CD200 Human, Sf9

What is human CD200 and what structural characteristics make it important for research?

Human CD200 is a type-1 membrane glycoprotein belonging to the immunoglobulin superfamily with two immunoglobulin domains. The mature protein consists of amino acids 31-232, with a molecular mass of approximately 24.8 kDa in its non-glycosylated form, though native glycosylated CD200 appears at 49-57 kDa on SDS-PAGE .

CD200 functions as an essential immunoregulatory molecule that interacts with its structurally related receptor (CD200R) expressed on immune cells. This interaction:

• Regulates myeloid cell activity

• Delivers inhibitory signals to macrophage lineage cells

• Attenuates inflammatory reactions

• Promotes immune tolerance

• Costimulates T-cell proliferation

The CD200-CD200R axis plays crucial roles in maintaining tissue homeostasis, particularly in immune-privileged sites like hair follicles and the central nervous system, preventing exaggerated immune responses that could lead to tissue damage .

What are the advantages of expressing CD200 in Sf9 cells compared to other expression systems?

The baculovirus-Sf9 expression system offers distinct advantages for CD200 production:

Researchers frequently choose Sf9 cells for CD200 expression when glycosylation is important but mammalian systems are too costly or low-yielding . The baculovirus-Sf9 system represents an excellent compromise for research-grade protein production.

How does CD200 expressed in Sf9 cells differ from the native human protein?

CD200 produced in Sf9 cells exhibits several key differences from native human CD200:

• Glycosylation patterns:

  • Sf9-expressed CD200 contains primarily high-mannose type N-glycans

  • Lacks complex glycans and terminal sialic acids found in human CD200

  • Results in slightly different migration patterns on SDS-PAGE

• Molecular characteristics:

  • When expressed in Sf9 cells, recombinant CD200 is typically fused to tags (His-tag or hIgG-fusion) for purification

  • The molecular mass is approximately 49.5-49.7 kDa for tagged, glycosylated CD200

  • Native CD200 in human tissues exists in membrane-bound form, while recombinant versions may include soluble domains only

• Functional differences:

  • Despite glycosylation differences, Sf9-expressed CD200 retains binding ability to CD200R

  • May show quantitative differences in binding affinity compared to native protein

  • Usually sufficient for most research applications including binding studies and functional assays

What are the optimal conditions for expressing human CD200 in Sf9 cells?

Based on optimization studies with Sf9 cells, the following parameters yield optimal CD200 expression:

For large-scale production, consider these additional factors:

• Addition of Pluronic-F68 (0.1%) to protect cells from shear stress

• Monitoring dissolved oxygen (maintain >30%)

• Supplementing with ZnSO₄ for certain constructs

• Optimizing feed percentage based on cell consumption rates

Post-infection, monitor protein expression via small-scale analysis before harvesting the entire culture to confirm successful production.

What purification strategies work best for CD200 expressed in Sf9 cells?

Effective purification of CD200 from Sf9 culture supernatant typically follows this workflow:

• Initial processing:

  • Harvest supernatant by centrifugation (1500×g, 30 minutes)

  • Clarify by further centrifugation (10,000×g, 10 minutes)

  • Optional filtration through 0.22 μm membrane

• Capture and purification:

  • For His-tagged CD200: Immobilized metal affinity chromatography (IMAC)

  • For Fc-fusion CD200: Protein A/G affinity chromatography

  • Proprietary chromatographic techniques may be employed for specific constructs

• Polishing steps:

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography for higher purity requirements

  • Endotoxin removal if needed for cell-based assays

• Buffer and storage:

  • Formulate in phosphate-buffered saline (pH 7.4) with 10% glycerol

  • For long-term storage, add carrier protein (0.1% HSA or BSA)

  • Aliquot to avoid multiple freeze-thaw cycles

  • Store at -20°C for extended periods

Typical yields range from 5-20 mg/L of culture, with purity >90-95% as determined by SDS-PAGE.

How can researchers validate the functionality of recombinant CD200 from Sf9 cells?

A comprehensive validation approach includes these complementary methods:

• Biochemical characterization:

  • SDS-PAGE to confirm molecular weight and purity

  • Western blot with anti-CD200 antibodies

  • N-terminal sequencing or mass spectrometry to verify protein identity

• Binding assays:

  • ELISA with recombinant CD200R or anti-CD200 antibodies

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Flow cytometry using CD200R-expressing cells

• Functional testing:

  • Inhibition of macrophage activation (measured by cytokine production)

  • Attenuation of inflammatory responses in mixed leukocyte reactions

  • T-cell proliferation assays (CD200 costimulates T-cell proliferation)

• Comparative analysis:

  • Side-by-side comparison with commercial CD200 standards

  • Testing against CD200 from different expression systems (E. coli, mammalian cells)

  • Dose-response studies to establish EC50 values

Functional CD200 should demonstrate specific binding to CD200R and exhibit immunomodulatory effects consistent with its role in regulating myeloid cell activity .

