ULBP3 Human, Sf9

What is ULBP3 and how is it produced in Sf9 cells?

ULBP3 (UL16 Binding Protein 3) belongs to a family of cell-surface proteins that function as ligands for the human NKG2D receptor. It is also known by alternative names including RaeT1N (retinoic acid early transcript), NKG2DL3, and ALCAN-gamma. The name ULBP derives from its identification as a ligand for human cytomegalovirus glycoprotein UL16 .

Production methodology in Sf9 cells involves:

• Cloning the ULBP3 gene (typically amino acids 30-217) into a baculovirus transfer vector

• Transfecting Sf9 cells with bacmid DNA to generate initial viral particles (V0)

• Amplification of viral supernatant in Sf9 cells for 72 hours at 27°C to create working viral stock (V1)

• Infection of High Five or Sf9 cells with the viral stock for protein expression

• Harvesting via ultracentrifugation (100,000 × g for 2 hours) to remove viral particles

• Purification using affinity chromatography based on fusion tags (commonly His-tag or IgG-Fc)

This baculovirus expression system allows for production of properly folded, glycosylated ULBP3 protein suitable for functional studies and structural analysis.

What is the molecular structure and properties of ULBP3 Human, Sf9?

ULBP3 Human Recombinant produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain with the following properties:

• Contains 430 amino acids (amino acids 30-217 of the native protein plus fusion tags)

• Molecular mass of approximately 49.3 kDa

• Typically fused to a 239 amino acid hIgG-His-Tag at C-terminus to facilitate purification

• Purified by proprietary chromatographic techniques to >95% purity as determined by SDS-PAGE

Structural characteristics:

• Distantly related to MHC class I proteins, but possesses only the alpha 1 and alpha 2 Ig-like domains

• Unlike MHC class I, has no capacity to bind peptide or interact with beta 2-microglobulin

• In its native form, anchored to the cell membrane via a GPI-linkage

• Functions as a ligand for the KLRK1/NKG2D receptor

The amino acid sequence includes: ADPDAHSLWY NFTIIHLPRH GQQWCEVQSQ VDQKNFLSYD CGSDKVLSMG HLEEQLYATD AWGKQLEMLR EVGQRLRLEL ADTELEDFTP SGPLTLQVRM SCECEADGYI RGSWQFSFDG RKFLLFDSNN RKWTVVHAGA RRMKEKWEKD SGLTTFFKMV SMRDCKSWLR DFLMHRKKRL EPTAPPTMAP GLEPKSCDKT HTCPPCPAPE L

What are the optimal storage and handling conditions for ULBP3 Human, Sf9?

For maximum stability and biological activity, ULBP3 Human, Sf9 requires specific storage and handling protocols:

Storage conditions:

• Short-term (2-4 weeks): 4°C

• Long-term: -20°C

• For extended storage periods, add carrier protein (0.1% HSA or BSA)

• Avoid multiple freeze-thaw cycles which can compromise protein integrity

Formulation characteristics:

• Typically provided as a sterile filtered colorless solution

• Standard concentration is 1mg/ml

• Buffer composition includes 10% glycerol and Phosphate-Buffered Saline (pH 7.4)

• Sample solutions may be provided at specific concentrations (e.g., 25 μL, 10mM)

Handling recommendations for experimental use:

• Determine optimal dilutions for each specific application through titration experiments

• Allow protein to equilibrate to room temperature before opening the vial

• Use aseptic technique when handling to prevent contamination

• For binding studies, consider potential interference from buffer components

These storage and handling practices ensure maximum retention of ULBP3's structural integrity and functional properties for reliable experimental results.

What is the function of ULBP3 in immune response?

ULBP3 plays significant roles in immune surveillance and regulation through several mechanisms:

• NKG2D receptor engagement: ULBP3 binds to the KLRK1/NKG2D receptor expressed on Natural Killer (NK) cells, NKT cells, gamma delta T cells, and CD8+ T cells .

