CD226 Human, Sf9

What is CD226 and what are its key structural characteristics?

CD226 belongs to the immunoglobulin superfamily containing 2 Ig-like domains of the V-set. It is a 65kDa glycoprotein expressed on NK cells, platelets, monocytes, and a subset of T cells. When produced in Sf9 cells using baculovirus expression systems, human CD226 is a single glycosylated polypeptide chain containing 471 amino acids (19-247a.a.) with a molecular mass of 53.2kDa, though it typically appears as 50-70kDa on SDS-PAGE due to glycosylation patterns .

The protein structure features immunoglobulin-like domains that facilitate binding interactions with ligands. The Immunoglobulin fold (Ig-fold) represents one of the most common protein folds in the human genome and typically forms a beta sheet sandwich barrel containing 7-9 strands . In CD226, this structural arrangement enables its interactions with binding partners and supports its immune regulatory functions.

What biological functions does CD226 perform in human immune cells?

CD226 serves multiple important functions in the immune system:

• It facilitates cellular adhesion of platelets and megakaryocytic cells to vascular endothelial cells .

• It plays a significant role in megakaryocytic cell maturation .

• CD226 functions as an activating receptor in Natural Killer (NK) cells. It belongs to a receptor family that includes the inhibitory receptor TIGIT, forming a balanced regulatory system for NK cell activity .

• It recognizes ligands from the nectin family of adhesion molecules, particularly nectin-2 and CD155 .

The regulation of NK cell activity involves integration of both activating and inhibitory signals acquired at the immunological synapse with potential target cells. CD226 contributes to this regulation through its interactions with nectin family members, ultimately influencing NK cell cytotoxicity against target cells .

Why is the Sf9-baculovirus expression system preferred for CD226 production?

The Sf9 insect cell-baculovirus expression vector system (IC-BEVS) offers several advantages for producing complex human proteins like CD226:

• Post-translational modifications: The system provides eukaryotic post-translational modifications, particularly glycosylation, which is crucial for CD226's proper folding and function .

• Efficiency: The IC-BEVS has emerged as a time- and cost-efficient production platform compared to mammalian expression systems .

• Scalability: The system allows for relatively high expression levels and can be scaled up for larger production needs.

• Protein quality: Sf9 cells can produce properly folded proteins with native-like structure, maintaining core functional properties of human CD226.

• Established protocols: There are well-established methodologies for expressing proteins in this system, including optimization strategies for enhancing protein yield and quality .

The baculovirus infection process involves a significant takeover of the host cell transcriptome, with an 8-fold increase in viral and transgene expression observed between 24 and 48 hours post-infection, making it an efficient system for recombinant protein production .

How does CD226 interact with nectin family members at the molecular level?

The interaction between CD226 and nectin family members occurs through specific molecular mechanisms:

Understanding these molecular interactions is crucial for developing strategies to modulate immune responses in various disease contexts.

What are the critical quality attributes of CD226 produced in Sf9 cells?

When producing CD226 in Sf9 cells, several critical quality attributes must be monitored:

The formulation of CD226 typically includes Phosphate Buffered Saline (pH 7.4) and 10% glycerol to maintain stability . For long-term storage, it is recommended to store at 4°C if the entire vial will be used within 2-4 weeks, or at -20°C for longer periods, preferably with the addition of a carrier protein (0.1% HSA or BSA) to prevent freeze-thaw degradation .

How can transcriptomic analysis inform optimization of CD226 production in Sf9 cells?

Transcriptomic analysis of Sf9 cells during recombinant protein production provides valuable insights for process optimization:

• Infection kinetics: RNA-seq data shows an 8-fold increase in reads mapping to baculovirus or transgene sequences between 24 and 48 hours post-infection (hpi), confirming the progressive takeover of the host cell transcriptome by baculovirus .

• Differential gene expression: Studies have identified 336 genes differentially expressed at 24 hpi (versus non-infected cells) and 4784 genes at 48 hpi (versus infected cells at 24 hpi), including genes such as dronc, birc5/iap5, and prp1 .

