HAVCR2 Human, Sf9
Description
Introduction to HAVCR2 Human, Sf9
HAVCR2 (Hepatitis A Virus Cellular Receptor 2), also known as TIM-3 (T-cell immunoglobulin and mucin-domain containing protein 3), is a transmembrane protein critical for immune regulation. It functions as an immune checkpoint, modulating T-cell responses, macrophage activation, and immunological tolerance . The recombinant human HAVCR2 protein expressed in Sf9 cells (a moth-derived cell line) is widely used in research to study immune mechanisms, cancer immunotherapy, and autoimmune diseases.
Protein Domains
HAVCR2 belongs to the TIM family and contains:
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IgV domain: A membrane-distal immunoglobulin-like domain with disulfide bonds forming a cleft for ligand binding (e.g., galectin-9, phosphatidylserine) .
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Mucin domain: A glycosylated region rich in serine, proline, and threonine, critical for protein stability and interactions .
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Transmembrane domain: Anchors the protein to the cell membrane.
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Cytoplasmic tail: Contains tyrosine residues that mediate intracellular signaling .
Amino Acid Sequence and Tags
Recombinant HAVCR2 variants differ in construct design:
| Source | Amino Acids | Tag | Molecular Mass |
|---|---|---|---|
| E. coli | 206 aa (aa 22–202) | N-terminal His-tag | 22.7 kDa |
| Sf9 | 423 aa (aa 22–202) | C-terminal hIgG-His-tag | 47.3 kDa |
The Sf9-expressed protein includes glycosylation, enhancing structural authenticity compared to bacterial systems .
Production Workflow
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Baculovirus Transduction: Sf9 cells are infected with a recombinant baculovirus encoding HAVCR2.
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Secretion: The protein is expressed as a soluble form, facilitated by the Sf9 system’s eukaryotic post-translational machinery .
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Purification:
Advantages of Sf9 Expression
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Glycosylation: Sf9 cells add complex carbohydrates, mimicking human protein modifications .
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Solubility: High yield due to insect cell secretion pathways .
Biochemical Properties of Recombinant HAVCR2
| Parameter | Value | Source |
|---|---|---|
| Purity | >85% (SDS-PAGE) | |
| Stability | Store at -20°C; avoid freeze-thaw cycles | |
| Activity | Binds galectin-9, phosphatidylserine, and CEACAM1 |
Key Differences Between Constructs:
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E. coli vs. Sf9: The Sf9 protein is larger (47.3 kDa vs. 22.7 kDa) due to glycosylation and tags .
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Functional Impact: Sf9-expressed HAVCR2 better mimics native ligand-binding capabilities .
Immunology and Autoimmunity
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T-cell Regulation: Studies use HAVCR2 to investigate Th1/Th17 suppression and Treg-cell function .
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Hemophagocytic Lymphohistiocytosis (HLH): Mutations in HAVCR2 are linked to HLH; recombinant protein aids in cytokine profiling (e.g., IL-1RA) .
Cancer Immunotherapy
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Checkpoint Blockade: HAVCR2 is co-expressed with PD-1 in exhausted T cells; dual blockade enhances antitumor responses .
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Antibody Development: Sf9-expressed HAVCR2 is used to screen therapeutic antibodies (e.g., LY3321367, MBG453) .
Neurological Disorders
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Alzheimer’s Disease: HAVCR2 variants are associated with late-onset sporadic Alzheimer’s; recombinant protein studies explore amyloid-β interactions .
Disease Associations
Ongoing Clinical Trials
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Combination Therapies: Anti-HAVCR2 monoclonal antibodies (e.g., TSR-022) are tested with anti-PD-1 drugs to overcome resistance .
Sf9 Cell Line Contamination
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Sf-rhabdovirus: A lepidopteran rhabdovirus identified in Sf9 cells poses biosafety risks, though no human transmission has been reported .
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Mitigation: Rigorous screening and virus-free cell lines are critical for therapeutic protein production .
