CD207 Human, sf9

What is CD207 (Langerin) and what are the advantages of expressing it in sf9 cells?

CD207, also known as Langerin, is a C-type lectin receptor primarily expressed by Langerhans cells and specific dendritic cell subsets. It functions as a pattern recognition receptor with roles in antigen capture, processing, and presentation. The protein contains carbohydrate recognition domains (CRDs) that bind specific glycan structures, particularly those containing mannose residues.

Expression of human CD207 in sf9 insect cells offers several significant advantages for research applications. The sf9 expression system provides high protein yields, proper folding of complex proteins, and the capacity to produce large quantities of recombinant protein. Unlike bacterial expression systems, sf9 cells can perform many post-translational modifications, including glycosylation, although with patterns distinct from mammalian cells. Additionally, the baculovirus expression vector system (BEVS) used with sf9 cells accommodates large gene inserts and can express multimeric protein complexes, making it ideal for structural and functional studies of CD207.

What vectors and constructs are most effective for CD207 expression in sf9 cells?

For optimal expression of human CD207 in sf9 cells, researchers typically utilize baculovirus transfer vectors containing strong promoters such as the polyhedrin (polh) or p10 promoters. Effective constructs often include:

• Full-length CD207 (including transmembrane domain) for cellular localization studies

• Extracellular domain constructs (usually amino acids 68-328) for functional binding studies

• CRD domain only (amino acids ~197-328) for crystallography and binding specificity studies

Addition of affinity tags significantly facilitates downstream purification, with hexahistidine (His6) tags being most common, typically positioned at either the N- or C-terminus. Some studies indicate that N-terminal tags may be preferable as they interfere less with the CRD function. Signal sequences, either native or insect-optimized (such as the honeybee melittin signal sequence), improve secretion and yield.

For optimal expression, researchers should consider codon optimization for sf9 cells, which can increase protein yields by 2-5 fold compared to native human sequences.

How can I verify proper folding and functionality of recombinant CD207 after expression?

Verification of proper folding and functionality for CD207 expressed in sf9 cells should employ multiple complementary approaches:

• Conformational Epitope Recognition: Using conformation-specific monoclonal antibodies in Western blotting or ELISA assays confirms proper folding. Antibodies targeting discontinuous epitopes are particularly informative.

• Calcium-Dependent Carbohydrate Binding: As a C-type lectin, properly folded CD207 exhibits calcium-dependent binding to specific carbohydrates. Solid-phase binding assays using mannose-coated surfaces can verify functionality, with binding abolished upon addition of EDTA (calcium chelator).

• Circular Dichroism (CD) Spectroscopy: This technique provides information about secondary structure content, with properly folded CD207 showing characteristic spectra with α-helix and β-sheet features.

• Thermal Shift Assays: Well-folded CD207 demonstrates a cooperative unfolding transition with a distinct melting temperature (Tm). Addition of calcium typically increases Tm by 3-5°C, indicating proper metal-binding capacity.

• Size Exclusion Chromatography (SEC): SEC profiles can verify the oligomeric state, with properly folded CD207 typically forming trimers in solution.

These complementary approaches provide robust verification of proper protein folding and function before proceeding to more specialized applications.

How do post-translational modifications of CD207 in sf9 cells differ from native human CD207?

CD207 expressed in sf9 cells exhibits important differences in post-translational modifications compared to native human protein, primarily in glycosylation patterns. These differences impact both structural and functional properties of the recombinant protein.

Key differences include:

• N-linked glycosylation: Sf9 cells produce primarily high-mannose and paucimannose structures, lacking complex mammalian-type glycans with sialic acid caps. Human CD207 has two N-glycosylation sites (N57 and N175), which in sf9 cells contain shorter glycan structures.

• O-linked glycosylation: Sf9 cells have limited O-glycosylation capacity compared to human cells, resulting in under-glycosylation of CD207 at potential O-glycosylation sites.

• Phosphorylation: While sf9 cells perform protein phosphorylation, the patterns differ from human cells. Mass spectrometry analysis shows that CD207 expressed in sf9 cells lacks phosphorylation at S89 and S153, which are modified in native protein.

These differences affect protein properties in several ways:

• Altered molecular weight and electrophoretic mobility (typically 2-4 kDa lower than mammalian-expressed CD207)

• Reduced stability in solution (approximately 15-20% reduction in half-life at 37°C)

• Modified binding affinity to certain glycan structures (particularly those containing sialic acid)

• Different immunogenicity profile in functional assays

Researchers can address these differences through glycoengineering approaches, including co-expression with mammalian glycosyltransferases or using modified sf9 cell lines with humanized glycosylation machinery.

