CD14 Mouse, Sf9

What is the structural characterization of mouse CD14 expressed in Sf9 cells?

Mouse CD14 produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 357 amino acids (specifically amino acids 16-366) with a calculated molecular mass of 38.3kDa. Due to glycosylation, it typically appears larger (40-57kDa) when analyzed by SDS-PAGE . The crystal structure reveals mouse CD14 as a horse-shoe shaped bent solenoid with an N-terminal hydrophobic cavity that provides a putative binding site for lipopolysaccharide (LPS) and other acylated ligands . This structural architecture is evolutionarily conserved, as similar horseshoe-shaped conformations have been observed in CD14 from other species, including human and ovine CD14 . The protein is commonly expressed with a 6 amino acid His tag at the C-Terminus to facilitate purification through chromatographic techniques .

How does Sf9-expressed mouse CD14 compare to native mouse CD14 in terms of functional activity?

Recombinant mouse CD14 (rmCD14) expressed in Sf9 cells retains key biological functions comparable to its native counterpart. Flow cytometric analyses demonstrate that recombinant CD14 maintains strong specific antigen-binding ability to CD14 receptors . Importantly, functional studies show that Sf9-expressed mouse CD14 effectively blocks LPS binding to cell surface receptors, causing significant reductions in positive cell percentage (from 98.37% to 1.35%) and mean fluorescence intensity (from 98.36 to 7.31) .

Both native and recombinant forms of soluble CD14 (sCD14) can induce B cell growth and differentiation at nanomolar concentrations, bypassing the physiological sequence of events that normally limit B cell activation . Additionally, mouse CD14 from Sf9 cells maintains its ability to function as a co-receptor for LPS recognition, evidenced by its capacity to mediate LPS-induced activation of Kupffer cells and other immune cells . These functional similarities make Sf9-expressed mouse CD14 a valuable tool for immunological research.

What methodology is recommended for expressing mouse CD14 in Sf9 insect cells?

Based on established protocols, mouse CD14 expression in Sf9 cells typically involves:

• Vector Construction: Mouse CD14 is often truncated (e.g., at amino acid 344) and linked with a histidine tag into a baculoviral expression vector such as pAcSG2 . This construction requires careful design to ensure proper expression while maintaining functional domains.

• Transfection and Selection: The recombinant baculovirus vector is introduced into Sf9 cells using methods such as the cotransfection method with Baculo-Gold, followed by selection and purification of recombinant clones .

• Cell Culture Conditions: Sf9 cells are typically cultivated in specialized insect cell media such as Excell medium under controlled temperature and pH conditions . The culture environment significantly impacts protein yield and quality.

• Expression Monitoring: Expression levels should be monitored over time to determine optimal harvest time, often using Western blot analysis with anti-CD14 or anti-His tag antibodies.

• Purification Strategy: Purification typically employs affinity chromatography leveraging the His-tag, followed by additional chromatographic steps such as gel filtration to achieve high purity .

Researchers should consider that factors including cell density at infection, multiplicity of infection, harvest timing, and addition of protease inhibitors during purification all significantly impact yield and quality of the final product.

How can the quality and purity of mouse CD14 expressed in Sf9 cells be assessed?

A multi-faceted approach to quality assessment is recommended:

• SDS-PAGE Analysis: Evaluate purity and apparent molecular weight. Mouse CD14 from Sf9 cells typically appears as a band at 40-57kDa due to glycosylation, larger than its calculated mass of 38.3kDa . Gradient gels (10-15%) provide optimal resolution.

• Western Blotting: Confirm identity using specific anti-CD14 antibodies or anti-His antibodies if a His-tag is present. This also helps detect potential degradation products.

• Chromatographic Profile Analysis: Analytical size exclusion chromatography can assess aggregation state and homogeneity. CD14 is typically purified through multiple chromatographic steps, including affinity chromatography using protein G sepharose beads or His-tag affinity resins .

• Functional Assays: Biological activity testing is crucial for confirming proper folding and function:

  • LPS binding assays using fluorescently-labeled LPS

  • Flow cytometry to assess binding to CD14-positive cells

  • Ability to block LPS-induced cytokine production in cellular assays

• Structural Integrity: Circular dichroism spectroscopy can provide information about secondary structure elements, ensuring proper protein folding.

These complementary approaches provide comprehensive quality assessment beyond simple purity determination, ensuring that the recombinant protein maintains its structural and functional characteristics.

What role does glycosylation play in mouse CD14 expressed in Sf9 cells?

Glycosylation significantly influences CD14 structure, function, and cellular processing:

Sf9-expressed mouse CD14 undergoes glycosylation, contributing approximately 2-19kDa to its apparent molecular weight, causing it to migrate at 40-57kDa on SDS-PAGE despite a calculated mass of 38.3kDa . N-linked glycosylation sites can be predicted using bioinformatic tools and confirmed experimentally .

