OLR1 Human, Sf9

What is OLR1 Human, Sf9 and what are its key properties?

OLR1 Human expressed in Sf9 cells is a recombinant form of human Oxidized Low Density Lipoprotein Receptor 1. It is produced as a single, glycosylated polypeptide chain containing 225 amino acids (residues 58-273) with a molecular mass of 25.8kDa, though it typically appears at approximately 28-40kDa when analyzed by SDS-PAGE due to post-translational modifications. The recombinant protein is fused to a 6 amino acid His-tag at the C-terminus and is purified using proprietary chromatographic techniques .

OLR1 is a type II membrane protein belonging to the C-type lectin family that functions as a cell-surface receptor for oxidized low-density lipoprotein (Ox-LDL). This protein plays a significant role in early atherosclerosis by facilitating the transformation of monocyte-derived macrophages to foam cells in atherosclerotic lesions. Additionally, OLR1 triggers the activation of the NF-κB signal transduction pathway .

What are the common synonyms and alternative names for OLR1?

OLR1 is known by numerous alternative names and identifiers in scientific literature:

This diversity of nomenclature reflects the protein's discovery in different contexts and its multiple functional roles in various biological systems .

How should OLR1 Human, Sf9 be stored to maintain its stability?

For optimal stability of OLR1 Human expressed in Sf9 cells, follow these storage recommendations:

• For short-term storage (2-4 weeks), store at 4°C if the entire vial will be used within this period.

• For longer periods, store frozen at -20°C.

• To enhance long-term stability, it is recommended to add a carrier protein such as 0.1% HSA (Human Serum Albumin) or BSA (Bovine Serum Albumin).

• Avoid multiple freeze-thaw cycles which can significantly reduce protein activity and integrity.

The protein is typically supplied in a formulation containing Phosphate Buffered Saline (pH 7.4) and 10% glycerol, which helps maintain its stability during storage .

What are the advantages and limitations of using Sf9 cells for expressing human OLR1?

Advantages of Sf9 Expression System:

• Sf9 cells derived from Spodoptera frugiperda can efficiently produce complex eukaryotic proteins with proper folding and post-translational modifications.

• The baculovirus expression system allows for high-level protein production compared to many mammalian expression systems.

• Sf9 cells can grow to high densities in suspension culture, enabling scalable production.

• The system supports proper glycosylation patterns, although they differ from human glycosylation.

• The recombinant protein produced has shown greater than 95% purity when assessed by SDS-PAGE .

Limitations:

• Glycosylation patterns in insect cells differ from human cells, which may affect protein function for certain applications.

• Sf9 cells contain endogenous retroviral-like particles that could potentially be expressed and might interfere with certain experimental outcomes or contaminate purified protein preparations .

• The recombinant OLR1 appears at 28-40kDa on SDS-PAGE despite having a calculated molecular mass of 25.8kDa, indicating variable post-translational modifications that may affect protein characteristics .

What considerations should be made regarding endogenous retroviral-like particles in Sf9 cells?

Researchers working with OLR1 expressed in Sf9 cells should be aware that these cells contain endogenous retroviral-like particles that can be constitutively expressed or induced under certain conditions. Studies have identified extracellular retroviral-like particles in Spodoptera frugiperda, from which Sf9 cells are derived .

Key considerations include:

• Purification protocols: Ensure robust purification procedures to minimize potential contamination with retroviral elements.

• Chemical induction assessment: Chemicals like 5-azacytidine (AzaC) and 5-iodo-2′-deoxyuridine (IUdR) can induce endogenous retroelements, which might affect protein production or purity .

• Infectivity concerns: While studies suggest limited infectivity risk for human cells, comprehensive quality control testing is advisable, especially for therapeutic applications.

• RT activity monitoring: The PERT (Product-Enhanced Reverse Transcriptase) assay can be used to monitor reverse transcriptase activity in cell culture supernatants as a marker for retroviral particle expression .

What are the validated applications for OLR1 Human, Sf9 in atherosclerosis research?

OLR1 Human, Sf9 has several validated applications in atherosclerosis research:

• Receptor-ligand binding studies: Recombinant OLR1 can be used to investigate binding kinetics with oxidized LDL and other potential ligands.

• Signal transduction analysis: Since OLR1 triggers the activation of the NF-κB pathway, the protein can be used to study downstream signaling events in atherosclerosis progression .

• Foam cell formation models: OLR1 facilitates the transformation of monocyte-derived macrophages to foam cells, making it valuable for in vitro models of early atherosclerotic lesion development .

• Drug discovery: The protein can be used in screening assays to identify compounds that inhibit OLR1-OxLDL interactions, potentially leading to novel therapeutics.

