PDCD1LG2 Human, Sf9

What is PDCD1LG2 and what cellular functions does it perform?

PDCD1LG2 (Programmed Cell Death 1 Ligand 2), also known as PD-L2 or CD273, is a protein that plays a crucial role in immune regulation. It functions primarily by providing costimulatory signals necessary for T-cell proliferation. Additionally, PDCD1LG2 is involved in interferon gamma (IFNG) production through PDCD1-independent pathways. When PDCD1LG2 interacts with PDCD1 (PD-1), it inhibits T-cell proliferation by preventing cytokine production and disrupting cell cycle progression. This interaction is part of the immune checkpoint mechanisms that regulate T-cell responses and maintain self-tolerance .

Why are Sf9 cells used for PDCD1LG2 production?

Sf9 cells, derived from Spodoptera frugiperda (fall armyworm) pupal ovarian tissue, are used for PDCD1LG2 production due to their ability to efficiently express recombinant proteins via the baculovirus expression system. This insect cell system offers several advantages for research applications: it can produce high yields of properly folded mammalian proteins, performs post-translational modifications similar to mammalian systems (though with differences in glycosylation patterns), and accommodates large recombinant proteins. Furthermore, Sf9 cells grow rapidly in suspension cultures at room temperature without requiring CO2 incubation, and the baculovirus system provides strong viral promoters that drive high-level protein expression .

What is the molecular structure of recombinant PDCD1LG2 produced in Sf9 cells?

PDCD1LG2 Human Recombinant produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 423 amino acids (specifically positions 20-200 of the native sequence). It has a calculated molecular mass of 47.7kDa, though it typically appears at approximately 40-57kDa when analyzed by SDS-PAGE due to glycosylation. The recombinant protein is expressed with a 239 amino acid hIgG-His tag at the C-terminus, which facilitates purification and detection. The complete amino acid sequence includes specific structural domains that mediate its interaction with PDCD1 and other binding partners .

How does the glycosylation pattern of PDCD1LG2 produced in Sf9 cells differ from mammalian-expressed protein, and what are the functional implications?

The glycosylation pattern of PDCD1LG2 produced in Sf9 insect cells differs significantly from mammalian-expressed protein, which has important functional implications. Sf9 cells primarily produce paucimannose N-glycans (Man₃GlcNAc₂) and high-mannose glycans, lacking the complex terminal galactosylation and sialylation found in mammalian systems. These differences can affect protein folding, stability, half-life, and most importantly, immune recognition properties.

For PDCD1LG2 specifically, altered glycosylation may modify its binding affinity to PDCD1 and potentially impact downstream signaling pathways. Researchers should consider these differences when using Sf9-produced PDCD1LG2 for binding studies, functional assays, or when developing therapeutic candidates. For certain applications requiring mammalian-like glycosylation, engineered Sf9 cell lines or alternative expression systems might be more appropriate .

What are the optimal strategies for ensuring proper folding and biological activity of PDCD1LG2 when expressed in the Sf9/baculovirus system?

Ensuring proper folding and biological activity of PDCD1LG2 in the Sf9/baculovirus system requires several strategic approaches. First, optimization of the expression construct is critical - incorporating a signal peptide that directs the protein to the secretory pathway can improve folding. The mCBA promoter has been shown to be effective for expressing complex proteins in Sf9 cells while minimizing cytotoxicity, as demonstrated in similar recombinant protein expression systems .

Time of harvest is another critical parameter - extending the expression time too long can lead to protein degradation by baculovirus-encoded proteases. Typically, harvesting at 48-72 hours post-infection provides optimal yield of properly folded protein. Additionally, lowering the temperature to 27°C during expression can slow protein synthesis, allowing more time for proper folding.

For purification, a multi-step approach is recommended: initial capture using affinity chromatography (utilizing the His-tag), followed by polishing steps such as ion exchange and size exclusion chromatography. The inclusion of stabilizing agents such as glycerol (10%) in the final buffer formulation helps maintain protein integrity, as seen in the standard PDCD1LG2 preparation .

How can researchers accurately assess the functionality of recombinant PDCD1LG2 in immunological assays?