N-linked glycosylation critically determines whether CD14 exists in a membranous or soluble form, thus directly influencing its cellular localization and biological function . This modification affects how CD14 interacts with its binding partners, including LPS and other bacterial components.

The glycosylation pattern in Sf9 cells differs from mammalian cells, characterized by simpler high-mannose type glycans rather than complex mammalian glycans. This distinction is important when interpreting functional studies, as these differences may affect certain aspects of CD14 function, particularly receptor interactions and protein stability.

Researchers should note that altered glycosylation patterns in Sf9-expressed CD14 may influence specific protein-protein interactions relevant to immune signaling pathways, though the core LPS-binding function appears to be preserved despite these differences.

How does the crystal structure of mouse CD14 from Sf9 cells inform our understanding of LPS binding mechanisms?

The crystal structure of mouse CD14 from Sf9 cells has provided critical insights into LPS recognition and binding:

The structure reveals a horseshoe-shaped bent solenoid architecture with a prominent N-terminal hydrophobic pocket that serves as the putative binding site for LPS and other acylated ligands . This pocket is lined with hydrophobic amino acids that can accommodate the lipid A portion of LPS, the component responsible for endotoxic activity.

Structural analysis has enabled identification of specific amino acid residues that participate in LPS binding. For example, flow cytometric analysis demonstrates that recombinant CD14 can dramatically block LPS binding to cells, reducing positive cell percentage by 98.63% (from 98.37% to 1.35%) and mean fluorescence intensity by 92.57% (from 98.36 to 7.31) . This functional data correlates with the structural features observed in the crystal structure.

The three-dimensional arrangement of leucine-rich repeats (LRRs) creates the framework of the binding pocket, with the concave surface forming the primary interaction interface . Computational prediction of these LRRs, combined with crystallographic data, has mapped the spatial organization of these motifs, explaining how CD14 can recognize diverse pathogen-associated molecular patterns beyond just LPS.

The CD14 structure has enabled molecular docking studies predicting interactions with various ligands, including non-bacterial pathogens. For instance, in silico studies with molecular docking revealed that CD14 can bind to structural proteins of the parasitic nematode Haemonchus contortus, specifically alpha and beta tubulin , expanding our understanding of CD14's role beyond bacterial recognition.

What structural and functional differences exist between human and mouse CD14 when expressed in Sf9 cells?

Comparative analysis reveals both similarities and important differences between human and mouse CD14:

Both human and mouse CD14 share the characteristic horseshoe-shaped bent solenoid architecture with an N-terminal hydrophobic cavity that serves as the putative binding site for LPS and other acylated ligands . This structural conservation reflects the evolutionary preservation of this essential innate immune receptor.

The human CD14 crystal structure (PDB ID: 4GLP) was determined using protein expressed in human 293F cells rather than Sf9 cells, allowing comparison with mouse CD14 from Sf9 cells . This cross-species, cross-expression system comparison has identified conserved domains critical for LPS binding while highlighting species-specific differences in peripheral regions that may interact with co-receptors or adapter molecules.

When expressed in Sf9 cells specifically, both human and mouse CD14 may exhibit differences in post-translational modifications compared to their native counterparts, particularly in glycosylation patterns. These differences could affect certain aspects of protein function, especially in complex biological systems where interactions with other cellular components are important.

Functional studies indicate that while both human and mouse CD14 can mediate LPS responses, their sensitivity to LPS and interactions with species-specific co-receptors (like MD-2 and TLR4) may differ, potentially affecting experimental outcomes when using mouse CD14 to model human immune responses.

How can mouse CD14 from Sf9 cells be applied to study host-pathogen interactions in different disease models?

Recombinant mouse CD14 from Sf9 cells offers versatile applications across multiple disease models:

Bacterial Meningitis Models: Mouse CD14 has been instrumental in elucidating the pathogenesis of bacterial meningitis caused by pathogens including Escherichia coli, Streptococcus pneumoniae, and Listeria monocytogenes . Studies demonstrate that soluble CD14 (sCD14) levels dramatically increase in cerebrospinal fluid during infection and that sCD14 acts as an inflammatory co-ligand in vivo, potentially contributing to disease progression. Sf9-expressed CD14 can be used to:

• Block CD14-dependent signaling pathways

• Study the kinetics of sCD14 release during infection

• Investigate the interaction between CD14 and different bacterial components

Parasitic Infection Models: Beyond its traditional role in bacterial recognition, research has revealed CD14's involvement in parasitic immunity. Molecular docking studies complemented by experimental validation showed that CD14 binds to alpha and beta tubulin of Haemonchus contortus . This interaction was confirmed through differential mRNA expression analysis, which revealed enhanced CD14 expression in infected sheep compared to healthy controls. Recombinant CD14 can be used to:

