Recombinant Human Apolipoprotein A-I (ApoA-I)

Recombinant Human Apolipoprotein A-I (ApoA-I) is widely used in research due to its central role in reverse cholesterol transport, its anti-inflammatory and antioxidant properties, and its relevance in cardiovascular and cancer biology.

Key reasons to use recombinant ApoA-I in research applications:

• Reverse Cholesterol Transport (RCT): ApoA-I is the main protein component of high-density lipoprotein (HDL) and is essential for mediating the efflux of cholesterol from peripheral tissues to the liver for excretion, a process that protects against atherosclerosis. Recombinant ApoA-I enables controlled studies of RCT mechanisms and HDL biogenesis in vitro and in vivo.

• Atherosclerosis and Cardiovascular Disease Models: Recombinant ApoA-I and its variants (such as ApoA-I Milano) have been shown in animal and human studies to reduce atherosclerotic plaque burden, promote plaque regression, and improve HDL functionality. This makes it a valuable tool for preclinical and translational research on cardiovascular therapies.

• Anti-inflammatory and Antioxidant Effects: ApoA-I exhibits anti-inflammatory actions by modulating immune cell activation, inhibiting pro-inflammatory cytokine production, and reducing oxidative stress in vascular tissues. These properties are relevant for studying inflammation-related diseases and vascular biology.

• Cancer Research: Altered ApoA-I levels are associated with cancer risk and progression. ApoA-I has been implicated in tumor suppression through immune modulation and anti-inflammatory mechanisms, making recombinant ApoA-I useful for investigating cancer pathogenesis and potential therapeutic strategies.

• Biomarker and Functional Studies: Recombinant ApoA-I is used as a standard or control in assays measuring HDL function, cholesterol efflux capacity, and as a biomarker in clinical and translational studies.

• Therapeutic Development: Recombinant ApoA-I is being explored in clinical trials as a therapeutic agent for cardiovascular disease, providing a platform for drug development and mechanistic studies.

• Protein Engineering and Structure-Function Analysis: Recombinant production allows for site-directed mutagenesis and structural studies, facilitating the investigation of ApoA-I’s functional domains and interactions with lipids and cellular receptors.

In summary, recombinant human ApoA-I is a versatile tool for research in lipid metabolism, cardiovascular disease, inflammation, immunity, and cancer, enabling mechanistic studies, therapeutic development, and biomarker discovery under controlled experimental conditions.

Yes, recombinant human Apolipoprotein A-I (ApoA-I) can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated for your specific assay system. Recombinant ApoA-I expressed in E. coli is commonly used as a calibrator in commercial ELISA kits, and its standard curves are shown to be parallel and comparable to those generated with natural human ApoA-I.

Key considerations and supporting details:

• Assay Calibration: ELISA kits designed for ApoA-I quantification often use recombinant human ApoA-I as the reference standard. These kits demonstrate that standard curves generated with recombinant ApoA-I are linear and parallel to those obtained with natural ApoA-I, allowing for accurate quantification of both recombinant and endogenous protein in biological samples.

• Specificity: Most sandwich ELISA kits for ApoA-I are validated to recognize both natural and recombinant forms of the protein, ensuring that recombinant ApoA-I is suitable for use as a standard.

• Purity and Activity: Recombinant ApoA-I used as a standard should be of high purity (typically >95% by SDS-PAGE and HPLC) and, while biological activity is not always required for ELISA calibration, the protein should be structurally intact and free from interfering contaminants.

• Validation: Before using recombinant ApoA-I as a standard in your own custom or in-house ELISA, it is essential to validate that the antibody pair in your assay recognizes the recombinant form equivalently to the native protein. This can be confirmed by generating standard curves and comparing their slopes and linearity with those from natural ApoA-I.

• Preparation: Recombinant ApoA-I should be reconstituted and diluted in the same buffer or diluent used for your assay standards to ensure consistency and minimize matrix effects.

• Limitations: Always ensure the recombinant standard is compatible with your assay’s detection antibodies and that it does not contain tags or modifications that could affect antibody binding. Some recombinant preparations may lack post-translational modifications present in native ApoA-I, but this typically does not affect ELISA quantification unless the antibodies are modification-specific.

Best practices:

• Use recombinant ApoA-I from a reliable source with documented purity and sequence.
• Validate the standard curve in your assay system, especially if using custom antibodies.
• Prepare and store standards according to recommended protocols to maintain protein integrity.

In summary, recombinant human ApoA-I is widely accepted and scientifically appropriate as a standard for ELISA quantification, provided assay-specific validation is performed.

Recombinant Human Apolipoprotein A-I (ApoA-I) has been validated in published research for a broad range of scientific and therapeutic applications, primarily in cardiovascular, metabolic, neurological, immunological, and biochemical studies.

