TANK Human (1-119)

What is TANK Human (1-119) and how is it structurally characterized?

TANK Human (1-119) is a truncated version of the full-length TANK protein, consisting of only the first 119 amino acids. It is a single, non-glycosylated polypeptide chain with a molecular mass of approximately 13.6 kDa when produced in E. coli expression systems . The amino acid sequence begins with MDKNIGEQLNKAYEAFRQACMDRDSAV and continues through position 119 of the native protein . This fragment represents the N-terminal region of TANK, which is important for protein-protein interactions in immune signaling pathways.

When working with this protein, researchers should note that the structure may vary slightly depending on the expression system used (E. coli versus yeast) and the presence of fusion tags (such as His-tags) that might affect protein folding or function .

What are the recommended storage conditions for TANK Human (1-119)?

For optimal stability of TANK Human (1-119), it is recommended to store the protein at -20°C to -80°C for long-term storage (up to 12 months for lyophilized form, 6 months for liquid form) . For working with the protein over shorter periods (2-4 weeks), 4°C storage is acceptable if the entire vial will be used .

To maintain protein stability, avoid repeated freeze-thaw cycles as these can lead to protein denaturation and loss of function. For long-term storage, the addition of a carrier protein (0.1% HSA or BSA) is recommended as a stabilizing agent . The protein is typically formulated in a Tris-based buffer with 50% glycerol, which helps maintain stability during storage .

How can I verify the purity and identity of TANK Human (1-119)?

Standard quality control measures for TANK Human (1-119) include:

• SDS-PAGE analysis to verify purity (typically >85-90% as indicated by manufacturers)

• Mass spectrometry to confirm the molecular weight of 13.6 kDa for the E. coli-expressed version

• Western blotting using specific anti-TANK or anti-tag antibodies (particularly for His-tagged versions)

• N-terminal sequencing to confirm the identity of the first several amino acids

• Functional assays to verify biological activity, such as protein-protein interaction studies with known TANK binding partners like TRAF2

If working with His-tagged versions, consider performing a test purification using Ni-NTA or similar affinity resins to confirm tag accessibility and protein folding .

What are the differences between E. coli and yeast expression systems for TANK Human (1-119)?

Both E. coli and yeast expression systems are used to produce TANK Human (1-119), with each offering distinct advantages:

E. coli Expression System:

• Typically yields a 13.6 kDa protein

• Advantages include higher yield, faster growth rates, and more economical production

• Lacks post-translational modifications, which may be advantageous for structural studies requiring homogeneous samples

• Suitable for applications where glycosylation is not required for function

Yeast Expression System:

• Produces a slightly larger protein (15.8 kDa as reported)

• Offers eukaryotic-like post-translational modifications, which may preserve certain functional characteristics

• May provide better folding for complex proteins

• Generally results in lower endotoxin levels compared to E. coli-derived proteins

The choice between these systems should be guided by the intended experimental application. For structural studies, E. coli-derived protein might be preferred, while for functional assays mimicking human cellular conditions, yeast-expressed protein might provide better activity .

How can I optimize the purification protocol for TANK Human (1-119)?

For optimal purification of TANK Human (1-119), consider the following methodological approach:

• Lysis Buffer Optimization: Use a Tris-based buffer (pH 8.0) containing 0.2M NaCl and reducing agents like 2mM DTT to maintain protein stability during extraction

• Affinity Chromatography: For His-tagged versions, use Ni-NTA or IMAC purification as the initial capture step. Optimize imidazole concentrations in binding and elution buffers (typically 20-40mM for binding, 250-500mM for elution)

• Secondary Purification: Follow with size-exclusion chromatography (SEC) to remove aggregates and achieve >90% purity

• Buffer Exchange: Consider dialyzing into a final storage buffer containing 50% glycerol for stability

• Quality Control: Implement SDS-PAGE analysis at each purification step to monitor purity progression

• Scale Considerations: For research applications requiring larger amounts, consider implementing automated chromatography systems while maintaining the same buffer conditions and purification principles

This multi-step approach typically yields protein with >90% purity suitable for most research applications .

What are the key binding partners of TANK Human (1-119) and how can these interactions be studied?