Construct Limitations
Product Specs
ADLSEVEYRA EVGQNAYLPC FYTPAAPGNL VPVCWGKGAC PVFECGNVVL RTDERDVNYW TSRYWLNGDF RKGDVSLTIE NVTLADSGIY CCRIQIPGIM NDEKFNLKLV IKPAKVTPAP TRQRDFTAAF PRMLTTRGHG PAETQTLGSL PDINLTQIST LANELRDSRL ANDLRDSGAT IRIGLEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKHHH HHH.
Q&A
What is HAVCR2 and what are its primary functions in the human immune system?
HAVCR2 (T-cell immunoglobulin and mucin domain-containing protein 3 or TIM-3) is a member of the immunoglobulin superfamily that plays crucial roles in immune regulation. It controls macrophage activation and inhibits T-helper type 1 lymphocyte (Th1)-mediated auto- and alloimmune responses while promoting immunological tolerance. This protein is increasingly recognized as an important immune checkpoint molecule with relevance to both autoimmunity and cancer research .
Why are Sf9 insect cells preferred for recombinant human HAVCR2 expression?
Sf9 insect cells derived from Spodoptera frugiperda provide several advantages for expressing human proteins like HAVCR2. This expression system allows for high-level production of properly folded, functional proteins with post-translational modifications. Unlike bacterial systems, Sf9 cells can handle complex mammalian proteins with multiple domains and disulfide bonds. Research has demonstrated that human proteins expressed in this system often retain their functional characteristics, making it an excellent choice for producing proteins for structural and functional studies .
What are the fundamental components of the baculovirus/Sf9 expression system?
The baculovirus/Sf9 expression system consists of:
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Sf9 insect cells as hosts
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Recombinant baculovirus containing the gene of interest (e.g., HAVCR2)
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Appropriate vectors with promoters for high-level expression
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Selection markers for identifying successfully infected cells
In this system, recombinant baculovirus carrying the HAVCR2 gene infects Sf9 cells, hijacking their protein synthesis machinery to produce the target protein. Full-length recombinant human HAVCR2 can be expressed with various tags (e.g., GST, His) to facilitate purification and detection .
How can researchers monitor baculovirus infection and HAVCR2 expression in Sf9 cells?
Flow cytometry provides an effective method for monitoring Sf9 cell infection by recombinant baculovirus. Using side scattered light coupled with green fluorescence detection after immunolabeling of the recombinant protein, researchers can precisely assess infection rates from approximately 60 hours post-infection. This technique also characterizes the two-step infection process (primary and secondary infection) in asynchronously infected cultures. For HAVCR2 specifically, immunofluorescence detection using antibodies against the protein or its fusion tags (GST, His) can monitor expression levels and localization .
What are the optimal conditions for expressing functional HAVCR2 in Sf9 cells?
Optimal expression conditions include:
| Parameter | Recommended Condition | Rationale |
|---|---|---|
| Cell density at infection | Mid-exponential phase | Cells show higher sensitivity to infection during this phase |
| Multiplicity of infection (MOI) | Variable (0.1-10) depending on scale | Higher MOI leads to synchronized infection but requires more virus |
| Time of harvest | 60-72 hours post-infection | Allows sufficient protein accumulation while minimizing degradation |
| Temperature | 27°C | Optimal for Sf9 cell growth and protein expression |
| Media supplements | None or low serum | Simplifies downstream purification |
Research indicates that cells infected during exponential growth phase show significantly better baculovirus infection rates compared to cells infected at the end of growth phase .
What purification strategies yield high-quality HAVCR2 from Sf9 cells?
Affinity chromatography using fusion tags represents the primary purification approach:
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GST-tagged HAVCR2: Purified using glutathione-based affinity chromatography with elution buffers containing reduced glutathione. This approach has been used for full-length HAVCR2 expressed with N-terminal GST tags .
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His-tagged HAVCR2: Purified using immobilized metal affinity chromatography (IMAC). For example, recombinant HAVCR2 with a 25 amino acid His-tag at the N-terminus can be purified using proprietary chromatographic techniques .
Additional purification steps may include size exclusion chromatography or ion exchange chromatography to achieve higher purity for structural or functional studies .
How can researchers verify the functionality of recombinant HAVCR2 after purification?
Functional validation of purified HAVCR2 can be performed through multiple approaches:
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Binding assays: Testing interaction with known ligands such as galectin-9, phosphatidylserine, or CEACAM1 using surface plasmon resonance (SPR) or ELISA-based methods.