Key validated applications include:

• Reverse Cholesterol Transport (RCT) and HDL Biogenesis: Recombinant ApoA-I is widely used to study and enhance RCT, a process critical for removing excess cholesterol from peripheral tissues and forming high-density lipoprotein (HDL) particles. It serves as a scaffold protein for reconstituted HDL (rHDL) particles in vitro, which are then used in cholesterol efflux assays and lecithin:cholesterol acyltransferase (LCAT) activity assays.

• Cardiovascular Research and Therapeutics: Recombinant ApoA-I has been validated in animal models and clinical studies for its cardioprotective effects, including improving endothelial function, reducing inflammation, and promoting cholesterol efflux. It is used in preclinical and clinical trials to assess its potential to reduce cardiovascular disease (CVD) risk and events, such as after acute myocardial infarction.

• Immunomodulation and Inflammation: Studies have demonstrated that ApoA-I exhibits anti-inflammatory and immune-modulatory properties, including inhibition of complement activation, reduction of pro-inflammatory cytokine production, and modulation of immune cell activation and migration. It has been used in models of sepsis, autoimmune disease, and cancer to investigate these effects.

• Cancer Research: Recombinant ApoA-I has been validated for its tumor suppressive activity in vitro and in vivo, including inhibition of apoptosis, reduction of oxidative stress, and modulation of immune responses in the tumor microenvironment.

• Neurological Disorders and Biomarker Studies: ApoA-I is used in research on Alzheimer’s disease (AD) and other neurological conditions, both as a biomarker and as a therapeutic agent. Exogenous administration in animal models has shown reduction of amyloid-beta (Aβ) levels, alleviation of neuroinflammation, and prevention of cognitive deficits.

• Biochemical and Structural Studies: Recombinant ApoA-I is employed in structural biology to elucidate its conformation, lipid-binding properties, and interactions with cellular receptors and transporters (e.g., ABCA1). It is also used in protein engineering and foam fractionation studies to optimize production and purification protocols.

• Drug Delivery and Protein Solubilization: rHDL particles formed with recombinant ApoA-I are validated as carriers for drug delivery and as substrates for solubilizing transmembrane proteins in biochemical assays.

Experimental protocols and assays where recombinant ApoA-I is validated:

• Cholesterol efflux assays (using ABCA1-expressing cells)
• LCAT activity assays
• SDS-PAGE and Western blotting for protein characterization
• ELISA for quantification and biomarker studies
• Cellular binding assays (e.g., liver cell binding)
• In vivo administration in animal models for therapeutic evaluation

Summary Table of Validated Applications

These applications are supported by peer-reviewed studies and validated experimental protocols, demonstrating the versatility of recombinant human ApoA-I in both basic and translational research.

To reconstitute and prepare Recombinant Human Apolipoprotein A-I (ApoA-I) for cell culture experiments, dissolve the lyophilized protein in sterile buffer—commonly phosphate-buffered saline (PBS) or sterile deionized water—to a concentration suitable for your application, typically ranging from 100 μg/mL to 1 mg/mL.

Step-by-step protocol:

• Reconstitution:

  • Add sterile PBS or sterile deionized water directly to the lyophilized ApoA-I protein.
  • For most cell culture applications, a final concentration of 100 μg/mL in PBS is recommended. Some protocols use 400 μg/mL in sterile deionized water, or 1 mg/mL in water.
  • Gently mix by pipetting or slow vortexing until fully dissolved. Avoid vigorous agitation to prevent protein denaturation.

• Sterility:

  • Use sterile reagents and aseptic technique throughout to prevent contamination.
  • If necessary, filter the reconstituted solution through a 0.2 μm sterile filter.

• Storage:

  • Store the reconstituted ApoA-I at 2–8 °C for up to one month, or at –20 °C to –70 °C for longer periods.
  • Avoid repeated freeze-thaw cycles to maintain protein integrity.

• Preparation for cell culture:

  • Dilute the stock solution to the desired working concentration using sterile cell culture medium or buffer.
  • If using for rHDL formation, incubate ApoA-I with phospholipids (e.g., DMPC) and sonicate as needed to clarify the solution and promote rHDL assembly.

Additional notes:

• ApoA-I is structurally resilient and refolds to its native conformation after denaturation, so gentle handling is sufficient for reconstitution.
• Confirm protein concentration using a suitable assay (e.g., BCA assay) before use.
• Ensure low endotoxin levels if using in sensitive cell culture systems; purification protocols typically yield low endotoxin ApoA-I.

Summary Table:

This protocol ensures that ApoA-I is properly reconstituted and prepared for use in cell culture experiments, maintaining its biological activity and structural integrity.