• Signal transduction initiation: Upon binding, ULBP3 activates multiple cascades:

  • Calcium mobilization in immune cells

  • JAK2, STAT5, ERK signaling pathways

  • PI3K kinase/Akt signal transduction pathway

  • Production of cytokines and chemokines

• Comparative signaling properties: ULBP3 has lower affinity for KLRK1/NKG2D compared to other family members (ULBP1 and ULBP2) and consequently stimulates weaker signaling responses .

• Stress-induced expression: ULBP3 expression increases on cells undergoing stress, viral infection, or malignant transformation, serving as a "danger signal" for immune recognition.

• Unique resistance to viral evasion: Unlike other NKG2D ligands (MICA, MICB, ULBP1, and ULBP2), ULBP3 appears resistant to downregulation by certain viral immune evasion proteins like Rh159, suggesting an evolutionarily preserved role in anti-viral immunity .

This combination of functions positions ULBP3 as a critical component in the detection and elimination of compromised cells while helping maintain immunological homeostasis.

How does ULBP3 interact with NKG2D receptors and what signaling pathways are activated?

The ULBP3-NKG2D interaction involves sophisticated molecular engagement that triggers multiple downstream signaling events:

Binding mechanism:

• ULBP3 engages the NKG2D homodimer through its alpha1 and alpha2 domains

• Lower binding affinity compared to ULBP1 and ULBP2 results in distinct signaling kinetics

• Glycosylation patterns preserved in Sf9-produced protein influence binding characteristics

Signal transduction cascade:

• Immediate effects:

  • Calcium flux: Rapid increase in intracellular calcium concentrations

  • Receptor clustering and phosphorylation of adaptor molecules

• Activated pathways:

  • JAK2/STAT5: Leads to transcriptional regulation of target genes

  • ERK pathway: Mitogen-activated protein kinase signaling affecting cell activation

  • PI3K/Akt: Promotes survival signals and metabolic reprogramming

• Functional outcomes:

  • Cytokine/chemokine production: Including interferon-gamma and TNF-alpha

  • Granule polarization: Mobilization of cytolytic granules toward immunological synapse

  • Cytotoxic effector function: Elimination of target cells expressing ULBP3

The differential signaling strength induced by ULBP3 compared to other family members suggests evolutionary adaptation toward fine-tuned immune responses, potentially allowing for graduated responses to different threat levels. These signal strength variations likely contribute to the complex regulation of NK cell activation thresholds and functional plasticity.

What experimental methods are best for studying ULBP3-NKG2D interactions?

Investigating ULBP3-NKG2D interactions requires multiple complementary approaches:

• Protein-Protein Interaction Analysis:

  • ELISA-based binding assays: Coat plates with recombinant ULBP3 (1 μg/200 μl/well) in PBS and incubate with NKG2D-expressing cells or recombinant protein

  • Immunoprecipitation: Mix tagged ULBP3 with NKG2D-IgG1-Fc fusion proteins and capture using magnetic beads coated with tag-specific antibodies

  • Surface Plasmon Resonance: Determine association/dissociation rates and binding affinity constants

  • Biolayer Interferometry: Measure real-time binding kinetics with less sample consumption

• Functional Cellular Assays:

  • CD107a degranulation assay: Measure NK cell degranulation upon exposure to ULBP3-expressing targets by incubating cells with fluorescently-labeled anti-CD107a antibodies

  • IFN-γ ELISA/ELISpot: Quantify cytokine production following NK cell activation

  • Cytotoxicity assays: Assess target cell lysis using flow cytometry-based or chromium release methods

  • Calcium flux assays: Measure immediate signaling response with calcium-sensitive dyes

• Competition Experiments:

  • Pre-incubate NK cells with recombinant ULBP3 at varying concentrations (10-20 μg)

  • Use anti-NKG2D blocking antibodies as positive controls

  • Compare responses between WT ULBP3 and mutated variants to map interaction sites

• Advanced Imaging:

  • Confocal microscopy: Visualize receptor clustering and immune synapse formation

  • FRET analysis: Measure molecular proximity between ULBP3 and NKG2D in real time

  • Super-resolution microscopy: Examine nanoscale organization of receptor complexes

• Signal Transduction Analysis:

  • Phospho-flow cytometry: Detect phosphorylation events in downstream signaling molecules

  • Western blotting: Track activation of JAK2, STAT5, ERK and PI3K/Akt pathways

  • Multiplexed kinase activity profiling: Assess multiple signaling pathways simultaneously

These methodologies provide complementary information on binding properties, functional consequences, and molecular mechanisms of the ULBP3-NKG2D interaction.

