CLEC2B Human, Sf9

What is CLEC2B and what are its main functions?

CLEC2B (C-Type Lectin Domain Family 2 Member B) belongs to the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily. This protein family shares a common protein fold and performs diverse functions including cell adhesion, cell-cell signaling, glycoprotein turnover, and plays significant roles in inflammation and immune response . CLEC2B is also known by several synonyms including Activation-induced C-type lectin (AICL), C-type lectin superfamily member 2 (CLECSF2), and IFN-alpha-2b-inducing-related protein 1 (IFNRG1) .

The CLEC2B gene is closely connected to other CTL/CTLD superfamily members on chromosome 12p13 in the natural killer gene complex region. It is primarily expressed in lymphoid tissues and in most hematopoietic cell types , suggesting critical functions in immune system regulation and inflammatory processes.

What are the structural characteristics of CLEC2B expressed in Sf9 cells?

CLEC2B expressed in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 366 amino acids (residues 26-149 plus tags) with a molecular mass of 41.7 kDa . Due to its glycosylation pattern, the protein typically appears at approximately 40-57 kDa when analyzed by SDS-PAGE . This size variation is characteristic of glycosylated proteins, as glycosylation affects protein mobility during electrophoresis.

When expressed in Sf9 cells, CLEC2B is commonly produced with additional tags to facilitate purification and detection. According to available data, it can be expressed with a 242 amino acid hIgG-His tag at the C-terminus . The amino acid sequence typically includes the core CLEC2B sequence plus these purification tags, resulting in a recombinant protein that maintains the structural domains necessary for functional studies while providing practical advantages for laboratory manipulation.

How does Sf9-expressed CLEC2B differ from E. coli-expressed CLEC2B?

There are several significant differences between CLEC2B expressed in Sf9 cells versus E. coli systems, which researchers should consider when selecting an expression system:

The most significant difference is that Sf9-expressed CLEC2B undergoes post-translational modifications, particularly glycosylation, which may be essential for proper protein folding, stability, and biological activity . The E. coli system produces a smaller, non-glycosylated version that may be suitable for applications where glycosylation is not critical or where higher protein yields are prioritized.

What is the optimal expression system for producing functional CLEC2B?

The optimal expression system for producing functional CLEC2B depends on the specific research requirements. Based on available data, both Sf9 baculovirus and E. coli systems have been successfully used to produce recombinant CLEC2B .

For applications requiring glycosylated CLEC2B that more closely resembles the native protein, the Sf9 baculovirus expression system is preferred . This system provides post-translational modifications, particularly glycosylation, which may be crucial for proper protein folding, stability, and biological activity, especially for proteins involved in immune system functions like CLEC2B.

When selecting an expression system, researchers should consider:

• The intended application (structural studies, functional assays, antibody production)

• The importance of post-translational modifications for the specific study

• Resource constraints (time, cost, equipment)

• Required protein yield and purity

How can Design of Experiments (DoE) principles be applied to optimize CLEC2B expression?

Design of Experiments (DoE) principles can significantly improve CLEC2B expression by systematically identifying optimal conditions. A methodical approach similar to the "Design of Transfections" (DoT) described in recent literature can be adapted for protein expression optimization .

When applying DoE to CLEC2B expression in Sf9 cells, researchers should follow these steps:

• Identify key factors affecting expression: For baculovirus-mediated expression, consider viral multiplicity of infection (MOI), cell density at infection, harvest time post-infection, temperature, and media composition.

• Select an appropriate experimental design:

  • Begin with a two-level full factorial design to screen for significant factors and their interactions

  • Progress to response surface designs like Box-Behnken or Central Composite Design for fine-tuning optimal conditions

• Define measurable response variables such as protein yield, purity, glycosylation pattern, and biological activity.

• Execute experiments according to the design matrix generated by statistical software such as Minitab .

