RAET1E Human, Sf9

What is RAET1E and why is it expressed in Sf9 cells?

RAET1E is a member of the MHC class I family located on chromosome 6q24.2-q25.3. It functions as a ligand for the NKG2D receptor expressed on various immune cells, playing crucial roles in innate and adaptive immune responses. RAET1E is involved in tumor immune surveillance by inducing the growth of anti-tumor cytotoxic lymphocytes .

When produced in Sf9 insect cells, RAET1E is typically expressed as a single, glycosylated polypeptide chain containing 204 amino acids (positions 31-225) with a molecular mass of 23.4kDa, though it appears at approximately 28-40kDa on SDS-PAGE due to glycosylation . The expression methodology employs baculovirus infection of Sf9 cells, which provides several advantages:

• High expression yields compared to mammalian systems

• Proper protein folding with essential post-translational modifications

• Cost-effective and scalable production process

• Ability to express proteins that may be toxic to mammalian cells

Sf9 cells have demonstrated successful expression of various membrane-associated and secreted proteins, making them suitable for RAET1E production for structural and functional studies .

How is RAET1E protein purified from Sf9 cell cultures?

Purification of RAET1E from Sf9 cell cultures follows a systematic approach tailored to tagged recombinant proteins. Based on the provided information, RAET1E is typically expressed with a 6-amino acid His tag at the C-terminus, which facilitates downstream purification .

The standard purification protocol involves:

• Harvesting infected Sf9 cells 48-72 hours post-infection with recombinant baculovirus

• Cell lysis using appropriate buffer systems containing mild detergents

• Clarification of lysate by centrifugation to remove cell debris

• Immobilized metal affinity chromatography (IMAC) using nickel or cobalt columns that bind the His-tagged protein

• Extensive washing to remove non-specifically bound proteins

• Elution of RAET1E using imidazole gradient or step elution

• Additional chromatographic steps (ion exchange, size exclusion) for further purification if needed

The purified RAET1E protein is typically formulated in Phosphate Buffered Saline (pH 7.4) with 10% glycerol as a stabilizing agent . The final purity of the protein should exceed 90% as determined by SDS-PAGE analysis . For immunological studies, confirmation of proper folding and biological activity through binding assays with NKG2D receptor is recommended.

What are the key structural differences between native human RAET1E and RAET1E expressed in Sf9 cells?

While RAET1E expressed in Sf9 cells maintains the same primary amino acid sequence as native human RAET1E, several structural differences exist due to the distinct cellular machinery for protein processing:

Glycosylation patterns:

• Native human RAET1E contains complex N-linked glycans with terminal sialic acid residues typical of mammalian cells

• Sf9-expressed RAET1E displays high-mannose or paucimannose N-glycans that lack sialic acid and have simpler structures

Membrane anchoring:

• Native RAET1E possesses a type I membrane-spanning sequence at its C-terminus, distinguishing it from other RAET1 proteins that have glycosylphosphatidylinositol (GPI) anchors

• Sf9-expressed RAET1E is typically engineered as a soluble protein with the transmembrane domain replaced by a His-tag for purification purposes

Protein mobility:
Based on studies with other proteins expressed in Sf9 cells, RAET1E might display different membrane mobility characteristics compared to mammalian expression systems. For example, LBR 1-238GFP showed high mobility in both ER and nuclear membranes in Sf9 cells but limited mobility at the nuclear rim in mammalian cells . This suggests different protein-protein interactions or membrane organization between the systems.

These structural differences should be considered when interpreting experimental results, particularly for immunological and binding studies where glycosylation and protein conformation may influence receptor interactions.

What post-translational modifications occur in RAET1E when expressed in Sf9 cells?

RAET1E undergoes several post-translational modifications when expressed in Sf9 cells, though these differ somewhat from those in mammalian systems:

N-linked glycosylation:
RAET1E is described as a glycosylated polypeptide in Sf9 cells . The glycans added by insect cells are typically high-mannose type rather than the complex glycans found in mammals. This accounts for the difference between the theoretical molecular weight (23.4 kDa) and the observed size on SDS-PAGE (28-40 kDa) .