• Biological processes: Functional annotation reveals enrichment of processes crucial for protein production, including:

  • Cell cycle regulation

  • Cell growth pathways

  • Protein folding mechanisms

  • Cellular amino acid metabolic processes

• Optimization targets: These transcriptomic insights identify potential cell engineering targets that could be modified to enhance production, such as:

  • Upregulating chaperones for improved protein folding

  • Modifying metabolic pathways to increase cellular resources for protein synthesis

  • Engineering cell death pathways to extend productive infection periods

By understanding these molecular changes during infection, researchers can rationally design bioprocess engineering strategies to maximize CD226 production yield and quality in the IC-BEVS system .

What is the optimal protocol for expressing CD226 in the Sf9-baculovirus system?

The successful expression of CD226 in Sf9 cells requires attention to several key methodological details:

• Cloning strategy:

  • Replace the native CD226 signal peptide with a mellitin signal sequence to ensure efficient secretion

  • Add a C-terminal tag (e.g., His-tag) to facilitate purification

  • Use appropriate restriction sites (such as BamH1) for insertion into a baculovirus transfer vector (e.g., pVTBac)

• Baculovirus generation:

  • Co-transfect the recombinant plasmid with Baculogold DNA into Sf9 cells

  • Purify recombinant baculoviruses through two rounds of plaque selection

  • Verify expression by Western blotting using an appropriate antibody (e.g., anti-His tag)

• Protein production:

  • Infect Sf9 cells at a multiplicity of infection (MOI) of 4 plaque forming units (pfu) per cell

  • Maintain cells in suspension culture for 48 hours post-infection

  • Harvest by centrifugation at 2000 x g at 4°C for 30 minutes

  • Filter the supernatant through a 0.22 μm membrane

This protocol has been successfully used to express CD96, which shares structural similarities with CD226, suggesting its applicability for CD226 production with appropriate modifications based on protein-specific requirements .

What purification strategies yield the highest quality CD226 from Sf9 expression systems?

A multi-step purification approach is recommended to obtain high-quality CD226 from Sf9 culture supernatants:

• Initial processing:

  • Dialyze filtered supernatant against PBS using a 10 kDa cut-off membrane

  • For His-tagged CD226, add Ni-NTA resin (approximately 2 ml per 1-liter culture) pre-equilibrated with PBS

  • Incubate overnight at 4°C on a rotary shaker

• Chromatography:

  • Transfer the resin to a column and wash with PBS

  • Elute bound protein with an imidazole gradient (10 mM, 25 mM, 50 mM, 250 mM, and 500 mM) in PBS

  • Based on purity assessment, pool the appropriate fractions (typically 100 or 250 mM imidazole)

• Final processing:

  • Dialyze the pooled fractions against PBS to remove imidazole

  • Concentrate using a 10 kDa molecular weight cut-off centrifugation membrane

  • Analyze by SDS-PAGE and Western blotting to confirm purity and identity

• Storage:

  • Store purified CD226 at 4°C for short-term use (2-4 weeks)

  • For long-term storage, add 10% glycerol, aliquot, and store at -20°C

  • Consider adding a carrier protein (0.1% HSA or BSA) to prevent adsorption and freeze-thaw damage

This purification strategy typically yields CD226 with greater than 90% purity as determined by SDS-PAGE, suitable for most research applications .

How can researchers validate the structural integrity and functionality of purified CD226?

Comprehensive validation of CD226 structural integrity and functionality requires multiple complementary approaches:

• Structural analysis:

  • SDS-PAGE to confirm molecular weight (expected 50-70 kDa due to glycosylation)

  • Western blotting with domain-specific antibodies

  • Mass spectrometry to verify protein identity and post-translational modifications

  • Circular dichroism to assess proper folding of immunoglobulin domains

• Binding studies:

  • Evaluate binding to known ligands such as nectin-2 and CD155

  • Compare binding affinity with literature values (typically in the micromolar range)

  • Assess binding specificity using competitive inhibition with antibodies

• Functional assays:

  • Test NK cell activation in response to CD226 engagement

  • Evaluate effects on cellular adhesion

  • Measure downstream signaling pathways

  • Compare activity to mammalian-expressed CD226 when possible

• Stability assessment:

  • Thermal stability studies

  • Assess aggregation propensity

  • Evaluate freeze-thaw stability

  • Determine compatibility with experimental buffers and conditions

These validation steps ensure that the purified CD226 maintains its native structure and function, providing reliable results in downstream applications and experiments.

What are common challenges in CD226 expression and purification, and how can they be addressed?