• Analyze results to:

  • Identify the most influential factors

  • Detect interactions between factors

  • Build predictive models of how factors affect CLEC2B expression

  • Generate response surface plots to visualize optimal conditions

• Validate the model by testing the predicted optimal conditions in replicate experiments .

This approach is more efficient than traditional one-factor-at-a-time methods, as it can identify complex interactions between variables that might otherwise be missed . The systematic optimization can lead to significantly improved yields and consistent protein quality.

What purification strategies yield the highest purity of Sf9-expressed CLEC2B?

Purification of Sf9-expressed CLEC2B typically involves several chromatographic steps to achieve high purity while maintaining protein functionality. Based on available information, CLEC2B expressed in Sf9 cells is purified using proprietary chromatographic techniques .

A comprehensive purification strategy for Sf9-expressed CLEC2B might include:

• Initial clarification: Centrifugation and filtration to remove cellular debris

• Capture step: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar resins to capture the His-tagged CLEC2B

• Intermediate purification: Size exclusion chromatography to separate monomeric CLEC2B from aggregates and other contaminants

• Polishing step: Ion exchange chromatography for further purification if needed

• Final formulation: Buffer exchange into a stabilizing formulation such as PBS with 10% glycerol

Throughout purification, samples from each step should be analyzed by SDS-PAGE and activity assays to monitor protein quality. The search results indicate that purities greater than 90% can be achieved for recombinant CLEC2B proteins .

Key considerations for successful purification include:

• Maintaining appropriate temperature (typically 4°C) throughout the process

• Including protease inhibitors to prevent degradation

• Optimizing elution conditions to maximize yield while maintaining purity

• Carefully selecting buffer components compatible with downstream applications

• Minimizing freeze-thaw cycles of the final product

What are the recommended storage conditions for maintaining CLEC2B stability?

Proper storage of CLEC2B is essential for maintaining protein stability and functionality over time. Based on available data, specific recommendations include:

For short-term storage (2-4 weeks):

• Store at 4°C if the entire vial will be used within this timeframe

• Keep the protein in its original formulation buffer

For long-term storage:

• Store frozen at -20°C

• Add a carrier protein (0.1% HSA or BSA) for enhanced stability

• Aliquot the protein solution to avoid multiple freeze-thaw cycles, which can lead to protein degradation

The CLEC2B protein solution is typically formulated with stabilizing agents appropriate to the expression system:

• Sf9-expressed CLEC2B: Phosphate Buffered Saline (pH 7.4) containing 10% glycerol

• E. coli-expressed CLEC2B: 20mM Tris-HCl buffer (pH 8.0), 2M UREA and 10% glycerol

These formulations help maintain protein stability, with glycerol serving as a cryoprotectant and the buffers maintaining optimal pH for protein stability. For researchers working with diluted CLEC2B solutions, adding carrier proteins like BSA can prevent protein adsorption to surfaces and further enhance stability.

What functional assays are most appropriate for characterizing Sf9-expressed CLEC2B?

Comprehensive characterization of Sf9-expressed CLEC2B requires multiple complementary assays to assess various aspects of protein structure and function. Based on CLEC2B's known properties as a C-type lectin involved in immune function, the following assays are recommended:

• Structural integrity assessment:

  • Circular Dichroism (CD) spectroscopy to confirm secondary structure elements

  • Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS) to determine oligomeric state

  • Thermal shift assays to evaluate protein stability under various conditions

• Glycosylation analysis:

  • Mass spectrometry to characterize glycan structures

  • Lectin blotting to detect specific glycan structures

  • Comparison with native CLEC2B glycosylation pattern if available

• Binding assays:

  • Surface Plasmon Resonance (SPR) to measure binding kinetics with potential ligands

  • ELISA-based binding assays with known or suspected interaction partners

  • Cell-based binding assays if CLEC2B interacts with cellular receptors

• Functional activity assays:

  • Assays measuring activation of downstream signaling pathways

  • Cell-based assays evaluating effects on inflammation or immune response

  • Cytokine induction assays, particularly related to IFN-alpha given the protein's alternative name (IFN-alpha-2b-inducing-related protein 1)

When developing these assays, researchers should include appropriate positive and negative controls and validate that the Sf9-expressed CLEC2B behaves similarly to the native protein. Dose-response experiments are particularly valuable for confirming specific biological activities.