How does viral immune evasion affect ULBP3 expression and function?

Viral immune evasion strategies targeting NKG2D ligands reveal important insights about ULBP3's unique properties:

• Differential susceptibility to viral countermeasures:

  • Research demonstrates that viral immune evasion protein Rh159 reduces surface expression of MICA, MICB, ULBP1, and ULBP2, but remarkably does not affect ULBP3

  • This selective targeting suggests evolutionary pressure has shaped both viral evasion strategies and host defenses, with ULBP3 potentially evolving resistance

• Mechanisms of viral evasion targeting other NKG2D ligands:

  • Intracellular transport interference: Viral proteins like Rh159 prevent trafficking of NKG2D ligands from ER to cell surface

  • ER retention: EndoH sensitivity of Rh159-affected ligands confirms their localization to early secretory compartments

  • Co-immunoprecipitation studies: Show direct binding between viral proteins and NKG2D ligands, but not with ULBP3

• Experimental approaches to investigate ULBP3 resistance:

  • Flow cytometry analysis: Demonstrates maintained ULBP3 surface expression in cells expressing viral evasion proteins, while other ligands show reduced expression

  • Pulse-chase metabolic labeling: Tracks protein maturation and transport through the secretory pathway

  • Immunoblotting with glycosidase treatment: Determines protein maturation status based on glycosylation patterns

• Functional consequences and research applications:

  • ULBP3's resistance to certain viral evasion mechanisms may make it a superior target for immunotherapeutic approaches in virus-associated malignancies

  • Understanding the molecular basis for this resistance could inform engineering of evasion-resistant versions of other NKG2D ligands

  • The "immune evasion profile" of tumors could be diagnostically relevant for selecting appropriate immunotherapeutic strategies

This unique characteristic of ULBP3 highlights its potential importance in maintaining immune surveillance during viral infections and suggests specialized evolutionary adaptation within the NKG2D ligand family.

What are the challenges in purifying ULBP3 from Sf9 cells and how can they be overcome?

Purification of ULBP3 from Sf9 cells presents several technical challenges requiring specific methodological solutions:

• Expression optimization challenges:

  • Challenge: Achieving sufficient yield and proper folding

  • Solution: Optimize viral titer (typical MOI of 80) and expression time; use specialized insect cell media; maintain expression at controlled temperature (27°C for 72h)

  • Methodological approach: Perform small-scale expression tests before scaling up; analyze protein expression kinetics to determine optimal harvest time

• Purification strategy challenges:

  • Challenge: Obtaining high purity (>95%) while maintaining native conformation

  • Solution: Multi-step purification protocol starting with affinity chromatography based on fusion tags (His-tag or Strep-tag II)

  • Methodological approach: For His-tagged ULBP3, use IMAC followed by size exclusion chromatography (SEC) in buffer containing 100 mM Tris/HCl (pH 8), 150 mM NaCl, and 1 mM EDTA

• Glycosylation heterogeneity:

  • Challenge: Insect cell glycosylation differs from mammalian patterns

  • Solution: Characterize glycosylation using endoglycosidase treatments (EndoH, PNGase F)

  • Methodological approach: Compare EndoH-treated versus untreated protein by SDS-PAGE to assess glycosylation state and homogeneity

• Protein stability issues:

  • Challenge: Maintaining activity during storage and avoiding aggregation

  • Solution: Formulate with stabilizing agents (10% glycerol in PBS, pH 7.4); add carrier proteins (0.1% HSA or BSA) for long-term storage

