CD247 Human, Sf9

What is the functional role of CD247 in T cell signaling?

CD247 (CD3 zeta chain) is an essential component of the T cell receptor (TCR) complex involved in signal transduction following antigen recognition. It contains immunoreceptor tyrosine-based activation motifs (ITAMs) that become phosphorylated upon TCR engagement, initiating downstream signaling cascades. Studies in CD247-deficient models demonstrate its critical importance, as deletion of CD247 in the Dahl SS rat resulted in an almost complete loss of circulating CD3+ T-lymphocytes, significantly reducing T cell function . This signaling pathway is critical for T cell activation, proliferation, and effector functions that orchestrate immune responses.

How does CD247 expression correlate with disease states?

CD247 expression shows significant correlation with disease progression in multiple conditions. In idiopathic pulmonary fibrosis (IPF), CD247 is significantly downregulated in patients compared to healthy controls in both blood and lung tissue samples . Lower CD247 expression correlates with reduced lung diffusion capacity (Dlco% predicted) and poorer clinical outcomes, including shorter transplant-free survival time . In hypertension research, CD247 deletion in Dahl SS rats led to significantly lower mean arterial blood pressure (134±1 vs 151±2 mmHg) and reduced renal damage compared to wild-type rats when fed high-salt diets . These findings suggest CD247 potentially serves as a biomarker for disease severity and prognosis.

What experimental models are available for studying CD247 function?

Several experimental systems have been developed to study CD247:

• Genetic knockout models: CD247−/− rat models show almost complete loss of circulating CD3+ T cells with significantly reduced T cell infiltration into tissues like kidneys

• Cell culture systems: Primary human T cells expressing CD247 can be used in co-culture experiments with target cells expressing other relevant proteins

• Recombinant expression systems: The Sf9 insect cell system offers advantages for expressing complex mammalian proteins like CD247 with proper folding and some post-translational modifications

The choice of model depends on whether you're investigating CD247's role in specific disease contexts or pursuing structural and functional characterization of the protein itself.

What are the key considerations when designing expression constructs for human CD247 in Sf9 cells?

When designing baculovirus expression constructs for human CD247 in Sf9 cells, researchers should consider:

• Codon optimization: Adapting human CD247 codons to match Sf9 preferences can significantly improve expression yields

• Signal sequences: Including an appropriate signal sequence (either native or insect-specific) ensures proper trafficking

• Purification tags: Strategic placement of affinity tags (His, FLAG, etc.) that don't interfere with protein folding or function

• Transmembrane domain handling: CD247 contains a transmembrane domain which can complicate expression and purification; consider truncating this domain for soluble protein production or including detergent-compatible purification strategies

• Co-expression options: Evaluating whether co-expression with other CD3 chain components improves stability and native conformation

The choice between full-length or domain-specific constructs should be guided by the specific research questions being addressed.

What purification strategies yield functional CD247 from Sf9 cultures?

A multi-step purification approach is typically necessary:

• Initial capture: Affinity chromatography using tag-based systems (Ni-NTA for His-tagged constructs)

• Intermediate purification: Ion-exchange chromatography to separate charged variants

• Polishing step: Size-exclusion chromatography to remove aggregates and ensure homogeneity

For membrane-associated forms of CD247, detergent selection is critical. Mild detergents like DDM or LMNG often preserve functionality better than harsher alternatives like SDS. Functional integrity can be assessed through binding assays with known interaction partners or antibodies targeting conformational epitopes.

How can researchers assess the functional activity of recombinant CD247?

Methods to evaluate CD247 functionality include:

• Binding assays: Biolayer interferometry or surface plasmon resonance to measure interactions with binding partners

• Phosphorylation assays: Western blotting with phospho-specific antibodies to detect ITAM phosphorylation

• Cell-based functional assays: Co-culture experiments where CD247-expressing T cells are monitored for activation markers (CD25 expression, IFN-γ secretion) when encountering target cells

• Structural integrity verification: Circular dichroism or thermal shift assays to confirm proper folding

Research has demonstrated that functional CD247 can dramatically enhance T cell responses to cancer cells when incorporated into bispecific antibody designs, as seen with the CD27xEGFR construct which increased T cell activation markers and IFN-γ secretion in co-culture experiments .

What role does CD247 play in immunotherapeutic approaches?

CD247 is increasingly incorporated into immunotherapeutic strategies:

• Bispecific antibodies: Constructs like CD27xEGFR leverage CD247 signaling to enhance T cell activation specifically in tumor microenvironments

• Checkpoint modulation: CD247 expression levels may influence responses to checkpoint inhibitor therapies

• Biomarker applications: CD247 expression in peripheral blood may serve as a prognostic indicator for diseases like IPF

The design of CD27xEGFR with its Fc-silent domain demonstrates how researchers can minimize potential toxicity by reducing Fc gamma receptor-mediated binding while maintaining CD247-dependent signaling capabilities . This approach allows targeted activation of T cells primarily in the tumor microenvironment.

How do post-translational modifications of CD247 differ between human cells and Sf9 expression systems?

Key differences in post-translational modifications include:

Researchers must validate whether these differences impact experimental outcomes, particularly for functional studies where post-translational modifications may be critical for activity.

What approaches can resolve contradictory findings about CD247 in different disease models?

Contradictory findings regarding CD247's role in disease pathology can be addressed through:

• Standardized quantification methods: Using consistent protocols for measuring CD247 expression across studies

• Cell type-specific analysis: Evaluating CD247 in specific immune cell subsets rather than bulk tissue or blood samples

• Temporal considerations: Assessing CD247 at multiple disease stages, as expression may change during progression

• Genetic background effects: Considering how different genetic backgrounds might influence CD247 function and expression

Single-cell RNA sequencing approaches have revealed that CD247 is primarily expressed in T cells and NK cells in human lung tissue, with expression patterns changing during disease progression from acute inflammation to fibrotic stages .

What are common challenges in achieving high-yield expression of functional CD247 in Sf9 cells?

Researchers frequently encounter these challenges:

• Low expression levels: Often resolved by optimizing MOI (multiplicity of infection), harvest timing, and culture conditions

• Protein aggregation: Mitigated through buffer optimization and addition of stabilizing agents

• Improper folding: Improved by co-expression with chaperones or lowering expression temperature

• Degradation during purification: Minimized by including protease inhibitors and optimizing purification speed

Empirical optimization is typically necessary, as each recombinant protein presents unique challenges.

How can researchers validate that Sf9-expressed CD247 accurately represents human protein function?

Validation approaches include:

• Comparative binding studies: Side-by-side evaluation of binding properties compared to native human CD247

• Secondary structure analysis: Circular dichroism to confirm similar structural elements

• Functional reconstitution: Testing whether the recombinant protein can restore function in CD247-deficient cell lines

• Phosphorylation pattern analysis: Comparing phosphorylation sites and kinetics after stimulation

A comprehensive validation approach employs multiple complementary methods to build confidence in the recombinant protein's representativeness.