How can I verify the proper folding and activity of recombinant CLEC2B?

Verifying proper folding and activity of recombinant CLEC2B involves a multi-faceted approach combining biophysical, biochemical, and functional analyses:

• Biophysical characterization:

  • Circular Dichroism (CD) spectroscopy to confirm secondary structure elements

  • Intrinsic fluorescence spectroscopy to assess tertiary structure

  • Size exclusion chromatography to confirm monomeric state or appropriate oligomerization

  • Limited proteolysis, which can indicate properly folded domains resistant to digestion

• Thermal stability analysis:

  • Differential Scanning Fluorimetry (DSF) or Thermal Shift Assay (TSA)

  • Differential Scanning Calorimetry (DSC)

  • These techniques reveal whether the protein has a cooperative unfolding transition characteristic of well-folded proteins

• Binding functionality:

  • Confirmation of calcium-dependent binding, typical for C-type lectins

  • Binding to known ligands or interaction partners

  • Comparison of binding kinetics with native protein if possible

• Glycosylation assessment:

  • Verification of glycosylation through glycan-specific staining or mass spectrometry

  • Comparison with glycosylation pattern of native CLEC2B

• Biological activity:

  • Cell-based assays demonstrating expected biological responses

  • Activity in signaling pathway activation assays

  • Comparison with positive controls and literature-reported activities

A well-folded and active CLEC2B should demonstrate stability under physiological conditions, exhibit expected binding properties, and elicit appropriate biological responses in functional assays. Any significant deviations might indicate improper folding or post-translational modifications that affect function.

How does post-translational modification in Sf9 cells impact CLEC2B binding properties?

Post-translational modifications, particularly glycosylation, in Sf9 cells can significantly impact CLEC2B binding properties and functionality. Researchers should be aware of these important considerations:

Sf9 cells produce proteins with insect-type glycosylation patterns, which differ from human glycosylation in several key ways:

• Insect cells primarily produce paucimannose N-glycans (Man₃GlcNAc₂)

• They lack the ability to produce complex, sialylated N-glycans typical in human proteins

• They utilize different enzymes for glycan processing

These differences could potentially impact CLEC2B binding properties in several ways:

• If CLEC2B itself depends on specific glycan structures for proper folding or stability

• If CLEC2B-ligand interactions are influenced by glycosylation of either partner

• If glycans on CLEC2B contribute to binding surface properties or solubility

For researchers conducting binding studies with Sf9-expressed CLEC2B, it's advisable to:

• Compare binding properties with CLEC2B expressed in mammalian systems when possible

• Consider enzymatic deglycosylation experiments to assess the role of glycans

• Use complementary approaches like site-directed mutagenesis to identify key binding residues independent of glycosylation

• Validate key findings with differently glycosylated versions of the protein

Understanding these potential impacts is crucial for interpreting binding data and translating in vitro findings to physiological contexts. If precise glycosylation is critical for the research question, researchers might consider alternative expression systems that produce human-like glycosylation patterns.

What are the comparative advantages of using different tags with CLEC2B expression?

The choice of protein tags can significantly impact CLEC2B expression, purification, and functionality. Based on the available information, CLEC2B has been expressed with different tags:

• Sf9-expressed CLEC2B with a 242 amino acid hIgG-His tag at C-Terminus

• E. coli-expressed CLEC2B with a 23 amino acid His-tag at N-terminus

Each tagging strategy offers distinct advantages and potential limitations:

The optimal tag choice depends on the specific research application:

• For structural studies: Smaller tags or cleavable tags are generally preferred

• For interaction studies: Consider tag location relative to binding domains

• For high-throughput expression: His-tags offer efficient purification

• For in vivo studies: Minimal tags or tag removal may be necessary

Researchers should consider validating key findings with differently tagged versions or untagged protein to ensure that observed properties are intrinsic to CLEC2B rather than tag-related artifacts.