• Map binding interfaces between CD14 and parasitic antigens

• Develop potential therapeutic interventions that disrupt these interactions

• Study species-specific differences in parasite recognition

Sepsis Models: CD14-deficient mice display blunted pro-inflammatory cytokine production and survive challenge with otherwise lethal doses of LPS . This protective effect highlights CD14's central role in inflammatory responses that can lead to septic shock. Sf9-expressed CD14 enables:

• Dose-dependent studies of CD14's contribution to cytokine storms

• Structure-function analysis through site-directed mutagenesis

• Development of CD14-targeting therapeutics

Liver Disease Models: CD14 mediates LPS activation of Kupffer cells (liver macrophages), implicating it in the pathogenesis of alcoholic liver disease and other hepatic conditions . Comparative studies using human and mouse Kupffer cells demonstrate that CD14 blockade significantly diminishes TNF-α production in response to LPS. Recombinant CD14 can facilitate:

• Cross-species comparison of CD14-dependent liver inflammation

• Mechanistic studies of alcohol-induced liver injury

• Therapeutic targeting of Kupffer cell activation

What methodological limitations exist when using Sf9-expressed mouse CD14 in immunological studies?

Several important limitations must be considered when designing experiments with Sf9-expressed mouse CD14:

Glycosylation Differences: Insect cells produce proteins with high-mannose type glycans rather than the complex glycans found in mammalian cells. This difference is particularly significant for CD14, as N-linked glycosylation influences whether it exists in membranous or soluble form . Researchers should validate findings from Sf9-expressed CD14 in mammalian systems when studying glycosylation-dependent functions.

Species-Specific Differences: Phylogenetic analysis indicates that mouse CD14 differs significantly from human CD14, with sheep CD14 showing greater homology to human CD14 . This evolutionary divergence can limit the translational relevance of findings from mouse CD14 studies, particularly when investigating human diseases or developing therapeutics.

Structural Variations: While the core structure of CD14 expressed in Sf9 cells remains intact, subtle structural differences may exist compared to native mouse CD14. These variations could affect binding affinities, protein-protein interactions, or downstream signaling cascades. Crystal structure analysis should be complemented with functional assays to validate biological activity.

Context-Dependent Function: Research demonstrates that anti-CD14 monoclonal antibodies effectively suppress TNF and IL-6 responses to low concentrations of LPS, but this suppression becomes less effective at higher LPS concentrations in the presence of plasma . This indicates that CD14 function is context-dependent and influenced by other plasma factors, a complexity that may not be fully captured in simplified experimental systems using recombinant CD14.

Application Constraints: For studies investigating CD14 in its membrane-bound form or in complex with other cellular components, soluble recombinant CD14 from Sf9 cells may not fully recapitulate the native biology. Membrane-associated functions, lipid raft interactions, and clustering behaviors may differ from the native context.

How do post-translational modifications of mouse CD14 in Sf9 cells affect its application in research?

Post-translational modifications significantly influence CD14 structure, function, and experimental applications:

Glycosylation Effects: Mouse CD14 produced in Sf9 cells undergoes glycosylation, adding approximately 2-19kDa to its apparent molecular weight . Insect cell glycosylation patterns differ from mammalian patterns, featuring high-mannose structures rather than complex glycans. This difference may alter:

• Protein stability and half-life in experimental systems

• Binding kinetics with LPS and other ligands

• Interactions with lectins and other glycan-binding proteins

• Immunogenicity when used in in vivo studies

Disulfide Bond Formation: Disulfide bonds are essential for CD14 folding and stability, ultimately affecting the 3D structure that is biologically active . Sf9 cells may form these bonds differently than mammalian cells, potentially affecting protein conformation. Researchers can use computational prediction tools to analyze disulfide bond patterns and compare them with experimental data to ensure proper folding.

GPI Anchor Considerations: Membrane-bound CD14 typically contains a glycosylphosphatidylinositol (GPI) anchor responsible for membrane attachment . Recombinant soluble CD14 from Sf9 cells lacks this modification, which may affect studies investigating membrane-dependent functions. Applications requiring membrane-bound CD14 may need additional engineering or alternative expression systems.

Leucine-Rich Regions: CD14 contains leucine-rich repeats (LRRs) and leucine zippers essential for pathogen recognition and potential dimerization . While the primary sequence containing these motifs is preserved in recombinant expression, subtle differences in folding or post-translational modification may affect their functional properties. Careful functional validation is required when studying these domains using Sf9-expressed CD14.

Research Application Considerations: When using Sf9-expressed mouse CD14 for specific applications, researchers should consider how post-translational modifications might affect experimental outcomes:

• For structural studies: Compare with native CD14 using techniques like circular dichroism

• For binding studies: Validate with multiple assay formats and include appropriate controls

• For in vivo applications: Consider potential immunogenicity of insect cell glycans

• For therapeutic development: Evaluate whether insect cell modifications affect target binding or pharmacokinetics