Recombinant Human ACE

Recombinant Human ACE (Angiotensin-Converting Enzyme) is used in research applications to study its enzymatic activity, role in cardiovascular and pulmonary physiology, and its therapeutic potential in diseases such as hypertension, heart failure, acute lung injury, and viral infections like COVID-19.

Key scientific reasons to use recombinant human ACE in research include:

• Controlled Enzymatic Studies: Recombinant ACE allows precise measurement of its activity, such as cleavage of fluorogenic peptide substrates, enabling detailed kinetic and mechanistic studies. This is essential for understanding substrate specificity, inhibitor screening, and drug development.

• Disease Modeling and Therapeutic Research: Recombinant ACE is critical for investigating the renin-angiotensin system (RAS) in models of cardiovascular, renal, and pulmonary diseases. It helps elucidate how ACE modulates levels of angiotensin peptides, influencing blood pressure, inflammation, fibrosis, and organ protection.

• COVID-19 and Viral Entry Studies: Recombinant ACE2 (a related enzyme) is widely used to study SARS-CoV-2 spike protein binding, viral entry mechanisms, and to develop neutralizing therapies. Recombinant ACE2 can block viral entry in vitro and reduce infection in animal models, making it a valuable tool for antiviral research.

• Bioassays and Functional Characterization: Recombinant ACE is used in bioassays to test the effects of drugs, antibodies, or small molecules on ACE activity, and to characterize its interactions with other proteins or ligands.

• Safety and Consistency: Recombinant production ensures high purity, batch-to-batch consistency, and eliminates variability associated with tissue-derived enzymes, which is crucial for reproducible experimental results.

• Clinical Translation: Recombinant human ACE and ACE2 have been evaluated in clinical trials for safety and efficacy in conditions such as acute respiratory distress syndrome (ARDS) and COVID-19, supporting translational research from bench to bedside.

Typical applications include:

• Enzymatic assays (e.g., substrate cleavage, inhibitor screening)
• Cell culture studies (e.g., signaling, viral infection models)
• Animal models of disease (e.g., hypertension, lung injury)
• Protein-protein interaction studies (e.g., spike protein binding)
• Therapeutic candidate evaluation (e.g., recombinant ACE2 as a decoy receptor for SARS-CoV-2)

Using recombinant human ACE provides a reliable, well-characterized tool for dissecting its biological functions and for developing new therapeutic strategies targeting the RAS pathway and related diseases.

Yes, recombinant human ACE can be used as a standard for quantification or calibration in ELISA assays, provided it is properly characterized and matches the assay’s requirements. Recombinant ACE is commonly used as a standard in commercial human ACE ELISA kits, where it enables the generation of a standard curve for quantitative measurement of ACE in biological samples.

Key considerations for using recombinant human ACE as an ELISA standard:

• Purity and Characterization: The recombinant ACE should be highly purified and its concentration accurately determined, typically by methods such as HPLC or spectrophotometry.
• Epitope Compatibility: The recombinant ACE must contain the relevant epitopes recognized by the capture and detection antibodies in your ELISA. Most commercial standards use the full-length or major extracellular domain (e.g., amino acids 30–1261).
• Matrix Matching: Standards should be prepared in a buffer that closely matches the sample matrix to minimize matrix effects and ensure accurate quantification.
• Validation: Confirm that the recombinant ACE behaves similarly to native ACE in your assay system, especially regarding antibody binding and detection. This is typically validated by the kit manufacturer, but for custom assays, you should verify linearity, recovery, and parallelism.

Typical protocol:

• Prepare serial dilutions of recombinant human ACE to cover the assay’s dynamic range (e.g., 0.78 ng/mL to 50 ng/mL).
• Run these standards in parallel with your samples to generate a standard curve.
• Use the curve to interpolate ACE concentrations in unknown samples.

Limitations:

• Recombinant standards may differ from native ACE in post-translational modifications, which can affect antibody recognition in rare cases. Most commercial kits are validated to ensure equivalence for quantification.
• Always use standards that match the format and specifications of your ELISA (e.g., same isoform, tag-free if required).

Summary Table: Recombinant Human ACE as ELISA Standard

In conclusion, recombinant human ACE is widely accepted and used as a standard for ELISA quantification, provided it is appropriately validated for your assay system.

Recombinant Human ACE (Angiotensin-Converting Enzyme) has been validated in published research for several key applications, primarily in the fields of cardiovascular biology, virology (notably SARS-CoV-2), and enzymology.