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Cell-based assays: Assessing HAVCR2's immunomodulatory effects on T cell activation and cytokine production.
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Structural integrity analysis: Using circular dichroism or thermal shift assays to confirm proper protein folding.
Research with other recombinant proteins expressed in Sf9 cells demonstrates that they retain functionality similar to their native counterparts. For example, studies show that recombinant olfactory receptors produced in this system maintained cellular activities upon odorant stimulation as measured by calcium imaging .
How do post-translational modifications of HAVCR2 in Sf9 cells compare to those in human cells?
While Sf9 cells perform many of the same post-translational modifications as mammalian cells, important differences exist:
| Modification Type | Sf9 Cells | Human Cells | Potential Impact on HAVCR2 |
|---|---|---|---|
| N-glycosylation | Simple, high-mannose type | Complex, sialylated | May affect binding to ligands and stability |
| O-glycosylation | Limited | Extensive | Could impact mucin domain function |
| Phosphorylation | Present but patterns differ | Cell-type specific | May alter signaling capabilities |
| Disulfide bonds | Generally preserved | Native pattern | Critical for immunoglobulin domain structure |
These differences should be considered when interpreting functional data, particularly for immune receptors like HAVCR2 where glycosylation can influence binding and signaling properties .
What challenges might researchers encounter when scaling up HAVCR2 production in Sf9 cells?
Scaling up HAVCR2 production from laboratory to larger scales presents several challenges:
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Infection dynamics: The asynchronous infection process becomes more pronounced in larger cultures, potentially reducing yield. Flow cytometric monitoring can help optimize infection parameters .
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Cell density effects: Research shows reduced sensitivity to baculovirus infection in cells at high densities or at the end of growth phase. Careful monitoring of growth curves and infection timing is essential .
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Oxygen transfer: Larger bioreactors require optimized aeration strategies that differ from small-scale shake flask cultures.
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Protein stability: During extended expression periods or processing of large volumes, protein degradation may become significant. Adding protease inhibitors (e.g., PMSF as used in some preparations) can help maintain protein integrity .
How should HAVCR2 constructs be designed for different research applications?
Design considerations vary based on the research goal:
| Research Application | Recommended Construct Design | Rationale |
|---|---|---|
| Structural studies | Truncated constructs (e.g., aa 22-202) with minimal tags | Removes flexible regions that may hinder crystallization |
| Functional assays | Full-length with cleavable tags | Preserves native structure after tag removal |
| Binding studies | IgV domain constructs | The IgV domain contains most ligand binding sites |
| Cell signaling research | Full-length with minimal modifications | Maintains transmembrane and cytoplasmic signaling domains |
For example, recombinant human HAVCR2 expressed in E. coli has been successfully produced as a single polypeptide chain containing 206 amino acids (aa 22-202), focusing on the extracellular domain with an N-terminal His-tag .
What are the most effective stabilization methods for purified HAVCR2?
Stabilizing purified HAVCR2 requires careful buffer formulation:
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Buffer components: Optimal stability has been achieved with buffers containing 20mM Tris-HCl (pH 8.0), 0.4M urea, and 10% glycerol for His-tagged constructs .
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Storage conditions: For GST-tagged HAVCR2, stability is enhanced in buffers containing 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM glutathione, 0.1 mM EDTA, 0.25 mM DTT, 0.1 mM PMSF, and 25% glycerol .
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Long-term preservation: Addition of carrier proteins (0.1% HSA or BSA) is recommended for extended storage, with storage at -20°C and avoidance of multiple freeze-thaw cycles .
How can researchers troubleshoot common expression issues with HAVCR2 in the Sf9 system?
When encountering low expression levels or non-functional protein:
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Sequence verification: Confirm the HAVCR2 sequence is correct and in-frame with any fusion tags.
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Infection monitoring: Use flow cytometry to verify successful infection rates. Studies show that precise assessment of infection extent is possible from 60 hours post-infection .
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Cell health: Ensure Sf9 cells are in exponential growth phase at infection, as research demonstrates significantly reduced baculovirus infection sensitivity in cells at the end of growth phase .