What strategies can resolve conflicting binding data between CD207 expressed in different systems?

Conflicting binding data between CD207 expressed in sf9 versus mammalian systems is a common challenge. A systematic troubleshooting approach includes:

• Expression System Comparison Study: Generate parallel preparations from sf9, mammalian (HEK293, CHO), and if possible, native sources. Analyze binding properties using identical assay conditions and multiple binding assay formats.

• Post-Translational Modification Analysis: Perform detailed glycomic and proteomic profiling to identify specific modifications present in each preparation. Mass spectrometry methods such as LC-MS/MS with electron-transfer dissociation can map differences with amino acid resolution.

• Binding Assay Validation: Determine if binding discrepancies are assay-dependent by employing multiple orthogonal techniques:

  • Surface plasmon resonance (SPR)

  • Isothermal titration calorimetry (ITC)

  • Glycan array screening

  • Cell-based binding assays

• Metal Ion Dependency: C-type lectins like CD207 require calcium for activity. Evaluate whether binding differences are related to metal ion concentration or chelation effects in different buffer systems.

• Oligomerization State Assessment: CD207 functions as a trimer. Analytical ultracentrifugation or size exclusion chromatography with multi-angle light scattering (SEC-MALS) can determine if oligomerization differences between expression systems contribute to binding variations.

When binding differences persist despite these approaches, researchers should consider reporting data from multiple expression systems with clear description of the limitations of each preparation, providing a more comprehensive understanding of CD207's binding properties.

How can I design CD207 variants with modified carbohydrate recognition domains while maintaining protein stability?

Designing CD207 variants with modified carbohydrate recognition domains requires balancing changes to binding specificity while preserving structural integrity. A systematic approach includes:

When expressing these variants in sf9 cells, it's critical to validate proper folding using the methods described in section 1.3. Expression temperature reduction (to 27°C instead of standard 28-30°C) often improves folding of challenging CD207 variants.

What are the optimal viral titers and infection protocols for maximizing CD207 yield in sf9 cells?

Maximizing CD207 yield in sf9 cells requires optimization of multiple infection parameters. Based on systematic studies, the following protocol yields optimal results:

• Cell Density at Infection: Infect sf9 cells at 1.5-2.0 × 10^6 cells/mL, which balances cell density with nutrient availability. Higher densities reduce per-cell yield due to nutrient limitation.

• Multiplicity of Infection (MOI): For CD207 expression, low MOI strategies (0.5-2 PFU/cell) generally outperform high MOI approaches. This reduces cellular stress and extends the productive infection period.

• Time of Harvest: Optimal harvest time for CD207 is typically 72-96 hours post-infection, significantly longer than for many other proteins. Extended expression time allows for proper folding and trimerization.

• Temperature Modulation: Reducing temperature to 27°C at 24 hours post-infection extends cell viability and improves protein quality, particularly for complex proteins like CD207 that require extensive folding.

• Media Supplementation: Addition of 0.1% Pluronic F-68 and 1% fetal bovine serum at the time of infection improves cell viability and increases CD207 yield by approximately 30%.

The table below summarizes the impact of different parameters on CD207 yield:

What purification methods work best for CD207 expressed in sf9 cells?

Purification of CD207 from sf9 expression systems requires a multi-step approach to address specific challenges, including proper trimer formation and separation from insect cell contaminants. The following optimized protocol yields high-purity, functional protein:

• Initial Clarification: Harvest supernatant (for secreted constructs) or lyse cells (for intracellular constructs) and clarify by centrifugation at 20,000 × g for 30 minutes, followed by 0.45 μm filtration.

• Immobilized Metal Affinity Chromatography (IMAC): For His-tagged CD207, use Ni-NTA resin with a shallow imidazole gradient (20-250 mM) in buffer containing 2 mM CaCl₂. The presence of calcium helps maintain protein structure during purification.

• Calcium-Dependent Lectin Affinity Chromatography: This orthogonal method exploits CD207's natural function. Use mannose-sepharose columns with calcium-containing binding buffer and EDTA-containing elution buffer. This step effectively separates functional from non-functional protein.

• Size Exclusion Chromatography (SEC): Critical for separating CD207 trimers from aggregates and monomers. Use Superdex 200 column in buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 2 mM CaCl₂.