Validated Applications in Published Research:

• Therapeutic and Preclinical Models:

  • Treatment of Acute Respiratory Distress Syndrome (ARDS): Recombinant human ACE2 has been tested in both animal models and clinical trials for its ability to treat ARDS by modulating angiotensin II levels and reducing lung injury.
  • COVID-19 Research: Recombinant human ACE2 has been used as a decoy receptor to block SARS-CoV-2 infection in cell culture, organoids, and animal models, and is under clinical evaluation for COVID-19 therapy.
  • Hypertension and Cardiovascular Disease Models: Recombinant ACE2 has been used in vivo to study its effects on blood pressure regulation, angiotensin II degradation, and prevention of angiotensin II-induced hypertension in animal models.

• Biochemical and Enzymatic Assays:

  • Enzyme Activity Assays: Recombinant ACE2 is widely used in vitro to study its enzymatic activity, substrate specificity, and inhibition by small molecules or natural compounds.
  • Pharmacokinetic and Pharmacodynamic Studies: Recombinant ACE2 has been administered to healthy volunteers to assess its pharmacokinetics, pharmacodynamics, safety, and tolerability.

• Molecular and Cell Biology:

  • Binding and Neutralization Assays: Used to assess binding affinity to viral spike proteins (e.g., SARS-CoV-2) and to evaluate neutralization potential in cell-based assays.
  • Protein-Protein Interaction Studies: Recombinant ACE2 is used in surface plasmon resonance (SPR), ELISA, and Western blot assays to study interactions with ligands, inhibitors, or viral proteins.

• Structural and Mechanistic Studies:

  • Structural Biology: Recombinant ACE2 is used for crystallography and structure-function analyses to understand its mechanism and interactions with substrates or inhibitors.

Summary Table of Validated Applications

Additional Notes:

• Recombinant human ACE2 is also used as a research tool to study the renin-angiotensin system and its role in various physiological and pathological processes.
• Engineered variants and fusion proteins of recombinant ACE2 have been developed to enhance stability, binding, or therapeutic potential, and these have been validated in both in vitro and in vivo models.

If you require details on a specific application or protocol, please specify the context (e.g., enzymatic assay, viral neutralization, animal model).

To reconstitute and prepare Recombinant Human ACE protein for cell culture experiments, first confirm whether your protein is supplied lyophilized or in solution. The following protocol outlines best practices for lyophilized recombinant ACE protein:

Reconstitution Steps:

1. Centrifuge the vial briefly before opening to ensure all powder is at the bottom and minimize loss.
2. Add sterile distilled water or the recommended buffer (e.g., PBS, pH 7.4, or assay buffer such as 50 mM MES, pH 6.5) to achieve the desired concentration, typically between 0.1–0.5 mg/mL for stock solutions.
3. Gently mix by inverting or slow pipetting. Avoid vortexing or vigorous pipetting, as this can denature the protein.
4. Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation.

Stabilization and Storage:

• For short-term storage (up to 1 week), keep the reconstituted protein at 2–8°C.
• For long-term storage, aliquot and freeze at –20°C to –80°C. Add a carrier protein (e.g., 0.1% BSA, 5% HSA, 10% FBS, or 5% trehalose) to stabilize and prevent adsorption or degradation.
• Avoid repeated freeze-thaw cycles by aliquoting.

Preparation for Cell Culture:

• Dilute the stock solution to the working concentration using cell culture medium or buffer compatible with your experiment. If using serum-free conditions, avoid animal-derived carrier proteins and use trehalose or other non-animal stabilizers.
• For direct addition to cell culture, ensure the final buffer composition is compatible with cell viability and function (e.g., isotonicity, pH 7.2–7.4).
• Typical working concentrations for functional assays range from 1–100 ng/mL, but optimize based on your experimental design.

Additional Notes:

• Always consult the product-specific Certificate of Analysis (CoA) for recommended reconstitution buffer and concentration, as these may vary by manufacturer and intended application.
• If the protein is used for enzymatic assays, follow the specific buffer and substrate recommendations (e.g., 50 mM MES, pH 6.5, for ACE activity assays).
• For cell-based assays, confirm endotoxin levels are sufficiently low (<1 EU/µg) to avoid confounding cellular responses.

Summary Table: Recombinant Human ACE Protein Reconstitution

This protocol ensures protein stability, activity, and compatibility for cell culture experiments. Always tailor final concentrations and buffer conditions to your specific experimental requirements.