fluo-4, Fura-2) can quantify intracellular calcium changes in TSPAN18-expressing or TSPAN18-knockdown cells following stimulation with agents like thrombin or histamine. Studies have shown 55-70% decreased Ca²⁺ mobilization in TSPAN18-knockdown endothelial cells .
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Patch-clamp electrophysiology: Direct measurement of Orai1 channel activity in the presence or absence of TSPAN18 can provide mechanistic insights into how TSPAN18 regulates channel function.
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NFAT reporter assays: As TSPAN18 overexpression induces 20-fold activation of Ca²⁺-responsive NFAT signaling, NFAT-luciferase reporters can serve as sensitive readouts of TSPAN18-mediated calcium signaling .
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Proximity ligation assays: These can detect and quantify TSPAN18-Orai1 interactions in intact cells, providing spatial information about their association.
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Surface biotinylation assays: These have demonstrated that Orai1 cell surface localization is reduced by 70% in TSPAN18-knockdown endothelial cells .
How can researchers effectively use CRISPR/Cas9 to generate TSPAN18 knockout cellular models?
CRISPR/Cas9 has been successfully employed to knockout tetraspanin genes in various cell types. For TSPAN18 specifically:
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Guide RNA design: Target early exons to ensure complete protein disruption. Multiple guide RNAs targeting different exons can increase knockout efficiency.
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Validation strategies:
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Western blotting to confirm protein absence
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Flow cytometry if suitable antibodies are available
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Functional assays such as calcium mobilization in response to thrombin or histamine (55-70% decrease expected in knockouts)
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von Willebrand factor release assays (90% reduction expected in knockout endothelial cells)
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Control considerations: Generate rescue lines re-expressing TSPAN18 to confirm phenotype specificity. This is especially important as researchers have used CRISPR/Cas9 to confirm tetraspanins as host cellular factors in various biological processes .
How does TSPAN18 regulate Orai1 trafficking and function at the molecular level?
TSPAN18's regulation of Orai1 involves multiple mechanisms:
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Trafficking regulation: TSPAN18 facilitates Orai1 transport to the plasma membrane, with Orai1 surface localization reduced by 70% in TSPAN18-knockdown endothelial cells . This suggests TSPAN18 may function as a molecular chaperone for Orai1.
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Molecular interaction: TSPAN18 directly interacts with Orai1, likely through its extracellular domains. This interaction may stabilize Orai1 at the cell surface or modify its conformation to enhance channel activity .
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Signaling amplification: TSPAN18 overexpression induces 20-fold activation of Ca²⁺-responsive NFAT signaling in an Orai1-dependent manner, suggesting it may enhance Orai1 channel opening probability or conductance .
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Tetraspanin web organization: Like other tetraspanins, TSPAN18 likely organizes membrane microdomains ("tetraspanin webs") that cluster Orai1 channels and associated signaling molecules to facilitate efficient calcium signaling.
Future research using techniques such as cryo-electron microscopy or hydrogen-deuterium exchange mass spectrometry could further elucidate the structural basis of TSPAN18-Orai1 interactions.
What comparative approaches can reveal functional conservation between bovine and human TSPAN18?
Understanding cross-species conservation of TSPAN18 function requires multi-faceted approaches:
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Sequence and structural analysis: Alignment of bovine and human TSPAN18 sequences, with particular attention to the EC2 domain that mediates most protein interactions. Mouse tetraspanin CD9, for example, shows approximately 90% homology to human CD9 .
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Cross-species complementation: Testing whether bovine TSPAN18 can rescue phenotypes in human TSPAN18-knockdown cells and vice versa.
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Binding partner conservation: Determining whether bovine and human TSPAN18 interact with the same repertoire of proteins, particularly Orai1.
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Functional conservation assessment: Comparing calcium signaling responses and von Willebrand factor release mechanisms between species.
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Domain swapping experiments: Creating chimeric proteins with domains from bovine and human TSPAN18 to identify regions critical for species-specific functions.
This type of comparative analysis is important because tetraspanin functions can show significant cross-species conservation, as demonstrated by the use of mouse models to study tetraspanin functions relevant to human biology .
How can recombinant TSPAN18 be used to dissect the mechanisms of bacterial infection?
Recent research has implicated tetraspanins in bacterial infection processes:
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Infection modulation: Tetraspanins CD9, CD63, and CD81 affect Burkholderia thailandensis-induced multinucleated giant cell (MNGC) formation in macrophages . Recombinant proteins corresponding to the EC2 domains of these tetraspanins inhibited MNGC formation .