TANK Human (1-119) interacts with several key proteins in the NF-κB signaling pathway:

• TRAF Proteins: TANK binds to TRAF1, TRAF2, and TRAF3, regulating their activity by sequestering them in a latent state in the cytoplasm

• TBK1 and IKBKE: TANK constitutively binds these kinases, playing a crucial role in antiviral innate immunity signaling

To study these interactions, researchers can employ:

• Co-immunoprecipitation (Co-IP): Using antibodies against TANK to pull down protein complexes, followed by western blotting for binding partners

• GST Pull-down Assays: With GST-tagged TANK (1-119) as bait to identify interaction partners

• Surface Plasmon Resonance (SPR): To quantitatively measure binding kinetics and affinities between TANK and its partners

• Yeast Two-Hybrid Screening: For identifying novel interaction partners

• FRET or BRET Assays: For studying protein-protein interactions in live cells

When designing these experiments, it's critical to consider that the 1-119 fragment may not contain all binding domains present in the full-length protein, potentially limiting some interaction studies .

How does TANK Human (1-119) contribute to NF-κB signaling regulation?

TANK Human (1-119) participates in NF-κB signaling regulation through several mechanisms:

• Negative Regulation of TRAF2-mediated Signaling: TANK inhibits TRAF2-mediated NF-κB activation triggered by CD40, TNFR1, and TNFR2 receptors

• LMP1 Signaling Inhibition: TANK blocks the binding of TRAF2 to LMP1 (Epstein-Barr virus latent membrane protein 1), thereby inhibiting LMP1-mediated NF-κB activation

• I-kappa-B-kinase Regulation: TANK functions as an adapter protein involved in IKK regulation, affecting downstream NF-κB activation

To experimentally investigate these functions:

• Use reporter gene assays with NF-κB responsive elements to measure pathway activation

• Employ gene knockdown/knockout approaches followed by reconstitution with TANK (1-119) to assess functional recovery

• Analyze phosphorylation status of pathway components (IκB, p65) by western blotting after TANK (1-119) overexpression

• Compare the effects of full-length TANK versus the 1-119 fragment to identify domain-specific functions

These methodological approaches can help elucidate whether the 1-119 fragment retains the full regulatory capabilities of the complete protein or represents a functionally distinct entity in the signaling cascade .

How can TANK Human (1-119) be used in structural biology studies?

TANK Human (1-119) offers several advantages for structural biology investigations:

• X-ray Crystallography: The non-glycosylated, homogeneous nature of E. coli-expressed TANK (1-119) makes it suitable for crystallization trials. Researchers should consider:

  • Screening different buffer conditions (pH range 6.5-8.5)

  • Testing various precipitants (PEG series, ammonium sulfate)

  • Adding stabilizing agents like glycerol or small molecular weight PEGs

  • Co-crystallization with binding partners to capture interaction interfaces

• NMR Spectroscopy: At 13.6 kDa, TANK (1-119) is within the suitable size range for solution NMR studies:

  • Isotopic labeling (15N, 13C) can be readily achieved in E. coli expression systems

  • 2D and 3D heteronuclear experiments can elucidate the structure

  • Titration experiments with binding partners can map interaction surfaces

• Cryo-EM: For studying larger complexes:

  • TANK (1-119) can be incorporated into reconstituted signaling complexes

  • Tag-based purification approaches can isolate intact complexes for structural analysis

When designing structural studies, researchers should consider using the His-tagged versions for initial purification, with the option to cleave the tag if it interferes with crystallization or proper folding .

How can I design inhibitors or modulators targeting TANK Human (1-119) interactions?

Developing modulators of TANK (1-119) interactions involves several strategic approaches:

• Structure-Based Drug Design:

  • Utilize crystal structures of TANK in complex with binding partners to identify interaction interfaces

  • Apply computational docking to screen virtual libraries for compounds that may disrupt protein-protein interactions

  • Design peptidomimetics based on the binding interface sequences of natural partners

• High-Throughput Screening Approaches:

  • Develop FRET/BRET-based assays to monitor TANK interactions with TRAF proteins

  • Implement AlphaScreen technology for detecting disruption of protein-protein interactions

  • Design split-luciferase complementation assays for cell-based screening

• Fragment-Based Drug Discovery:

  • Screen small molecular fragments (MW < 300 Da) that bind to TANK (1-119)

  • Use NMR, SPR, or thermal shift assays to detect binding

  • Link or grow promising fragments into more potent molecules

• Validation Methods:

  • Confirm target engagement using cellular thermal shift assays (CETSA)

  • Verify functional effects with pathway-specific reporter assays

  • Assess selectivity against related TRAF-interacting proteins

This approach parallels successful strategies used for other signaling proteins, such as the development of small-molecule inhibitors for tankyrase 1 (TNKS1), where crystal structures revealed binding modes that informed further optimization .