Assessing the functionality of recombinant PDCD1LG2 requires a multi-faceted approach targeting its binding properties and biological effects. Primary binding assays should include surface plasmon resonance (SPR) or bio-layer interferometry (BLI) to determine binding kinetics with PDCD1 (PD-1), yielding kon, koff, and KD values that can be compared to literature standards.

Cell-based functional assays are essential for confirming biological activity. These include T-cell proliferation assays where PDCD1LG2 should demonstrate inhibitory effects when PD-1 is expressed. Additionally, assays measuring IL-2 and IFN-γ production by activated T-cells in the presence of PDCD1LG2 can quantify its immunosuppressive function.

Flow cytometry-based binding assays using PD-1-expressing cells can confirm proper protein conformation. An important control is comparing the activity of Sf9-produced PDCD1LG2 with mammalian-expressed protein to account for glycosylation differences. Finally, competitive binding assays using known PD-1/PD-L2 blocking antibodies can verify specificity of the interaction and proper epitope presentation .

What is the optimal protocol for expressing PDCD1LG2 in Sf9 cells using the baculovirus system?

The optimal protocol for expressing PDCD1LG2 in Sf9 cells using the baculovirus system involves several critical steps. Begin by cloning the PDCD1LG2 gene (amino acids 20-200) into a baculovirus transfer vector containing appropriate promoters. The mCBA (modified chicken beta-actin) promoter has shown effectiveness for complex protein expression while minimizing cytotoxicity during protein production .

For bacmid generation, transform the transfer vector into DH10Bac-competent cells and plate on blue-white screening LB agar containing appropriate antibiotics (50 μg/ml kanamycin, 7 μg/ml gentamicin, 10 μg/ml tetracycline, 100 μg/ml Blu-gal, and 40 μg/ml IPTG). After 48 hours incubation at 37°C, pick white colonies for verification by colony PCR. Extract the recombinant bacmid DNA using a high-quality plasmid purification kit .

Transfect Sf9 cells with the purified bacmid DNA using a liposome-based transfection reagent when cells are at approximately 70% confluence. Collect the P1 viral stock after 4-5 days of incubation at 27°C. Amplify this stock by infecting fresh Sf9 cells at MOI = 0.1 to generate high-titer P2 viral stock. Confirm viral titer using plaque assay, ensuring titers >1 × 10^8 PFU/ml .

For protein expression, infect Sf9 cells at MOI = 1-2 when in mid-logarithmic growth phase. Harvest cells 48-72 hours post-infection, depending on expression kinetics. Purify the His-tagged PDCD1LG2 using nickel affinity chromatography followed by additional purification steps to achieve >95% purity .

What purification strategy provides the highest yield and purity of functional PDCD1LG2?

A multi-step purification strategy is essential for obtaining high-yield, high-purity functional PDCD1LG2 from Sf9 cell culture. Begin with clarification of the cell culture supernatant through centrifugation (10,000 × g for 30 minutes) followed by filtration through a 0.45 μm membrane to remove cellular debris.

The initial capture step utilizes immobilized metal affinity chromatography (IMAC) leveraging the C-terminal His-tag on the recombinant PDCD1LG2. Pre-equilibrate a nickel or cobalt resin column with binding buffer (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4), load the clarified supernatant, wash extensively, and elute with an imidazole gradient (up to 500 mM).

Follow with ion exchange chromatography, typically using a Q Sepharose column, to remove contaminants with different charge properties. As a final polishing step, size exclusion chromatography on a Superdex 200 column separates any remaining aggregates or degradation products from the monomeric PDCD1LG2.

Throughout purification, maintain buffers at pH 7.4 and include protease inhibitors to prevent degradation. The final preparation should be formulated in phosphate-buffered saline with 10% glycerol as a stabilizer, achieving >95% purity as verified by SDS-PAGE. This approach typically yields 4-6 mg of purified PDCD1LG2 per liter of Sf9 culture .

How can researchers validate the structural integrity and biological activity of purified PDCD1LG2?

Validating both structural integrity and biological activity of purified PDCD1LG2 requires a comprehensive analytical approach. Begin with SDS-PAGE under reducing and non-reducing conditions to assess purity (should exceed 95%) and evaluate potential disulfide-linked aggregates. Circular dichroism (CD) spectroscopy provides information about secondary structure elements, which should match the expected profile for PDCD1LG2.

Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) determines molecular weight, oligomeric state, and detects potential aggregation. Thermal shift assays (differential scanning fluorimetry) assess protein stability and proper folding through measurement of melting temperature.

For specific binding activity, perform ELISA or surface plasmon resonance (SPR) assays using recombinant PDCD1 (PD-1) as the binding partner. Determine binding kinetics (kon, koff) and calculate the equilibrium dissociation constant (KD), which should be in the nanomolar range for properly folded PDCD1LG2.

Cell-based assays provide the ultimate validation of biological activity. Co-culture PDCD1LG2 with activated T cells expressing PD-1 and measure suppression of T-cell proliferation and cytokine production. Compare results to a reference standard of known activity, such as commercially available PDCD1LG2 produced in mammalian cells. Flow cytometry can also be used to confirm binding to PD-1 expressed on cell surfaces .

How can PDCD1LG2 Human produced in Sf9 cells be used in cancer immunotherapy research?

PDCD1LG2 Human produced in Sf9 cells serves as a valuable tool in cancer immunotherapy research through several experimental applications. First, it can be used to screen and characterize potential therapeutic antibodies targeting the PD-1/PD-L2 pathway. Researchers can perform binding and competition assays to identify antibodies that block the PDCD1LG2/PD-1 interaction, which represents a potential immune checkpoint blockade strategy.

In functional studies, recombinant PDCD1LG2 can be used to investigate the immunosuppressive microenvironment of various tumor types. By incubating tumor-infiltrating lymphocytes with PDCD1LG2, researchers can assess T-cell exhaustion mechanisms and identify cancer types that might respond to PD-1/PD-L2 blockade therapies. This approach helps in understanding why certain tumors respond to checkpoint inhibitors while others are resistant.

Additionally, PDCD1LG2 can be employed in combination therapy studies. Recent research has shown that combining immune checkpoint inhibition with other treatments, such as those inducing immunogenic cell death (like pyroptosis), can enhance anti-tumor effects. For example, studies have demonstrated that rAAV-P2 treatment combined with anti-PD-L1 therapy significantly improved tumor regression compared to either treatment alone .

What are common challenges in working with Sf9-expressed PDCD1LG2 and how can they be addressed?

Researchers working with Sf9-expressed PDCD1LG2 commonly encounter several challenges that require specific solutions. Glycosylation heterogeneity is a primary concern, as Sf9 cells produce paucimannose N-glycans rather than complex mammalian glycans. This can be addressed by enzymatic deglycosylation followed by re-analysis if glycosylation impacts the experimental readout, or by expressing PDCD1LG2 in engineered Sf9 cell lines with humanized glycosylation pathways.

Protein aggregation during storage is another common issue. To mitigate this, include 10% glycerol in storage buffers, maintain protein at concentrations below 1 mg/mL, avoid freeze-thaw cycles by preparing single-use aliquots, and store at -80°C for long-term or at 4°C (no more than 1 week) for short-term use.

Batch-to-batch variability can compromise experimental reproducibility. Implement rigorous quality control protocols including SDS-PAGE, Western blotting, and functional binding assays for each production batch. Maintaining a reference standard from a well-characterized batch allows for comparative analysis.

Insect cell-derived contaminants may co-purify with PDCD1LG2 and interfere with immunological assays. Additional purification steps such as hydroxyapatite chromatography or immunoaffinity chromatography using anti-PDCD1LG2 antibodies can remove these contaminants. Always include appropriate negative controls (such as similarly purified products from non-transformed Sf9 cells) in immunological experiments .

How does the inclusion of the hIgG-His tag affect PDCD1LG2 functionality, and when should tag removal be considered?

The 239 amino acid hIgG-His tag fused to the C-terminus of PDCD1LG2 produced in Sf9 cells has significant implications for protein functionality that researchers must consider. This fusion tag serves dual purposes: the hIgG portion enhances protein solubility and stability during expression, while the His portion facilitates purification via metal affinity chromatography.

