CD274 Human, Sf9

What is CD274 Human Recombinant protein and what is its role in immune regulation?

CD274, also known as Programmed Death-Ligand 1 (PD-L1), is a critical immune checkpoint molecule that plays a dual role in immune regulation. The recombinant human CD274 produced in Sf9 Baculovirus cells is a single glycosylated polypeptide chain containing 462 amino acids (19-238 a.a.) with a molecular mass of 52.5kDa, though it typically appears at approximately 50-70kDa on SDS-PAGE due to glycosylation . CD274 participates in costimulatory signaling essential for T-cell proliferation and the production of interleukin-10 (IL10) and interferon-gamma (IFNG) through a PDCD1-independent and IL2-dependent pathway . Importantly, when CD274 interacts with PDCD1 (PD-1), it inhibits T-cell proliferation and cytokine production, which represents its immunosuppressive function . This dual functionality makes CD274 a key target in cancer immunotherapy research.

Why is the Sf9 expression system preferred for producing CD274 Human Recombinant protein?

The Sf9 Baculovirus expression system offers several methodological advantages for CD274 Human Recombinant protein production that make it particularly suitable for research applications. This insect cell system enables proper post-translational modifications, especially glycosylation patterns that more closely resemble those of mammalian cells compared to bacterial systems . For CD274 specifically, glycosylation is critical for maintaining proper protein folding and biological activity. The Sf9 system allows for high expression levels of complex proteins while maintaining their native conformation. For experimental procedures requiring CD274 human recombinant protein, researchers should note that proteins produced in this system typically require purification by proprietary chromatographic techniques to achieve >95% purity as determined by SDS-PAGE . When designing experiments, consider that the molecular size on SDS-PAGE will appear at approximately 50-70kDa due to glycosylation, rather than the theoretical molecular weight of 52.5kDa .

How should CD274 Human Recombinant protein from Sf9 cells be stored and handled to maintain optimal activity?

Proper storage and handling of CD274 Human Recombinant protein is essential for maintaining its biological activity in research applications. The protein solution (typically formulated at 1mg/ml) contains Phosphate Buffered Saline (pH 7.4) and 10% glycerol . For short-term storage (2-4 weeks), refrigeration at 4°C is sufficient if the entire vial will be used within that timeframe . For longer-term storage, the protein should be kept frozen at -20°C . To prevent protein degradation during extended storage periods, researchers should add a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) . Multiple freeze-thaw cycles significantly reduce protein activity and should be strictly avoided; therefore, aliquoting the protein solution before freezing is strongly recommended . When designing experiments, researchers should account for potential activity loss during storage and validate protein functionality before critical experiments.

What methodologies should be employed to evaluate the interaction between CD274 Human Recombinant protein and its binding partners in vitro?

When investigating CD274 interactions with binding partners such as PD-1 (PDCD1), researchers should employ multiple complementary methodologies to ensure comprehensive characterization. Surface plasmon resonance (SPR) offers quantitative binding kinetics data, where purified CD274 Human Recombinant protein from Sf9 cells should be immobilized on a sensor chip with its binding partners flowing over in solution. For cellular interaction studies, co-immunoprecipitation assays using anti-His tag antibodies (targeting the 239 amino acid hIgG-His tag at the C-Terminus of the recombinant protein) can pull down CD274 protein complexes . ELISA-based binding assays provide another approach, requiring careful blocking (2% BSA recommended) to prevent non-specific binding. When designing these experiments, researchers should note that the glycosylation status of CD274 from Sf9 cells may differ slightly from native human CD274, potentially affecting binding kinetics. Control experiments comparing binding properties with mammalian cell-derived CD274 may be necessary to validate key findings. Additionally, researchers should consider that CD274 can function in both PDCD1-dependent and PDCD1-independent pathways, necessitating experimental designs that can distinguish between these mechanisms .

What are the optimal conditions for functional assays using CD274 Human Recombinant protein from Sf9 cells?

When designing functional assays with CD274 Human Recombinant protein from Sf9 cells, several critical parameters must be optimized for reliable results. The protein solution, containing PBS (pH 7.4) with 10% glycerol, should be equilibrated to room temperature before use . For T-cell inhibition assays, a dose-response analysis is essential, typically starting with concentrations ranging from 0.1-10 μg/mL of CD274 recombinant protein. The assay medium should be supplemented with 2-5% heat-inactivated FBS rather than serum alternatives to maintain proper protein stability. When assessing CD274's inhibitory function on T-cell activation, plate-bound or bead-coupled anti-CD3/CD28 antibodies provide more consistent T-cell stimulation than soluble antibodies. For analyzing the PDCD1-independent costimulatory effects of CD274, researchers should include IL-2 in the culture medium (typically at 10-20 IU/mL) while blocking PDCD1 with neutralizing antibodies to isolate this specific pathway . Importantly, researchers should validate the functionality of each protein batch using positive controls such as established T-cell lines known to respond to CD274 signaling before proceeding with critical experiments.