What are the experimental considerations when investigating TANK (1-119) in antiviral immunity pathways?

When studying TANK (1-119) in antiviral immunity contexts, researchers should consider these methodological approaches:

• Viral Infection Models:

  • Compare responses in cells overexpressing TANK (1-119) versus full-length TANK

  • Use RNA viruses (e.g., VSV, Sendai virus) to trigger RIG-I-dependent pathways where TANK functions

  • Measure type I interferon production as a functional readout of pathway activation

• Signaling Complex Analysis:

  • Investigate the formation of the TBK1/IKBKE/TANK complex during viral infection

  • Use proximity ligation assays to visualize complex formation in situ

  • Employ TANK (1-119) as a potential dominant-negative to disrupt endogenous complexes

• Phosphorylation Studies:

  • Analyze the phosphorylation status of TANK (1-119) following viral infection

  • Identify kinases responsible for TANK modification

  • Create phospho-mutants to assess the functional importance of specific residues

• Transcriptional Profiling:

  • Compare the transcriptional landscape in cells expressing TANK (1-119) versus full-length TANK following viral challenge

  • Focus on interferon-stimulated genes (ISGs) and NF-κB target genes

  • Use ChIP-seq to map the recruitment of transcription factors to relevant promoters

• In Vivo Applications:

  • Consider transgenic models expressing TANK (1-119) to study the in vivo relevance

  • Challenge with appropriate viral pathogens to assess immune response modifications

  • Analyze tissue-specific effects that may not be apparent in cell culture models

These experimental approaches should be carefully controlled, particularly when comparing the truncated 1-119 fragment to the full-length protein, as the fragment may exhibit unique properties or dominant-negative effects.

What are common challenges in working with TANK Human (1-119) and how can they be addressed?

Researchers working with TANK Human (1-119) may encounter several technical challenges:

• Protein Solubility Issues:

  • Problem: Aggregation or precipitation during purification or storage

  • Solution: Optimize buffer conditions by testing different pH ranges (7.0-8.5), salt concentrations (150-300mM NaCl), and adding stabilizing agents (glycerol, reducing agents like DTT or TCEP)

  • Method: Perform dynamic light scattering to monitor aggregation states in different buffers

• Inconsistent Activity in Functional Assays:

  • Problem: Batch-to-batch variation in activity

  • Solution: Implement rigorous quality control testing, including activity-based assays comparing to reference standards

  • Method: Develop quantitative binding assays (SPR, BLI) to ensure consistent interaction with known partners

• Expression Yield Variability:

  • Problem: Low or inconsistent yields from expression systems

  • Solution: Optimize codon usage for the expression host, test different induction conditions (temperature, inducer concentration, duration)

  • Method: Compare multiple constructs with different fusion tags or solubility enhancers

• Tag Interference with Function:

  • Problem: His-tag or other fusion tags interfering with binding or activity

  • Solution: Create constructs with cleavable tags or with tags positioned at different termini

  • Method: Compare the activity of tagged versus tag-cleaved protein in functional assays

• Endotoxin Contamination (E. coli-expressed protein):

  • Problem: Endotoxin affecting cellular assays

  • Solution: Implement additional purification steps (Triton X-114 phase separation, polymyxin B columns)

  • Method: Use LAL testing to verify endotoxin reduction to acceptable levels

How can I design experiments to distinguish between functions of TANK (1-119) and full-length TANK?