  • Methodological approach: Test different buffer conditions; monitor aggregation using dynamic light scattering; assess functionality after various storage periods

• Functional verification:

  • Challenge: Confirming biological activity of purified protein

  • Solution: Implement binding assays with recombinant NKG2D or NKG2D-expressing cells

  • Methodological approach: ELISA-based binding assays using 1 μg/200 μl/well coating concentration ; flow cytometry-based binding assays; functional assays measuring NK cell activation

• Endotoxin contamination:

  • Challenge: Removing endotoxin for cellular assays and in vivo applications

  • Solution: Include endotoxin removal steps; test final preparation with LAL assay

  • Methodological approach: Incorporate ion exchange chromatography or specialized endotoxin removal resins in purification workflow

Sample purification protocol based on search results:

• Harvest Sf9/High Five cells 72h post-infection

• Remove viral particles by ultracentrifugation (100,000 × g, 2h)

• Filter supernatant through 0.22 μm filter

• Apply to StrepTactin Sepharose column or Ni-NTA based on tag

• Wash extensively to remove non-specifically bound proteins

• Elute with biotin (for Strep-tagged protein) or imidazole (for His-tagged protein)

• Perform SEC in 100 mM Tris/HCl (pH 8), 150 mM NaCl, and 1 mM EDTA

• Assess purity by SDS-PAGE (target >95%)

How can ULBP3 be utilized in cancer immunotherapy research?

ULBP3's role in immune surveillance makes it a promising target for cancer immunotherapy research:

• Enhancing tumor immunogenicity:

  • Methodology: Engineer tumor cells to overexpress ULBP3 using viral vectors or CRISPR/Cas9

  • Experimental approach: Compare NK cell-mediated killing of wild-type versus ULBP3-overexpressing tumor cells using degranulation assays (CD107a)

  • Rationale: Overcoming tumor immune evasion by increasing NK cell recognition signals

• Bispecific engager development:

  • Methodology: Design fusion proteins linking ULBP3 with tumor-targeting antibody fragments

  • Experimental design: Test constructs using co-immunoprecipitation to confirm binding to both NKG2D and tumor antigens

  • Analysis: Measure NK cell recruitment and activation against antigen-positive tumor cells

  • Functional assessment: Compare cytotoxicity against tumor cells with varying antigen expression levels

• Investigating soluble ULBP3 dynamics:

  • Research question: Does soluble ULBP3 act as an immunoevasin similar to exosomal ULBP3?

  • Methodology: Pre-incubate NK cells with recombinant ULBP3 (10-20 μg) before exposure to target cells

  • Measurement: Assess changes in NK cell degranulation, cytokine production, and cytotoxicity

  • Control comparison: Use NKp30-specific antibodies (p30-15, 10 μg/ml) as comparative control

• Exploiting ULBP3's resistance to viral evasion:

  • Research application: Target ULBP3-mediated immune activation specifically in virus-associated cancers

  • Rationale: ULBP3 maintains surface expression even when Rh159 and similar viral proteins downregulate other NKG2D ligands

  • Experimental approach: Compare efficacy of ULBP3-targeting strategies versus other NKG2D ligands in virus-positive tumors

  • Analysis method: Flow cytometry to quantify ligand expression; functional assays to measure NK cell activation

• NKG2D-ULBP3 signaling modulation:

  • Research focus: Exploit ULBP3's weaker NKG2D signaling properties for specific therapeutic applications

  • Methodology: Compare signaling patterns induced by ULBP3 versus other NKG2D ligands using phospho-specific antibodies

  • Application: Design ULBP3 variants with modified binding affinity or signaling properties

  • Therapeutic potential: Develop approaches that enhance helpful immune responses while minimizing potential immunopathology

Each of these research directions requires carefully controlled experiments using properly characterized recombinant ULBP3 produced in Sf9 cells, with appropriate controls to ensure scientific rigor and reproducibility.