Tag removal should be considered for applications requiring native-like protein behavior, particularly when:

• Conducting structural studies (X-ray crystallography, cryo-EM)

• Performing precise binding kinetics measurements

• Developing therapeutic candidates

• Using the protein in in vivo experiments

For tag removal, incorporate a specific protease cleavage site (such as TEV or PreScission) between PDCD1LG2 and the tag. Following initial purification, perform on-column cleavage followed by reverse affinity chromatography to separate the cleaved protein from the tag. Always validate that tag removal does not compromise protein stability, and compare the activity of tagged versus untagged protein in functional assays to determine the impact of the tag on your specific application .

How do Sf9-produced and mammalian cell-produced PDCD1LG2 compare in structural and functional properties?

Sf9-produced and mammalian cell-produced PDCD1LG2 exhibit notable differences in post-translational modifications that influence their structural and functional properties. The primary distinction is in glycosylation patterns: Sf9 cells generate predominantly paucimannose N-glycans (Man₃GlcNAc₂) and high-mannose structures, while mammalian cells produce complex N-glycans with terminal galactose and sialic acid residues. This differential glycosylation affects several properties of the protein.

The mammalian-produced PDCD1LG2 typically displays longer serum half-life due to terminal sialic acids that protect against clearance by asialoglycoprotein receptors. Binding studies reveal that while both proteins bind to PD-1, mammalian-produced PDCD1LG2 often shows 1.5-2.5 fold higher binding affinity in surface plasmon resonance assays, likely due to glycosylation-mediated conformational effects.

What insights can functional studies with recombinant PDCD1LG2 provide about tumor immune evasion mechanisms?

Functional studies with recombinant PDCD1LG2 have provided critical insights into tumor immune evasion mechanisms. Research demonstrates that upregulation of PDCD1LG2 in tumor microenvironments contributes significantly to immunosuppression through multiple pathways. When tumors overexpress PDCD1LG2, the interaction with PD-1 on tumor-infiltrating lymphocytes inhibits TCR-mediated activation, leading to diminished cytokine production, particularly IL-2 and IFN-γ.

Experiments using recombinant PDCD1LG2 in co-culture systems reveal that PD-1/PD-L2 signaling induces T-cell exhaustion, characterized by progressive loss of proliferative capacity and effector functions. This process involves altered metabolic programming of T cells, shifting from glycolysis to oxidative phosphorylation, which impairs their anti-tumor activity.

Interestingly, RNA-seq and single-cell analyses of tumor infiltrating lymphocytes treated with immunotherapies show that disrupting the PD-1/PD-L2 axis increases the expression of chemokines like CCL5, CXCL9, and CXCL10, which promote infiltration of CD4+ and CD8+ T cells into tumors. This explains why combination therapies that induce immunogenic cell death (like pyroptosis) while simultaneously blocking immune checkpoints show synergistic effects, as demonstrated in studies combining rAAV-P2 treatment with anti-PD-L1 therapy .

How can PDCD1LG2 be incorporated into experimental designs investigating combination immunotherapy approaches?

Incorporating PDCD1LG2 into experimental designs investigating combination immunotherapy approaches requires strategic planning across multiple experimental systems. In vitro studies should begin with co-culture systems where tumor cells, antigen-presenting cells, and T cells interact in the presence of recombinant PDCD1LG2, with or without blocking antibodies. This allows for mechanistic understanding of how PD-L2 blockade affects cellular cross-talk within the tumor microenvironment.

For advanced in vitro models, three-dimensional tumor spheroids or organoids incorporating both tumor and immune cells can be treated with combinations of PDCD1LG2 blocking agents and other immunotherapeutic approaches, such as CAR-T cells, cytokine therapies, or agents inducing immunogenic cell death. Flow cytometry and multiplex cytokine analysis can quantify changes in immune activation.

In mouse models, combining anti-PD-L2 antibodies with other treatment modalities has shown promising results. For example, studies demonstrated that rAAV-P2 treatment (which induces pyroptosis in tumor cells) combined with anti-PD-L1 therapy significantly improved tumor regression compared to either treatment alone. This approach altered the tumor microenvironment by increasing lymphocyte infiltration and upregulating chemokine genes including CCL5, CXCL9, and CXCL10.

These experimental designs should also include comprehensive immune profiling through techniques like single-cell RNA sequencing and cytometry by time of flight (CyTOF) to characterize changes in the composition and functional state of tumor-infiltrating immune cells following combination therapy. Such analyses can identify specific immune cell subsets that mediate therapeutic responses and potential resistance mechanisms .