Disulfide bond formation:
The RAET1E amino acid sequence contains cysteine residues that likely form disulfide bonds critical for proper protein folding and stability . Sf9 cells possess the necessary oxidizing environment in their secretory pathway to support disulfide bond formation.

Signal peptide processing:
The mature RAET1E protein expressed in Sf9 cells contains amino acids 31-225 , indicating that the signal peptide (amino acids 1-30) is properly cleaved during processing.

C-terminal modification:
When expressed with a C-terminal His-tag, the RAET1E sequence terminates with six histidine residues (HHHHHH) as shown in the amino acid sequence provided: "ADPHSLCFNF TIKSLSRPGQ PWCEAQVFLN KNLFLQYNSD NNMVKPLGLL GKKVYATSTW GELTQTLGEV GRDLRMLLCD IKPQIKTSDP STLQVEMFCQ REAERCTGAS WQFATNGEKS LLFDAMNMTW TVINHEASKI KETWKKDRGL EKYFRKLSKG DCDHWLREFL GHWEAMPEPT VSPVNASDIH WSSSSLPDHH HHHH" .

Researchers should be aware that while Sf9 cells perform many post-translational modifications essential for RAET1E functionality, they lack some mammalian-specific modification capabilities. For critical applications requiring mammalian-like modifications, validation against mammalian-expressed proteins may be necessary.

How stable is RAET1E protein produced in Sf9 cells during storage and handling?

The stability of RAET1E protein produced in Sf9 cells is an important consideration for experimental planning. According to available information, several factors affect stability:

Storage conditions:

• RAET1E from Sf9 cells is typically formulated in Phosphate Buffered Saline (pH 7.4) with 10% glycerol as a stabilizing agent

• For long-term storage, addition of carrier proteins (0.1% HSA or BSA) is specifically recommended

• Multiple freeze-thaw cycles should be avoided to maintain protein integrity

Temperature considerations:
The precise stability profile for RAET1E is not detailed in the search results, but based on similar recombinant proteins:

• Short-term storage (days to weeks): 4°C with appropriate preservatives

• Medium-term storage (months): -20°C in aliquots to avoid freeze-thaw cycles

• Long-term storage (>1 year): -80°C with carrier proteins added

Stability assessment methods:
For researchers working with RAET1E, stability can be monitored using:

• Size-exclusion chromatography to detect aggregation

• SDS-PAGE to assess degradation

• Functional binding assays with NKG2D receptor to confirm biological activity

• Circular dichroism (CD) spectroscopy to monitor secondary structure changes

Handling recommendations:

• Work on ice when preparing experiments

• Use freshly thawed aliquots when possible

• Include protease inhibitors during purification and handling

• Validate protein activity before critical experiments

The purity of the RAET1E preparation (greater than 90% as determined by SDS-PAGE ) will also impact stability, as contaminants like proteases can reduce shelf life. Proper handling and storage conditions as outlined above will help ensure consistent experimental results when working with Sf9-expressed RAET1E protein.

How do E2F transcription factors regulate RAET1E expression in different cellular contexts?

E2F transcription factors play a critical role in regulating RAET1E expression, establishing a direct link between cell proliferation and immune surveillance mechanisms. Research has demonstrated that E2F transcription factors directly activate transcription of Raet1 family genes, including Raet1e .

The molecular mechanism involves:

• Direct binding of E2F transcription factors to specific motifs in the RAET1E promoter region

• Transcriptional activation coupled with cell cycle progression

• Coordination with post-transcriptional regulatory mechanisms

Nuclear run-on transcription assays have shown that proliferating fibroblasts exhibit substantially higher Raet1e transcription compared to serum-starved (quiescent) fibroblasts . This indicates that transcriptional activation is a primary regulatory mechanism linking cell proliferation status to RAET1E expression.

Different cellular contexts demonstrate varied patterns of E2F-dependent RAET1E regulation:

Cancer cells:

• Deregulated E2F activity contributes to constitutive RAET1E expression

• This enhanced expression makes cancer cells more visible to the immune system through NKG2D recognition

• Expression levels may correlate with proliferation rates and malignancy

Normal proliferating cells:

• Controlled E2F activity induces transient RAET1E expression

• This expression appears to play roles in normal immune communication during tissue growth and healing

• Evidence shows that wound healing was delayed in mice lacking NKG2D, suggesting functional relevance of this pathway

These findings raise important questions about how the immune system distinguishes between normal proliferation (as in wound healing) and pathological proliferation (as in cancer) despite both involving RAET1E expression. The answer likely involves differences in expression intensity, duration, and coordination with other stress signals that collectively determine immune response thresholds.

