Recombinant Human ACE-2 (His-tag)

Recombinant human ACE2 with a His-tag offers several compelling advantages for research applications, combining functional utility with practical experimental benefits.

Biological Activity and Functional Relevance

Recombinant human ACE2 (His-tag) is biologically active and maintains its essential enzymatic and binding functions. The protein functions as a carboxypeptidase that converts angiotensin I to angiotensin 1-9 and angiotensin II to angiotensin 1-7, making it valuable for studying cardiovascular homeostasis and the renin-angiotensin system. Additionally, ACE2 serves as a functional receptor for human coronaviruses including SARS-CoV and SARS-CoV-2, making it particularly relevant for viral infection studies and therapeutic development.

The biological activity is rigorously validated through functional ELISA assays, where the protein demonstrates measurable binding to spike proteins with EC50 values in the 20-90 ng/mL range.

Advantages of the His-Tag

The His-tag provides multiple practical benefits that enhance research efficiency:

Affinity Purification: His-tags enable rapid and cost-effective protein purification using widely available nickel or cobalt resins, eliminating the need for expensive proprietary resins. This accessibility makes large-scale protein preparation economically feasible.

Small Size and Minimal Interference: The His-tag is remarkably small, preventing interference with protein folding, structure, and biological function. This ensures that the tagged protein behaves authentically in experimental systems.

Immunodetection Flexibility: The His-tag facilitates detection through Western blotting, ELISA, and immunohistochemistry using anti-His antibodies, reducing dependence on expensive target-specific antibodies. This is particularly advantageous when working with novel targets or designing multiplexed experiments requiring flexibility in antibody selection.

Low Immunogenicity: His-tags exhibit relatively low immunogenicity, eliminating the need for tag removal before using the protein as an immunogen for antibody production.

Quality and Purity Standards

Recombinant human ACE2 (His-tag) is produced to high quality specifications, with purity levels exceeding 85-95% as determined by SDS-PAGE analysis. Endotoxin levels are maintained below 1 EU/µg, ensuring suitability for sensitive biological applications.

Research Applications

The protein is suitable for diverse experimental approaches including functional ELISA, surface plasmon resonance (SPR), SDS-PAGE, Western blotting, immunoprecipitation, immune blocking assays, and binding studies. These applications span from basic mechanistic studies of the renin-angiotensin system to coronavirus research and therapeutic development.

Yes, recombinant human ACE-2 with a His-tag can be effectively used as a standard for quantification and calibration in ELISA assays. This application is well-established and widely supported in the scientific literature.

Suitability as an ELISA Standard

Recombinant ACE-2 proteins with His-tags are specifically designed and validated for use as calibration standards in ELISA protocols. The His-tag does not interfere with the protein's biological activity or its recognition by antibodies used in sandwich ELISA formats, making it an ideal choice for standardization purposes.

Key Characteristics for Standardization

Biological Activity and Binding Properties

The recombinant ACE-2 protein maintains full biological activity, which is critical for accurate quantification. The protein demonstrates measurable binding affinity to relevant ligands, such as SARS-CoV-2 spike protein RBD, with EC₅₀ values typically in the range of 20-90 ng/mL. This functional activity ensures that your standard behaves similarly to native ACE-2 in your assay system.

Molecular Weight and Oligomeric State

The protein typically exists as a homodimer with a molecular weight of approximately 170-220 kDa when analyzed by size exclusion chromatography with multi-angle light scattering (SEC-MALS). Understanding the oligomeric state is important for accurate concentration calculations and standard preparation.

Purity and Quality

Recombinant ACE-2 proteins are typically supplied with high purity (>85-95% by SDS-PAGE), and low endotoxin levels (<1 EU/µg), which are essential criteria for reliable ELISA standards. These specifications ensure minimal interference from contaminants in your calibration curve.

Practical Considerations

When using recombinant ACE-2 as your ELISA standard, consider the following:

• Formulation: The protein is often supplied with or without carrier proteins like bovine serum albumin (BSA). For ELISA applications, formulations with BSA are generally recommended to maintain protein stability and prevent non-specific adsorption to assay surfaces.