How should researchers approach experimental design when investigating CD274 amplification as a biomarker for immunotherapy response?

Investigating CD274 amplification as a potential biomarker for immunotherapy response requires careful experimental design that addresses several methodological considerations. Based on comprehensive analysis of 16,013 cancer samples, CD274 amplification prevalence varies significantly across cancer types, with cervical cancer showing the highest rate (3.26%), followed by head and neck cancers (2.78%) . For lung cancer subtypes, CD274 amplification was identified in 0.77% of adenocarcinomas, 2.77% of squamous cell carcinomas, 1.98% of small cell lung cancers, and 7.89% of large cell lung cancers . When designing biomarker studies, researchers should:

• Implement appropriate sample size calculations based on the expected prevalence in the target cancer type

• Include parallel assessment of established biomarkers (TMB, MSI, PD-L1 expression by IHC) for comprehensive analysis

• Stratify analyses by cancer type and histological subtype

• Control for potential confounding genomic alterations that show higher mutation rates in CD274-amplified samples

What controls and validation methods are necessary when using CD274 Human Recombinant protein in binding and functional studies?

Rigorous controls and validation methods are essential when utilizing CD274 Human Recombinant protein from Sf9 cells in research applications. For binding studies, researchers should implement the following controls: (1) a non-binding control protein with similar size and tags (e.g., irrelevant His-tagged protein produced in Sf9 cells) to account for non-specific interactions; (2) a blocking control using anti-CD274 neutralizing antibodies to confirm binding specificity; and (3) a positive control utilizing known CD274 binding partners such as PDCD1 (PD-1) recombinant protein . For functional assays, validation should include: (1) confirmation of protein activity through assessment of T-cell proliferation inhibition; (2) dose-response experiments to establish EC50/IC50 values; and (3) comparison with mammalian-expressed CD274 to account for potential differences in post-translational modifications. Protein quality should be verified before experiments using SDS-PAGE to confirm the expected molecular weight range (50-70kDa), and Western blotting with anti-CD274 and anti-His antibodies to verify identity . For long-term studies, researchers should implement lot-to-lot consistency testing, as expression in Sf9 cells may introduce batch variability in glycosylation patterns that could affect functional outcomes.

What factors might explain the heterogeneous associations between CD274 amplification and other immunotherapy biomarkers?

The heterogeneous associations between CD274 amplification and established immunotherapy biomarkers reflect the complex molecular landscape of cancer. Analysis across multiple cancer types revealed distinct patterns of association between CD274 amplification and tumor mutational burden (TMB), microsatellite instability (MSI), and PD-L1 expression . Several factors may explain this heterogeneity:

• Cancer-specific genomic landscapes: Different cancer types have unique mutational signatures and chromosomal instability patterns that may influence the co-occurrence of CD274 amplification with other genomic alterations.

• Methodological variations: Different assays for TMB calculation, MSI detection, and PD-L1 expression assessment may contribute to inconsistent associations.

• Biological mechanisms: CD274 amplification may arise through different mechanisms across cancer types – some driven by inflammatory responses and others by genomic instability.

• Sample purity considerations: Tumor sample purity and heterogeneity affect the accurate detection of genomic alterations, potentially obscuring true associations.

• Functional consequences: CD274 amplification may have different functional impacts on immune evasion mechanisms depending on the cellular context and existing immune microenvironment.

Researchers should account for these factors when interpreting correlations between CD274 amplification and other biomarkers, particularly when designing predictive models for immunotherapy response. Cancer-specific analyses rather than pan-cancer approaches may provide more meaningful insights into these complex associations .

How do differences in CD274 expression between tissue samples and circulating immune cells impact biomarker development?