• Endotoxin Removal: For cell-based applications, additional endotoxin removal using Triton X-114 phase separation or specialized endotoxin removal columns is recommended. High-quality CD207 preparations should contain <0.1 EU/mg protein.

The table below compares recovery and purity across purification steps:

The final purified CD207 should be stored in buffer containing 2 mM CaCl₂ to maintain stability, with glycerol (10%) added for freezing. Aliquots stored at -80°C remain stable for at least 6 months with minimal loss of activity.

What analytical techniques best characterize the structural integrity of purified CD207?

Comprehensive characterization of CD207 structural integrity requires multiple complementary analytical techniques that address primary, secondary, tertiary, and quaternary structure:

• Mass Spectrometry (MS):

  • Intact protein MS verifies molecular weight and detects potential truncations or modifications

  • Peptide mapping with LC-MS/MS confirms sequence coverage (>95% coverage recommended)

  • Glycopeptide analysis characterizes site-specific glycosylation patterns

• Spectroscopic Methods:

  • Circular dichroism (CD) spectroscopy quantifies secondary structure content

  • Fluorescence spectroscopy monitors tertiary structure through intrinsic tryptophan fluorescence

  • Fourier-transform infrared spectroscopy (FTIR) provides complementary secondary structure information

• Hydrodynamic Methods:

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) determines absolute molecular weight and oligomeric state

  • Analytical ultracentrifugation (AUC) provides detailed characterization of oligomerization equilibria

  • Dynamic light scattering (DLS) monitors size distribution and potential aggregation

• Thermal Stability Assessment:

  • Differential scanning calorimetry (DSC) measures thermal transitions and domain stability

  • Thermal shift assays with environmentally sensitive dyes track unfolding transitions

  • Comparison of Tm values in presence/absence of calcium (should increase by 3-5°C with calcium)

• Functional Characterization:

  • Surface plasmon resonance (SPR) measures binding kinetics to known ligands

  • Isothermal titration calorimetry (ITC) determines thermodynamic binding parameters

  • Glycan array screening evaluates binding specificity across diverse carbohydrate structures

The table below summarizes key structural parameters for properly folded CD207:

Deviations from these parameters may indicate structural issues that could affect experimental outcomes. Maintaining calcium in all buffers (2 mM CaCl₂) is critical for preserving CD207 structural integrity during analysis.

How can I design experiments to investigate CD207's role in antigen presentation using proteins expressed in sf9 cells?

Investigating CD207's role in antigen presentation requires carefully designed experiments that bridge biochemical properties with cellular functions. The following experimental approaches utilize sf9-expressed CD207 effectively:

• Antigen-CD207 Conjugate Preparation:

  • Chemically couple purified CD207 to model antigens (e.g., ovalbumin, fluorescent proteins) using heterobifunctional crosslinkers

  • Verify conjugate formation by SDS-PAGE, Western blotting, and size exclusion chromatography

  • Confirm maintenance of binding activity using mannose-affinity chromatography

• Cell-Based Trafficking Studies:

  • Fluorescently label CD207-antigen conjugates and track internalization in dendritic cells

  • Use confocal microscopy with organelle markers to determine subcellular localization at different time points

  • Compare trafficking patterns of wild-type CD207 versus binding-deficient mutants (e.g., E285Q)

• Antigen Processing Assessment:

  • Deliver CD207-antigen complexes to dendritic cells and measure antigen degradation using antibodies specific to intact versus processed epitopes

  • Monitor co-localization with proteolytic compartments using fluorescent reporters of protease activity

  • Compare processing efficiency between CD207-targeted and non-targeted antigen delivery

• T-Cell Activation Assays:

  • Load dendritic cells with CD207-targeted antigens and measure subsequent T-cell activation using:

    • T-cell proliferation assays (CFSE dilution)

    • Cytokine production (ELISA, intracellular cytokine staining)

    • Expression of activation markers (CD69, CD25, CD71)

  • Compare responses between different targeting strategies and against soluble antigen controls

• In Vivo Targeting Studies:

  • Administer fluorescently labeled CD207-antigen conjugates in vivo and track targeting to Langerhans cells and CD207+ dendritic cells

  • Assess subsequent immune responses by measuring antigen-specific T-cell populations

  • Compare responses between wild-type and CD207 knockout models to confirm specificity

For these experiments, it's essential to include appropriate controls:

• Binding-deficient CD207 mutants (E285Q, N287D) to confirm lectin-dependent effects

• Calcium chelation controls (EGTA treatment) to verify C-type lectin activity

• Comparison with other targeting molecules (e.g., DEC-205, DC-SIGN) to establish CD207-specific effects

What approaches can effectively study CD207-glycan interactions with recombinant proteins?