• Concentration Range: Commercial ELISA kits typically establish calibration ranges between 0.0625-4 ng/mL or 0.156-10 ng/mL, depending on the assay sensitivity. Your standard should cover the expected concentration range of your samples.

• Lot-to-Lot Consistency: Quality-controlled recombinant proteins demonstrate consistent performance across multiple production lots, which is essential for reproducible standardization.

The His-tag itself serves as an additional advantage, as it can be detected using anti-His antibodies if needed for verification purposes, and it does not significantly alter the protein's native structure or function in binding assays.

Recombinant Human ACE-2 (His-tag) has been validated for a wide range of applications in published research, primarily focusing on its role in SARS-CoV-2 and cardiovascular biology. Key applications include:

• Binding Assays: Used to measure binding affinity to SARS-CoV-2 Spike protein, including the receptor-binding domain (RBD), in functional ELISA and surface plasmon resonance (SPR) assays.
• Neutralization Assays: Employed to assess viral neutralization of SARS-CoV-2 and its variants, both in vitro and in vivo.
• Enzyme Activity Assays: Validated for catalytic activity as a carboxypeptidase, cleaving substrates such as angiotensins I and II, and fluorogenic peptide substrates.
• Cell Culture Studies: Applied in cell-based assays to study ACE-2’s role in viral entry, receptor function, and modulation of the renin-angiotensin system.
• Protein-Protein Interaction Studies: Used in immunoprecipitation and immune blocking assays to investigate interactions with other proteins, such as amino acid transporters.
• Western Blotting: Validated for detection and quantification of ACE-2 protein expression.
• SDS-PAGE: Used for purity and molecular weight analysis.
• Flow Cytometry and Fluorescence Staining: Applied in studies using fluorescently labeled ACE-2 (e.g., Alexa Fluor® 488) for detection and visualization.
• Animal Models: Utilized in preclinical studies to evaluate therapeutic efficacy, including administration in mouse models of SARS-CoV-2 infection.

These applications highlight the versatility of Recombinant Human ACE-2 (His-tag) in both basic research and translational studies related to infectious diseases and cardiovascular physiology.

To reconstitute and prepare Recombinant Human ACE-2 (His-tag) protein for cell culture experiments, dissolve the lyophilized protein in sterile distilled water or buffer to a concentration between 0.1–0.5 mg/mL. Use gentle pipetting to avoid foaming and ensure complete dissolution.

Essential steps and considerations:

• Reconstitution:

  • Add sterile distilled water or PBS (pH 7.4) to the lyophilized protein to achieve the desired concentration (commonly 0.1–0.5 mg/mL).
  • Gently mix by pipetting or slow vortexing until fully dissolved. Avoid vigorous agitation to prevent protein denaturation.
  • If the protein does not dissolve easily, allow it to sit at room temperature for a few minutes, then gently mix again.

• Buffer selection:

  • For cell culture applications, use a buffer compatible with your cells, such as PBS (pH 7.4) or cell culture medium without serum or additives that may interfere with ACE-2 activity.
  • If the protein is reconstituted in water, dilute further in the appropriate buffer before adding to cells.

• Carrier protein (optional):

  • Addition of BSA (0.1–1 mg/mL) can help stabilize the protein and prevent adsorption to tube walls, especially at low concentrations.

• Sterility:

  • Use sterile techniques throughout. Filter the reconstituted protein through a 0.22 μm filter if sterility is required for cell culture.

• Storage:

  • Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can reduce activity.
  • Store aliquots at −80°C for long-term use or at 4°C for short-term use (up to one week).

• Endotoxin:

  • Confirm the endotoxin level is suitable for cell culture (<1 EU/μg is typical for recombinant ACE-2).

• Application:

  • Dilute the protein in cell culture medium immediately before use. Optimize the final concentration based on experimental requirements and published activity data (e.g., EC50 for spike protein binding is 20–90 ng/mL).

Summary Table: Preparation Steps

Always consult the specific Certificate of Analysis or product datasheet for your batch to confirm recommended reconstitution and storage conditions.