• Structural studies: Purified OLR1 can be used for crystallography and other structural biology techniques to elucidate binding mechanisms.

How can OLR1 Human, Sf9 be detected in experimental systems?

Several validated detection methods for OLR1 Human, Sf9 include:

Immunodetection methods:

• Western blotting using specific antibodies such as Goat Anti-Human LOX-1/OLR1 Antigen Affinity-purified Polyclonal Antibody .

• Immunohistochemistry (IHC) has been validated on human placenta samples, with specific staining localized to cytotrophoblasts using anti-LOX-1/OLR1 antibodies .

• Immunocytochemistry/Immunofluorescence for cellular localization studies.

Molecular detection:

• Quantitative PCR for detecting OLR1 expression at the mRNA level, which has been used successfully in studies generating stable prostate cancer cell lines with LOX-1 overexpression and shRNA against OLR1 .

• His-tag detection systems can be employed to specifically identify the recombinant protein due to its 6 amino acid His-tag at the C-terminus .

Functional assays:

• OxLDL binding assays

• NF-κB activation reporter assays

What controls should be included when working with OLR1 Human, Sf9?

When designing experiments with OLR1 Human, Sf9, include these essential controls:

• Negative controls:

  • Empty vector-transfected Sf9 cells to account for background expression

  • Isotype control antibodies in immunodetection methods

  • Cells expressing irrelevant C-type lectins to control for non-specific lectin activity

• Positive controls:

  • Native human OLR1 (if available)

  • Known OLR1 expressing tissues (placenta samples have shown reliable expression)

  • Validated OLR1 overexpression systems in human cells

• Technical controls:

  • Purification tag controls (His-tag only proteins)

  • Loading controls for Western blots (β-actin, GAPDH)

  • RT-PCR housekeeping gene controls

• Functional controls:

  • Blocking antibodies to confirm specificity of OLR1-ligand interactions

  • Competitive inhibition with known OLR1 ligands

  • Dose-response curves for OxLDL binding

How can OLR1 Human, Sf9 be used in studies of receptor-mediated endocytosis?

OLR1 Human, Sf9 can be employed in sophisticated endocytosis studies using these approaches:

• Fluorescently labeled OxLDL trafficking:

  • Use fluorescently labeled OxLDL to track endocytic uptake and intracellular routing in cells expressing recombinant OLR1.

  • Time-lapse confocal microscopy can visualize the dynamic process of internalization.

• Endocytic pathway dissection:

  • Employ specific inhibitors of clathrin-mediated endocytosis (e.g., chlorpromazine) versus lipid raft-dependent pathways (e.g., methyl-β-cyclodextrin) to determine the mechanism of OLR1-mediated internalization.

  • Co-localization studies with markers of early endosomes (EEA1), late endosomes (Rab7), and lysosomes (LAMP1) can define the intracellular fate of internalized ligands.

• Structure-function analysis:

  • Mutational studies of the lectin-like domain can identify critical residues for ligand binding versus internalization.

  • Truncation variants expressed in Sf9 cells can help distinguish between binding and internalization functions.

• Live-cell assays:

  • TIRF microscopy to visualize membrane recruitment of endocytic machinery during OLR1-mediated uptake.

  • pH-sensitive fluorophores to track endosomal acidification following receptor internalization.

What approaches can be used to investigate the role of OLR1 in NF-κB signaling pathway activation?

To investigate OLR1's role in NF-κB signaling, researchers can employ these methodological approaches:

• Phosphorylation cascade analysis:

  • Western blotting for phosphorylated IκB-α to assess canonical NF-κB activation

  • Immunoprecipitation of signaling complexes to identify OLR1-associated adaptor proteins

  • Phospho-specific antibody arrays to broadly profile signaling changes

• Transcriptional activation assays:

  • Luciferase reporter assays with NF-κB response elements

  • ChIP assays to detect NF-κB subunit binding to target gene promoters

  • RNA-seq to comprehensively profile NF-κB target gene expression changes

• Inhibitor studies:

  • Use specific inhibitors of the NF-κB pathway (IKK inhibitors, proteasome inhibitors) to confirm OLR1-dependent activation

  • Compare effects of various OLR1 ligands (OxLDL, advanced glycation end products, apoptotic cells) on pathway activation

• Domain-specific effects:

  • Express OLR1 mutants lacking specific domains to identify regions required for NF-κB activation

  • Determine if the cytoplasmic domain alone can activate signaling when oligomerized

• Co-receptor analysis:

  • Investigate potential interactions with toll-like receptors or other pattern recognition receptors that may synergize with OLR1 in activating NF-κB

What methods can be used to study OLR1 glycosylation patterns in Sf9 versus human cells?