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Experimental approach: Similar approaches could be employed with recombinant bovine TSPAN18:
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Cellular factor analysis: Like other tetraspanins that mediate bacterial internalization and membrane fusion, TSPAN18 might play roles in specific infection processes . Researchers have demonstrated that tetraspanins are host cellular factors mediating internalization and membrane fusion during B. pseudomallei infection .
How should researchers address contradictory findings regarding TSPAN18 function across different experimental models?
When faced with contradictory data regarding TSPAN18 function:
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Consider expression levels: TSPAN18 is expressed by endothelial cells at several-fold higher levels than most other cell types . Functional significance may therefore differ between cell types based on expression levels.
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Examine model-specific factors:
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Cell type (primary vs. immortalized)
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Species differences (bovine vs. human vs. mouse)
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Knockout vs. knockdown approaches (complete absence vs. partial reduction)
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Acute vs. chronic manipulation (developmental compensation may occur in knockout models)
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Analyze context-dependent interactions: TSPAN18's functional impact may depend on the presence of specific binding partners that vary between experimental systems.
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Statistical robustness assessment: Evaluate sample sizes, statistical methods, and effect sizes across studies. Some genetic association studies of TSPAN18, for example, have shown p-values near significance thresholds (p = 0.05) that warrant careful interpretation .
What statistical approaches are most appropriate for analyzing TSPAN18 knockout phenotypes?
| Phenotype Measure | Recommended Statistical Method | Sample Size Considerations | Control Comparisons |
|---|---|---|---|
| Bleeding time | Non-parametric tests (Mann-Whitney) | N ≥ 10 mice per group | Wild-type; heterozygous; rescue |
| Thrombus formation | ANOVA with post-hoc tests | N ≥ 8 mice per group | Include sham-operated controls |
| Ca²⁺ mobilization | Repeated measures ANOVA | Minimum 30 cells across 3+ independent experiments | Include Orai1 knockdown as positive control |
| Von Willebrand factor release | Two-way ANOVA (genotype × stimulation) | Minimum 3 biological replicates with technical triplicates | Include dose-response curves |
Published studies have observed several key phenotypes in TSPAN18 knockout models that require specific statistical approaches:
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Bleeding phenotypes: TSPAN18-knockout mice lose on average 6-fold more blood in tail-bleed assays compared to controls . Given the typical variability in bleeding assays, non-parametric statistical methods are often most appropriate.
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Thrombosis models: The 60% reduction in thrombus size in deep vein thrombosis models and 50% reduction in platelet deposition following myocardial ischemia-reperfusion injury require careful statistical handling of outliers and consideration of multiple variables .
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Von Willebrand factor measurements: The 45% reduction in histamine-induced plasma von Willebrand factor in TSPAN18-knockout mice represents a substantial effect size that should be detectable with moderate sample sizes .
What new technologies could advance our understanding of TSPAN18's role in calcium signaling?
Emerging technologies offer new opportunities for TSPAN18 research:
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Super-resolution microscopy: Techniques like STORM or PALM can visualize TSPAN18-Orai1 nanoscale organization in the plasma membrane, potentially revealing how TSPAN18 organizes Orai1 clusters.
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Optogenetic calcium channel control: Optogenetic tools could help distinguish direct TSPAN18 effects on Orai1 from indirect regulatory mechanisms.
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Single-cell proteomics: Analysis of the TSPAN18 interactome at the single-cell level could reveal cell-specific interaction partners.
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Organ-on-chip models: These could provide more physiologically relevant systems to study TSPAN18 function in endothelial cells under flow conditions.
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CRISPR activation/inhibition screens: CRISPRa/CRISPRi approaches could identify genes that modify TSPAN18-dependent phenotypes, expanding our understanding of the broader signaling network.
How might therapeutic targeting of TSPAN18 be developed based on current research findings?
TSPAN18 research suggests several potential therapeutic approaches:
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Thrombosis management: Since TSPAN18-knockout mice show 60% reduced thrombus size in deep vein thrombosis models , TSPAN18 inhibitors might have antithrombotic potential with a novel mechanism of action.
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Bleeding disorder treatment: Conversely, TSPAN18 activators might help manage bleeding disorders by enhancing von Willebrand factor release from endothelial cells.
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Inflammatory response modulation: TSPAN18's role in endothelial calcium signaling in response to inflammatory mediators suggests potential applications in inflammatory vascular conditions.
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Targeted delivery approaches:
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EC2 domain-derived peptides
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Monoclonal antibodies against specific TSPAN18 epitopes
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Small molecule modulators of TSPAN18-Orai1 interaction
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Future development would require careful consideration of potential off-target effects on other calcium signaling pathways and extensive pre-clinical testing in relevant disease models.