Researchers may encounter several challenges when working with CD226 in the Sf9 system:

Transcriptomic studies of Sf9 cells during baculovirus infection reveal significant changes in expression of genes involved in protein folding and cellular amino acid metabolism, suggesting that harvest timing critically affects protein quality . Monitoring these cellular responses can help identify optimal production conditions for each batch.

How does glycosylation in Sf9 cells affect CD226 functionality compared to mammalian-expressed protein?

Glycosylation differences between insect and mammalian expression systems have important implications for CD226 functionality:

• Glycosylation patterns:

  • Sf9 cells produce proteins with high-mannose type N-glycans

  • Mammalian cells produce complex, sialylated N-glycans

  • Sf9-expressed CD226 lacks terminal sialylation and galactosylation present in human CD226

• Functional impacts:

  • Core recognition functions are generally preserved

  • Binding kinetics may differ due to glycan influences on protein conformation

  • Half-life in experimental systems may be reduced due to recognition of non-human glycan structures

  • Immunogenicity in in vivo systems may differ from native human CD226

• Experimental considerations:

  • Include mammalian-expressed controls where possible for comparative studies

  • Interpret binding affinity measurements in context of glycosylation differences

  • Consider enzymatic deglycosylation to assess the contribution of glycans to observed functions

  • Be cautious when extrapolating quantitative results to in vivo human systems

While Sf9-expressed CD226 maintains core structural features and binding capabilities, researchers should acknowledge these glycosylation differences when designing experiments and interpreting results, particularly for studies focusing on fine binding kinetics or in vivo applications .

What experimental controls are essential when using CD226 in NK cell functional assays?

When designing NK cell functional assays using CD226, several critical controls must be included:

• Target cell controls:

  • Verify expression levels of CD226 ligands (nectin-2, CD155) on target cells

  • Include ligand-negative cells as controls

  • Consider K562 cells, which are commonly used as susceptible targets for NK cell cytotoxicity assays but require verification of nectin expression

• Receptor specificity controls:

  • Include blocking antibodies against CD226

  • Test related receptors (TIGIT, CD96) to distinguish overlapping functions

  • Use domain-specific mutants to map functional regions

• Signal validation controls:

  • Monitor downstream signaling events (calcium flux, cytokine production)

  • Include positive controls for NK activation (IL-18, IL-21)

  • Be aware that IL-18 can cause increased death of human NK cell lines through Fas-FasL interactions and TNF-α secretion

• System controls:

  • Include buffer-only and irrelevant protein controls

  • Test for endotoxin contamination which could affect NK function

  • Verify NK cell viability throughout the assay period

• Comparative controls:

  • When possible, compare Sf9-expressed CD226 with mammalian-expressed versions

  • Include commercially available CD226 as reference standard

  • Test different batches to ensure reproducibility

These controls ensure that observed effects can be specifically attributed to CD226 function rather than experimental artifacts or confounding factors, particularly important given the complex regulation of NK cell activity through multiple receptor-ligand interactions .

What emerging technologies could enhance CD226 production and characterization in Sf9 systems?

Several emerging technologies offer potential improvements for CD226 production and characterization:

• Cell engineering approaches:

  • CRISPR-Cas9 modification of Sf9 cells to humanize glycosylation pathways

  • Genetic optimization to enhance protein folding machinery

  • Metabolic engineering to increase cell productivity and longevity

  • Integration of feedback-regulated promoters for controlled expression

• Process intensification:

  • Continuous bioprocessing platforms for increased yields

  • Advanced bioreactor designs with real-time monitoring

  • Perfusion culture systems to remove inhibitory byproducts

  • Machine learning algorithms for process parameter optimization based on transcriptomic data

• Analytical advancements:

  • Single-molecule techniques for structure-function relationships

  • Advanced mass spectrometry for comprehensive post-translational modification mapping

  • Cryo-electron microscopy for high-resolution structural determination of CD226-ligand complexes

  • Automated high-throughput functional screening platforms

• Computational approaches:

  • Molecular dynamics simulations to predict impact of insect versus mammalian glycosylation

  • In silico design of expression constructs optimized for Sf9 cells

  • Prediction of critical quality attributes based on process parameters

These technological advances could significantly improve production efficiency, protein quality, and functional characterization of CD226, enabling more detailed studies of its role in immune regulation and potential therapeutic applications.