Comparative glycosylation analysis between OLR1 expressed in Sf9 cells versus human cells can be approached using:

What are common challenges in OLR1 Human, Sf9 expression and purification?

Researchers commonly encounter these challenges when working with OLR1 Human, Sf9:

• Expression level variability:

  • Passage number of Sf9 cells can affect expression levels

  • Baculovirus titer optimization is critical for consistent expression

  • Monitor infection efficiency using reporter genes or viral plaque assays

• Protein aggregation:

  • OLR1 may form aggregates during expression or purification

  • Addition of mild detergents or optimizing buffer conditions can reduce aggregation

  • Consider step-wise dialysis when removing detergents

• Proteolytic degradation:

  • Add protease inhibitors throughout purification

  • Optimize purification speed to minimize exposure to proteases

  • Consider affinity chromatography approaches that enable rapid purification

• Batch-to-batch variation:

  • Standardize culture conditions (cell density, time of harvest)

  • Implement rigorous quality control testing (SDS-PAGE, activity assays)

  • Maintain reference standards from successful preparations

• Endotoxin contamination:

  • Implement endotoxin testing for preparations intended for cell-based assays

  • Use endotoxin removal methods if contamination is detected

How can researchers assess the functional activity of purified OLR1 Human, Sf9?

To assess the functional activity of purified OLR1 Human, Sf9, implement these validation methods:

• Ligand binding assays:

  • ELISA-based binding assays with immobilized OLR1 and labeled OxLDL

  • Surface Plasmon Resonance (SPR) to determine binding kinetics (kon, koff, KD)

  • Fluorescence Anisotropy to measure binding in solution

• Cell-based functional assays:

  • Transfection of OLR1-negative cells with purified protein to restore OxLDL uptake

  • NF-κB reporter assays to confirm signaling activation

  • Foam cell formation assays with macrophages

• Structural integrity assessment:

  • Circular Dichroism (CD) spectroscopy to confirm secondary structure

  • Thermal shift assays to assess protein stability

  • Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS) to verify oligomeric state

• Comparative activity metrics:

  • Compare activity to native OLR1 from human tissues where available

  • Establish specific activity values (activity units per mg protein)

  • Create standard curves with positive control preparations

How can OLR1 Human, Sf9 be utilized in studying cardiovascular diseases beyond atherosclerosis?

OLR1 Human, Sf9 can be applied to research in various cardiovascular conditions:

• Myocardial ischemia/reperfusion injury:

  • OLR1 mediates oxidative stress responses in cardiomyocytes

  • Investigate protective effects of OLR1 blocking agents in ex vivo heart models

  • Study OLR1-dependent inflammatory signaling in cardiac tissue

• Hypertension mechanisms:

  • Examine OLR1's role in endothelial dysfunction and vascular tone regulation

  • Investigate OLR1-mediated effects on nitric oxide production

  • Assess OLR1-dependent vascular smooth muscle cell proliferation

• Thrombosis and platelet function:

  • Study interactions between OLR1 and platelets in thrombosis models

  • Investigate OLR1's role in platelet activation and aggregation

  • Examine potential OLR1-directed antithrombotic strategies

• Heart failure progression:

  • Analyze OLR1's contribution to cardiac remodeling

  • Study OLR1-mediated apoptotic signaling in cardiomyocytes

  • Investigate OLR1 as a biomarker for heart failure progression

What experimental approaches can be used to study OLR1's role in cancer using the Sf9-expressed protein?

OLR1 Human, Sf9 can be applied to cancer research using these methodological approaches:

• Cancer cell transformation studies:

  • OLR1 overexpression and knockdown studies in prostate cancer cell lines have been validated

  • Compare effects of recombinant OLR1 protein treatment versus genetic manipulation

  • Assess transformation phenotypes (proliferation, invasion, colony formation)

• Tumor microenvironment modeling:

  • Co-culture systems with cancer cells and macrophages/endothelial cells

  • Investigate how OLR1-OxLDL interactions affect tumor-associated macrophage polarization

  • Study paracrine signaling between OLR1-expressing cells in the tumor microenvironment

• Metastasis mechanisms:

  • Cell migration and invasion assays with OLR1-modulated cells

  • Matrix metalloproteinase expression and activity assessment

  • In vivo metastasis models with OLR1-overexpressing or knockdown cells

• Therapeutic targeting approaches:

  • Development of neutralizing antibodies using the recombinant protein as immunogen

  • Small molecule inhibitor screening against the purified protein

  • Assessment of combination approaches targeting OLR1 and other cancer pathways