How can I troubleshoot low expression yields of CLEC2B in Sf9 cells?

Low expression yields of CLEC2B in Sf9 cells can result from various factors in the baculovirus expression system. A systematic troubleshooting approach, inspired by Design of Experiments methodology , can help identify and resolve issues:

• Viral stock quality issues:

  • Verify viral titer using plaque assays or qPCR

  • Prepare fresh viral stocks from master stocks

  • Ensure proper storage conditions for viral stocks (-80°C, protected from light)

  • Consider preparing a new viral stock if current stock is more than 6-12 months old

• Cell culture conditions:

  • Verify cell viability (>95%) before infection

  • Optimize cell density at infection (typically 1-2×10^6 cells/ml)

  • Check for mycoplasma contamination

  • Ensure proper maintenance of Sf9 cell line (passage number, media quality)

  • Consider using log-phase cells for infection

• Infection parameters:

  • Optimize MOI (typically 1-10 for protein expression)

  • Adjust time of harvest post-infection (typically 48-72 hours)

  • Consider temperature shifts (27-28°C is standard, but reduced temperature sometimes increases yield)

  • Ensure adequate oxygenation during culture

• Construct design issues:

  • Check for rare codons and consider codon optimization for insect cells

  • Verify signal sequence and tag placement

  • Ensure correct reading frame and sequence

  • Consider optimizing the promoter or adding enhancer elements

• Protein stability/toxicity:

  • Check for proteolytic degradation by adding protease inhibitors

  • Consider co-expression of chaperones

  • Test expression of smaller domains if the full-length protein is problematic

  • Analyze expression kinetics to determine optimal harvest time

Implementing a systematic DoE approach similar to the "Design of Transfections" described in the literature can help identify the most significant factors affecting CLEC2B expression and their optimal settings, providing a more efficient path to improved yields than changing one variable at a time.

What are the emerging applications of CLEC2B in immunological research?

CLEC2B, as a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily involved in inflammation and immune response , is being investigated in several emerging areas of immunological research:

• Innate immune recognition: As a C-type lectin expressed in hematopoietic cells, CLEC2B likely plays a role in pattern recognition and innate immune activation pathways, potentially recognizing specific molecular patterns associated with pathogens or cellular stress.

• Cell-cell communication in immune responses: Given its alternative name (Activation-induced C-type lectin), CLEC2B may be involved in the regulation of immune cell activation and intercellular signaling, particularly in lymphoid tissues where it is preferentially expressed .

• Inflammation regulation and resolution: The involvement in inflammation suggests potential roles in diseases with inflammatory components, such as autoimmune disorders, where CLEC2B might serve as a therapeutic target or biomarker.

• Cytokine induction pathways: The synonym IFN-alpha-2b-inducing-related protein 1 (IFNRG1) suggests a role in interferon responses, which are critical for antiviral immunity and immunomodulation.

Researchers using Sf9-expressed CLEC2B can investigate these functions through:

• Identifying binding partners and signaling pathways activated by CLEC2B

• Studying its expression patterns during different immune challenges

• Developing blocking or activating antibodies targeting CLEC2B

• Creating cell and animal models with modified CLEC2B expression or function

These emerging applications highlight the importance of having access to well-characterized recombinant CLEC2B proteins for advancing our understanding of immune system regulation.

How can structural biology techniques be applied to Sf9-expressed CLEC2B?

Structural biology techniques can provide valuable insights into CLEC2B function, mechanism, and potential therapeutic targeting. Sf9-expressed CLEC2B is compatible with several structural biology approaches, each with specific considerations:

A strategic combination of these complementary approaches can provide comprehensive structural information about CLEC2B, enabling researchers to correlate structure with biological functions in inflammation and immune response.