Studying CD207-glycan interactions requires a multi-faceted approach that combines glycan array screening, biophysical binding studies, and structural analysis. The following methodological framework provides comprehensive characterization:

• Glycan Array Screening:

  • Employ mammalian and pathogen-focused glycan arrays (250-600 structures) to identify binding candidates

  • Use fluorescently labeled CD207 at multiple concentrations (1-50 μg/mL)

  • Include calcium-dependent controls (± EDTA) to confirm C-type lectin activity

  • Compare wild-type CD207 with point mutants affecting the carbohydrate recognition domain

• Solution-Phase Binding Characterization:

  • Isothermal titration calorimetry (ITC) provides thermodynamic parameters (ΔH, ΔS, KD)

  • Surface plasmon resonance (SPR) yields kinetic constants (kon, koff) and affinity values

  • Microscale thermophoresis (MST) offers an alternative for challenging glycan interactions

  • Perform measurements across different buffer conditions (pH, ionic strength) to identify optimal binding environments

• Structure-Function Studies:

  • X-ray crystallography of CD207-glycan complexes reveals atomic-level binding details

  • NMR spectroscopy with isotopically labeled CD207 identifies binding interfaces through chemical shift perturbations

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) maps conformational changes upon glycan binding

• Competitive Binding Assays:

  • Develop microplate-based competition assays using immobilized reference ligands

  • Determine IC50 values for different glycan structures to establish relative affinities

  • Create comprehensive glycan binding profiles across structural families

The table below summarizes binding parameters for key glycan structures:

For more complex glycans or those available in limited quantities, frontal affinity chromatography coupled with mass spectrometry (FAC-MS) offers an alternative approach requiring minimal material while providing reliable affinity constants.

What strategies can overcome low expression or aggregation of CD207 in sf9 cells?

Low expression yields or aggregation of CD207 in sf9 cells represents a common challenge that can be addressed through systematic optimization:

• Construct Optimization:

  • Truncation series: Test multiple constructs with different domain boundaries to identify optimal soluble fragments

  • Signal sequence optimization: Compare native signal sequence with insect-optimized sequences (e.g., honeybee melittin, gp67)

  • Codon optimization: Implement sf9-optimized codons, particularly avoiding rare codons in the N-terminal region

• Expression Condition Modifications:

  • Temperature reduction: Shift to 27°C post-infection to slow protein production and improve folding

  • Cell density adjustment: Lower initial density to 1.2-1.5×10⁶ cells/mL to reduce metabolic stress

  • Feeding strategy: Implement fed-batch approach with glucose and yeastolate supplementation

  • Harvest timing: Extend expression period to 96-120 hours with monitoring for optimal yield/quality balance

• Fusion Partners and Solubility Enhancers:

  • Thioredoxin (TrxA) fusion: Particularly effective for improving CD207 CRD domain solubility

  • Maltose-binding protein (MBP): Enhances solubility while providing additional purification option

  • SUMO tag: Improves folding and can be removed with specific proteases

  • Addition of 5-10% glycerol and 0.1% Pluronic F-68 to culture media reduces aggregation

• Co-expression Strategies:

  • Molecular chaperones: Co-express with insect cell chaperones (e.g., Hsp70, calreticulin)

  • Folding enzymes: Co-express with protein disulfide isomerase (PDI) to facilitate disulfide bond formation

  • Glycosylation enhancers: Co-express with glycosyltransferases to improve glycosylation efficiency

• Aggregation Prevention During Purification:

  • Buffer optimization: Include 2 mM CaCl₂ in all buffers to stabilize the CRD domain

  • Detergent screening: Low concentrations (0.01-0.05%) of non-ionic detergents (Tween-20, CHAPS) reduce aggregation

  • Arginine supplementation: Addition of 50-100 mM arginine to buffers significantly improves stability

  • Stabilizing additives: Trehalose (5-10%) or sucrose (5-10%) enhance stability during storage

The table below summarizes improvement strategies and their typical impact:

Implementation of these strategies in combination can transform a poorly expressing CD207 construct into one with high yields of properly folded protein suitable for downstream applications.