To differentiate between the functions of TANK (1-119) and the full-length protein, consider these experimental approaches:

• Domain Mapping Studies:

  • Express multiple truncation constructs (1-119, 120-425, full-length) to map functional domains

  • Perform complementation assays in TANK-knockout cell lines

  • Use these constructs in binding assays to identify domain-specific interactions

• Competitive Binding Assays:

  • Test whether TANK (1-119) can compete with full-length TANK for binding to partners

  • Implement ELISA-based or fluorescence polarization competition assays

  • Determine binding affinities (Kd values) for both proteins with common partners

• Cellular Localization Analysis:

  • Compare subcellular distribution using fluorescently tagged constructs

  • Perform fractionation studies followed by western blotting

  • Assess co-localization with binding partners in different cellular compartments

• Differential Signaling Outcomes:

  • Monitor downstream signaling events (NF-κB activation, IRF3 phosphorylation)

  • Compare phosphorylation patterns of signaling intermediates

  • Analyze transcriptional responses using reporter assays or RNA-seq

• Structure-Function Correlation:

  • Generate point mutations in conserved residues within the 1-119 region

  • Test these mutations in both the truncated and full-length contexts

  • Identify residues that may have context-dependent functions

These comparative approaches can reveal whether TANK (1-119) represents a functional domain with independent activity or requires the context of the full-length protein for proper function.

What emerging technologies might advance our understanding of TANK Human (1-119)?

Several cutting-edge technologies hold promise for expanding our understanding of TANK Human (1-119):

• Cryo-Electron Tomography:

  • Could visualize TANK-containing complexes in their native cellular environment

  • May reveal previously unknown structural arrangements and interactions

  • Particularly valuable for studying membrane-proximal signaling complexes

• AlphaFold and Other AI-Based Structure Prediction:

  • Can predict structures of TANK (1-119) in complex with binding partners

  • Helps generate hypotheses about interaction interfaces for experimental validation

  • Enables virtual screening of compounds that might modulate TANK functions

• Proximity Labeling Proteomics (BioID, APEX):

  • Identify novel interaction partners of TANK (1-119) in living cells

  • Compare interactomes of truncated versus full-length protein

  • Map the dynamic changes in protein-protein interactions during immune activation

• CRISPR-Based Genomic Screens:

  • Identify genes that synthetically interact with TANK in immune signaling

  • Discover new pathway components or regulatory mechanisms

  • Generate cell lines with endogenously tagged TANK for physiological studies

• Single-Cell Multi-omics:

  • Analyze cell-to-cell variability in TANK-dependent signaling

  • Correlate TANK activity with transcriptional and proteomic profiles

  • Identify rare cell populations with distinct TANK-mediated responses

These technologies, when applied to TANK research, could resolve current knowledge gaps and potentially reveal new therapeutic opportunities targeting TANK-dependent pathways.

How might TANK Human (1-119) be utilized in developing novel immunomodulatory approaches?

TANK Human (1-119) represents a potential platform for developing innovative immunomodulatory strategies:

• Targeted Protein Degradation:

  • Design TANK-targeting PROTACs (Proteolysis Targeting Chimeras) to selectively degrade TANK in specific contexts

  • Develop immunoTACs that combine TANK-binding elements with immune cell recruiters

  • These approaches could provide more selective modulation than conventional inhibitors

• Engineered Protein Scaffolds:

  • Create TANK (1-119)-based decoy proteins that compete for binding partners

  • Develop bispecific molecules linking TANK domains to other signaling components

  • These engineered proteins could redirect signaling pathways in therapeutic directions

• Cell-Penetrating Peptides:

  • Identify short peptide sequences from TANK (1-119) that mediate key interactions

  • Conjugate these to cell-penetrating sequences to create pathway-specific inhibitors

  • Such peptides could offer advantages in specificity over small molecules

• mRNA-Based Therapeutics:

  • Develop modified mRNA encoding TANK (1-119) or dominant-negative variants

  • Target delivery to specific immune cell populations

  • This approach could achieve transient modulation of immune signaling

• Combination Approaches:

  • Pair TANK-targeted therapies with existing immunomodulators (checkpoint inhibitors, cytokines)

  • Identify synergistic combinations through high-throughput screening

  • Such combinations may achieve more selective immune modulation with fewer side effects

These approaches leverage our understanding of TANK's structural and functional properties to create novel therapeutic modalities with potential applications in autoimmune diseases, inflammatory conditions, and viral infections.