What experimental approaches can differentiate between RAET1D and RAET1E expression in pathological conditions?

Differentiating between RAET1D and RAET1E expression in pathological conditions requires precise experimental approaches due to their sequence similarity. Research on experimental autoimmune encephalomyelitis (EAE) provides a methodological framework applicable to other conditions .

Gene expression differentiation methods:

• Quantitative RT-PCR with isoform-specific primers:

  • Design primers targeting unique regions of RAET1D vs. RAET1E mRNA

  • This approach revealed that microglia express only Raet1d while macrophages express both Raet1d and Raet1e in EAE models

  • Requires rigorous validation with recombinant standards to ensure specificity

• In situ hybridization with isoform-specific probes:

  • Allows visualization of specific transcripts in tissue sections

  • Can be combined with immunofluorescence for cell type identification

  • Enables spatial analysis of expression patterns within lesions

• Cell sorting followed by expression analysis:

  • FACS isolation of specific cell populations (e.g., microglia vs. macrophages)

  • Subsequent analysis of RAET1D/RAET1E expression by qRT-PCR

  • This methodology revealed cell type-specific expression patterns in EAE

Regulation and induction patterns:

Research has shown that in the context of EAE:

• Raet1d and Raet1e genes are induced early upon disease onset

• They reach maximal expression at the peak of pathology

• Myeloid cells (macrophages and microglia) are major cellular sources of Raet1 transcripts

• Interestingly, only Raet1d expression is induced in microglia, whereas macrophages express both Raet1d and Raet1e

• M-CSF (macrophage colony-stimulating factor) was identified as a major factor controlling RAE-1 expression in microglia

These findings highlight the importance of cell type-specific analysis when studying RAET1 family members in pathological conditions. The differential expression and regulation of RAET1D and RAET1E suggest distinct roles in disease processes, which may inform therapeutic approaches targeting these pathways.

How can cross-linking studies with RAET1E in Sf9 cells reveal its interaction partners?

Cross-linking studies with RAET1E expressed in Sf9 cells provide a powerful approach to identify and characterize its interaction partners under controlled conditions. Based on methodologies described for similar proteins, a systematic approach can be developed .

Methodological framework for RAET1E cross-linking studies:

• Expression system preparation:

  • Generate recombinant baculoviruses encoding RAET1E with appropriate tags (His-tag, T7-tag)

  • Prepare Sf9 microsomal membranes loaded with RAET1E

  • Express potential interaction partners in the same system or add purified candidates

• Cross-linking reagent selection:

  • BS3 (Bis(sulfosuccinimidyl)suberate) with 11.4-Å spacer arm is suitable for many applications

  • Selection should be based on the amino acid composition at potential interaction interfaces

  • Lysine-specific reagents are commonly used as RAET1E contains multiple lysine residues

• Cross-linking reaction protocol:

  • After translation/expression, isolate membrane fractions by centrifugation

  • Resuspend membrane pellet in appropriate cross-linking buffer

  • Split samples into control and experimental groups

  • Add cross-linking reagent at optimized concentration

  • Quench reaction and solubilize complexes

• Complex isolation and analysis:

  • Purify cross-linked complexes via affinity chromatography (Talon purification for His-tagged proteins)

  • Analyze by SDS-PAGE, Western blotting, and mass spectrometry

  • Perform immunoprecipitation with antibodies against suspected partners

This methodology can be applied to investigate several critical aspects of RAET1E biology:

Potential applications:

• Receptor-ligand interactions:

  • Map the precise binding interface between RAET1E and NKG2D receptor

  • Identify potential co-receptors or accessory molecules

• Trafficking and membrane integration:

  • Study interactions with components of the secretory pathway

  • Investigate membrane integration mechanisms, potentially involving Sf9 importin-α-16 as seen with other proteins

• Regulatory interactions:

  • Identify proteins that regulate RAET1E stability or turnover

  • Study interactions that might modulate signaling functions

The Sf9 cell system offers particular advantages for these studies, including high expression levels, reduced background from endogenous mammalian proteins, and the ability to co-express multiple proteins for complex formation analysis.