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Expression construct optimization: If initial constructs yield poor results, consider altering tag position or type. Both N-terminal GST tags and His-tags have been successfully used with HAVCR2.
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Harvest timing optimization: Expression peaks can vary based on promoter and construct design. Monitor expression at different time points (48-96 hours) to determine optimal harvest time.
How does HAVCR2 expression in Sf9 cells compare to other expression systems?
Different expression systems offer distinct advantages:
| Expression System | Advantages for HAVCR2 | Limitations | Best Applications |
|---|---|---|---|
| Sf9/Baculovirus | High yield, proper folding, some PTMs | Glycosylation differs from human | Structural studies, functional assays |
| E. coli | Fast, inexpensive, high yield | Limited PTMs, potential folding issues | Truncated constructs, binding studies |
| Mammalian cells | Native-like PTMs, authentic folding | Lower yield, more expensive | Cell signaling studies, therapeutic development |
| Cell-free systems | Rapid expression, membrane protein compatibility | Reduced functionality, low yield | Initial screening, toxic proteins |
The choice depends on research priorities. For HAVCR2, E. coli expression has been successful for the extracellular domain (aa 22-202) , while Sf9 cells have been used for full-length expression with N-terminal tags .
What reconstitution methods are most effective for functional studies of membrane-associated HAVCR2?
For membrane-associated proteins expressed in Sf9 cells, successful reconstitution approaches include:
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Liposome reconstitution: Integrating purified protein into artificial membrane vesicles to study transmembrane functions. Research with other membrane proteins like transporters has shown that proteins purified from Sf9 cells retain functionality after reconstitution .
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Nanodiscs: Incorporating HAVCR2 into nanoscale phospholipid bilayers scaffolded by membrane scaffold proteins, providing a more native-like membrane environment.
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Detergent micelles: Maintaining HAVCR2 in detergent micelles that mimic membrane environments, which can be suitable for binding studies and some functional assays.
These approaches allow researchers to study membrane-associated aspects of HAVCR2 function, which might be particularly relevant for its role in immune cell signaling and regulation .
Product Science Overview
HAVCR2 was first described in 2002 by Vijay Kuchroo and colleagues during a screen to identify differentially expressed molecules between Th1 and Th2 cells . It is a cell surface molecule expressed on IFNγ producing CD4+ Th1 and CD8+ Tc1 cells . The protein plays a critical role in modulating both innate and adaptive immune responses . It is generally accepted to have an inhibitory function, although some reports suggest that its activity may be influenced by the cellular context and the respective ligand .
HAVCR2 belongs to the TIM family of cell surface receptor proteins. These proteins share a similar structure, with an extracellular region consisting of a membrane-distal single variable immunoglobulin domain (IgV), a glycosylated mucin domain of variable length located closer to the membrane, a transmembrane region, and an intracellular stem . The IgV domain is formed by two antiparallel beta sheets linked by disulfide bridges between four conserved cysteines . The extracellular portion of the IgV domain may also be glycosylated, and this glycan-binding site is recognized by the carbohydrate domain of ligands such as galectin-9 (Gal-9) .
HAVCR2 is a critical negative regulator in the immune system, acting as a negative checkpoint in peripheral tolerance and innate immune and inflammatory responses . It regulates macrophage activation and inhibits T-helper type 1 lymphocyte (Th1)-mediated auto- and alloimmune responses, promoting immunological tolerance . The receptor is expressed by a wide range of cells, including T lymphocytes, innate immune cells such as monocytes, natural killer (NK) cells, and dendritic cells (DC), as well as cancer stem cells .
HAVCR2 has been successfully targeted to treat several solid and hematogenous malignancies, including melanoma, acute myeloid leukemia (AML), and myelodysplastic syndromes (MDS) . Its role as a checkpoint inhibitor makes it a valuable target in cancer immunotherapy, similar to other checkpoint inhibitors such as PD-1 and CTLA-4 .
The human recombinant form of HAVCR2, produced in Sf9 cells, is used in various research and clinical applications. Sf9 cells, derived from the fall armyworm Spodoptera frugiperda, are commonly used in the baculovirus expression system for producing recombinant proteins. This system allows for high-level expression and proper post-translational modifications, making it suitable for producing functional recombinant proteins.