The discordance between CD274 expression in tumor tissue samples versus circulating immune cells presents a significant challenge for comprehensive biomarker development. While tissue-based CD274 assessment focuses primarily on tumor cell expression, studies have demonstrated that CD274 expression on peripheral blood monocytes and dendritic cells (DCs) independently correlates with immunotherapy outcomes . High CD274 expression on blood monocytes and DC subtypes is associated with poor response to PD-1 inhibitor therapy and reduced survival in lung cancer patients . This creates a complex scenario where different cellular compartments may provide complementary or sometimes contradictory predictive information. Methodologically, researchers should consider implementing dual assessment approaches that evaluate both tumor and immune cell CD274 expression. Flow cytometry analysis of peripheral blood monocytes (particularly CD14+HLA-DR++CD16+ intermediate monocytes), CD1c+ myeloid DCs, and CD303+ plasmacytoid DCs provides valuable immune information that complements tissue-based assessments . The inverse correlation observed between CD274 expression on DCs and DC quantities/lymphocyte counts in peripheral blood suggests complex immune regulation mechanisms that should be considered when developing comprehensive biomarker panels . This multi-compartment approach may improve prediction accuracy and provide insights into resistance mechanisms to immune checkpoint inhibition.

What novel applications of CD274 Human Recombinant protein from Sf9 cells could advance immunotherapy research?

CD274 Human Recombinant protein from Sf9 cells offers several innovative applications that could significantly advance immunotherapy research beyond traditional uses. One promising direction involves developing biomaterial-based delivery systems incorporating CD274 protein to create localized immunosuppressive microenvironments for studying T-cell exhaustion mechanisms or testing combination therapies. The recombinant protein could also be utilized to develop protein-based biosensors for real-time monitoring of anti-PD-L1 antibody distribution in preclinical models. Researchers could exploit the costimulatory function of CD274 in PDCD1-independent pathways to develop novel immunomodulatory approaches that selectively enhance beneficial immune responses while limiting autoimmunity . Additionally, the 239 amino acid hIgG-His tag at the C-terminus provides an opportunity to create orientation-controlled protein arrays for high-throughput screening of molecules that modulate CD274's dual functions . Structural biology approaches using the highly purified recombinant protein could identify allosteric regulatory sites distinct from the PDCD1 binding interface, potentially leading to novel small molecule modulators. These applications leverage the high purity (>95%) and defined composition of the Sf9-derived recombinant protein to expand our understanding of CD274 biology and therapeutic targeting strategies.

How might integrated analysis of CD274 amplification with other genomic alterations advance precision immunotherapy?

Integrated analysis of CD274 amplification with co-occurring genomic alterations represents a promising approach to refine patient selection for immunotherapy. The comprehensive genomic profiling of CD274-amplified tumors has revealed distinct mutational signatures across cancer types, with certain genes showing significantly higher mutation rates in CD274-amplified samples compared to wild-type . For example, in lung cancer, 24 genes showed higher mutation rates in CD274-amplified samples, while EGFR mutations were more frequent in CD274 wild-type samples . Future research should focus on:

• Developing integrated predictive models that incorporate CD274 amplification status with mutation profiles of cancer-specific driver genes

• Investigating the functional consequences of co-occurring mutations on the tumor immune microenvironment

• Designing combination therapy approaches that target both CD274-mediated immune evasion and specific co-mutated pathways

• Exploring differential treatment responses based on specific patterns of co-occurring genomic alterations

This integrated approach could substantially improve patient stratification beyond single-biomarker models. The observed cancer-specific patterns of co-mutations in CD274-amplified samples suggest that tailored therapeutic strategies may be required for different cancer types and subtypes . Methodologically, machine learning approaches applied to multi-omic datasets will likely be necessary to fully leverage these complex genomic relationships for clinical decision-making.

What technological advances are needed to better characterize the functional impact of CD274 variants and post-translational modifications?

Advancing our understanding of CD274 variants and post-translational modifications requires several technological innovations. Currently, the Sf9 expression system produces CD274 with glycosylation patterns that approximate but do not perfectly match native human patterns . Development of engineered Sf9 cell lines with humanized glycosylation machinery would provide more physiologically relevant recombinant proteins for functional studies. Single-molecule techniques like atomic force microscopy combined with fluorescence resonance energy transfer (FRET) could elucidate how specific glycosylation patterns affect CD274-PDCD1 binding dynamics at the molecular level. For comprehensive characterization of post-translational modifications (PTMs), advanced mass spectrometry approaches including electron transfer dissociation (ETD) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) should be implemented to map modification sites and their impact on protein conformation. CRISPR-based approaches to introduce specific CD274 variants or modify PTM sites in isogenic cell lines would allow precise assessment of their functional consequences. Additionally, the development of site-specific antibodies recognizing distinct CD274 PTMs would enable better characterization of these modifications in patient samples. These technological advances would collectively improve our understanding of structure-function relationships in CD274 biology and potentially identify novel therapeutic approaches targeting specific variants or modifications.