How do glycosylation differences in Sf9-produced CD200 impact its interaction with CD200R?

The glycosylation pattern of Sf9-produced CD200 has specific implications for CD200-CD200R interactions:

The biological significance of these differences depends on the specific research question and experimental system.

What are the challenges in using CD200-CD200R interaction studies for developing immunotherapeutic approaches?

CD200-CD200R pathway modulation presents several research challenges:

Researchers must carefully consider these factors when designing CD200-targeted intervention strategies.

How can CRISPR-Cas9 genome editing be applied to study CD200 function?

CRISPR-Cas9 technology offers powerful approaches for CD200 research:

• Cell line development strategies:

  • Generate CD200 knockout cell lines to study loss-of-function effects

  • Create reporter cell lines with fluorescent tags on CD200 or CD200R

  • Introduce point mutations to disrupt specific CD200-CD200R interactions

• Animal model applications:

  • Generate CD200 knockout mice to study systemic effects

  • Create point mutation animal models that specifically disrupt CD200-CD200R binding

  • Recent studies have used CRISPR-Cas9 to introduce precise mutations in CD200 (F69A) and its receptor that selectively break their interaction

• Mechanistic insights from genome editing:

  • CRISPR-engineered C. elegans with mutations disrupting LAT-1–TOL-1 interaction (functionally similar to CD200-CD200R) showed defects in brood size and embryo viability

  • Similar approaches in mammalian systems can reveal developmental and homeostatic functions

  • Such precise genetic disruptions avoid confounding effects seen in complete gene knockouts

• Technical considerations:

  • Design highly specific sgRNAs targeting CD200 or CD200R genes

  • Validate editing efficiency with T7 endonuclease assays

  • Test multiple sgRNAs to identify optimal targeting sequences

  • Consider knock-in strategies for introducing tagged or mutated versions of CD200

These genome editing approaches provide more definitive insights into CD200 function than traditional antibody blocking or overexpression studies.

What controls should be included when studying CD200-CD200R interactions in experimental systems?

Robust experimental design for CD200 studies requires these controls:

• Protein quality controls:

  • Glycosylation analysis to confirm proper post-translational modification

  • Thermal stability assays to verify proper folding

  • Endotoxin testing to exclude LPS contamination effects

• Binding specificity controls:

  • Isotype-matched control proteins expressed in the same system

  • CD200 pre-blocked with soluble CD200R

  • Competition assays with anti-CD200 antibodies

  • Dose-response studies to establish quantitative relationships

• Cellular assay controls:

  • CD200R-deficient cells to confirm receptor specificity

  • Comparison between different cell types expressing varying levels of CD200R

  • Positive controls using established CD200R ligands or agonistic antibodies

• In vivo experiment controls:

  • Appropriate isotype controls for antibody-based studies

  • Vehicle controls for recombinant protein administration

  • Genetic background controls for knockout or transgenic models

  • Age and sex-matched animals to control for those variables

These controls help distinguish CD200-specific effects from experimental artifacts and ensure reproducible results.

How should researchers interpret apparently contradictory findings about CD200 function in different experimental systems?

When navigating conflicting data about CD200 function:

• Consider context-dependent effects:

  • CD200 function varies across tissues and disease states

  • In cancer, high CD200 expression is associated with immunosuppression

  • In autoimmune conditions, CD200 pathway activation is generally beneficial

  • CD200 deficiency leads to chronic inflammation in the CNS but may enhance anti-tumor immunity

• Evaluate methodological differences:

  • Expression system variations (E. coli vs. Sf9 vs. mammalian cells)

  • Different fusion tags or protein constructs

  • Varied experimental readouts (binding vs. functional assays)

  • In vitro versus in vivo experimental approaches

• Analyze species-specific differences:

  • Human and mouse CD200-CD200R interactions have similar but not identical properties

  • C. elegans LAT-1–TOL-1 interaction provides evolutionary insight but may differ functionally

  • These differences can explain some apparently contradictory findings

• Reconcile contradictions through integrative analysis:

  • Consider relative expression levels of CD200 and CD200R

  • Evaluate the temporal dynamics of CD200-CD200R interaction

  • Assess the contribution of soluble versus membrane-bound CD200

  • Examine the specific cellular context and microenvironment