What are the technical challenges in studying RAET1E mobility using FRAP assays in Sf9 cells versus mammalian cells?

Fluorescence Recovery After Photobleaching (FRAP) assays provide valuable insights into protein mobility within cellular membranes. Research with other membrane proteins has revealed significant differences in mobility between Sf9 and mammalian cells that would likely apply to RAET1E studies .

System-specific mobility differences:

Studies with LBR (Lamin B Receptor) showed that LBR-GFP fusions had limited mobility at the nuclear rim in mammalian CHO-K1 cells but high mobility in both ER and nuclear membranes in Sf9 cells . This suggests RAET1E might display similar system-dependent mobility patterns due to:

• Different membrane lipid compositions

• Varying protein-protein interaction networks

• Distinct cytoskeletal organization

• Absence of mammalian-specific retention mechanisms in insect cells

Technical considerations for FRAP experiments with RAET1E:

• Construct design:

  • Create comparable RAET1E-GFP fusion proteins for both expression systems

  • Consider the position of the GFP tag to minimize interference with trafficking signals

  • Include appropriate controls with known mobility characteristics

• Imaging parameters:

  • For Sf9 cells: Adjust laser power and scan settings for smaller cell size

  • Account for optimal temperature differences (27°C for Sf9 vs. 37°C for mammalian cells)

  • Standardize expression levels to avoid artifacts from overexpression

• Data analysis challenges:

  • Develop appropriate mathematical models to account for different membrane geometries

  • Consider the impact of cell morphology on diffusion measurements

  • Establish system-specific correction factors for accurate comparison

According to research with LBR, FRAP analyses in Sf9 cells showed approximately equal and high mobility in both ER and nuclear membranes, while mammalian cells showed differential mobility between these compartments . This suggests fundamental differences in membrane protein behavior between expression systems that researchers must consider when interpreting RAET1E mobility data.

The biological significance of these differences lies in understanding how RAET1E distribution and dynamics relate to its immune recognition functions. Different mobility patterns may reflect system-specific protein-protein interactions that influence RAET1E clustering, surface expression, and ultimately its ability to engage NKG2D receptors on immune cells.

How does glycosylation of RAET1E in Sf9 cells impact its binding affinity to NKG2D receptors?

Glycosylation differences between RAET1E expressed in Sf9 cells versus mammalian cells can significantly impact its binding affinity to NKG2D receptors. This is a critical consideration for functional studies using recombinant proteins.

Glycosylation characteristics comparison:

RAET1E is described as a glycosylated polypeptide when expressed in Sf9 cells . The glycosylation profile differs from mammalian cells in several important ways:

Impact on NKG2D binding:

The altered glycosylation can affect binding through:

• Direct effects on binding interface:

  • If glycans are located near the receptor-binding surface, different glycoforms may alter accessibility

  • The simpler glycans in Sf9-expressed RAET1E may reduce steric hindrance in some cases

  • Conversely, mammalian-specific glycan structures might contribute positively to binding

• Conformational effects:

  • Glycosylation contributes to protein folding and stability

  • Different glycoforms may subtly alter the three-dimensional structure

  • This could indirectly affect the presentation of key binding residues

Methodological approaches to assess glycosylation impact:

• Comparative binding studies:

  • Surface Plasmon Resonance (SPR) to determine kinetic parameters

  • Bio-Layer Interferometry (BLI) for real-time binding analysis

  • Cell-based assays to measure functional outcomes of receptor engagement

• Enzymatic deglycosylation:

  • Treatment with endoglycosidases to remove or modify glycans

  • Comparison of binding parameters before and after treatment

  • Site-directed mutagenesis to eliminate specific glycosylation sites

For researchers using Sf9-expressed RAET1E in immunological studies, it's crucial to validate findings with mammalian-expressed proteins or deglycosylated controls to distinguish protein-protein interactions from glycan-mediated effects. This is particularly important when investigating the fine specificity of NKG2D recognition, as subtle differences in binding kinetics can significantly impact downstream immune responses.