ACE2 (18-740) Human

What is ACE2 (18-740) Human and why is it important for research?

ACE2 (18-740) refers to the extracellular domain (amino acids 18-740) of human Angiotensin-converting enzyme 2, which plays a central role in vascular, renal, and myocardial physiology. Unlike its homolog ACE, ACE2 converts angiotensin I to Ang1-9 and angiotensin II to Ang1-7. This domain is critically important because it contains the catalytic site responsible for these conversions and serves as the primary binding site for SARS coronaviruses. In knockout studies, ace2-/ace2- mice demonstrate severely reduced cardiac contractility, highlighting ACE2's importance in heart function regulation . The recombinant ACE2 (18-740) protein has become an essential tool for researchers studying cardiovascular physiology, COVID-19 pathogenesis, and therapeutic development against SARS-CoV-2.

What expression systems are optimal for producing high-quality ACE2 (18-740) Human?

Mammalian expression systems are strongly preferred for producing high-quality ACE2 (18-740) Human recombinant protein. Commercial sources typically use HEK 293 cells for expression, as seen in multiple product descriptions . Mammalian systems are essential because ACE2 requires proper glycosylation and other post-translational modifications for correct folding and function. When evaluating expression quality, researchers should expect >95% purity by SDS-PAGE analysis and endotoxin levels below 1 EU/μg . Alternative expression systems like bacterial or insect cells may yield higher quantities but typically produce proteins with altered glycosylation patterns that might not fully recapitulate the native human ACE2 functions, particularly for binding studies with viral proteins.

How should ACE2 (18-740) Human be stored and handled to maintain activity?

For optimal preservation of ACE2 (18-740) Human activity, the recombinant protein should typically be stored at -80°C for long-term storage, with working aliquots kept at -20°C to minimize freeze-thaw cycles. When handling the protein for experiments, it should be thawed gently on ice and maintained at 4°C during experimental preparations. The buffer composition is crucial for stability – typically PBS or HEPES-based buffers with controlled pH (7.2-7.4) are recommended, potentially with low concentrations of stabilizing agents. For certain applications like Surface Plasmon Resonance assays, the protein may require desalting using appropriate molecular weight cutoff columns (e.g., Zeba Spin 7K MWCO columns) . Researchers should validate protein activity after any modification to storage or handling protocols by assessing enzymatic function or binding capabilities.

How can ACE2 (18-740) be engineered for enhanced binding to SARS-CoV-2 variants?

Engineering ACE2 (18-740) for enhanced viral binding requires strategic amino acid substitutions at the ACE2/SARS-CoV-2 interface. Research has identified three key mutations—T27L, H34V, and N90E (collectively termed the "LVE" variant)—that dramatically improve binding affinity across multiple SARS-CoV-2 variants . The T27L and H34V substitutions optimize interactions with SARS-CoV-2 RBD amino acids 473 and 456, respectively, while N90E eliminates an N-linked glycosylation site that creates steric hindrance at the binding interface . Molecular modeling using software like Protean 3D with DFIRE scoring algorithms can predict optimal mutations based on stabilizing interactions. The effectiveness of these engineered variants is remarkable, with binding affinities improving from the nanomolar range (for wild-type ACE2) to the picomolar or even femtomolar range for engineered variants, representing up to 7,000-fold enhancement in binding strength for certain SARS-CoV-2 variants .

What methodologies can accurately measure binding affinities between ACE2 (18-740) and SARS-CoV-2 spike proteins?

Surface Plasmon Resonance (SPR) provides the most accurate and comprehensive analysis of binding affinities between ACE2 (18-740) and SARS-CoV-2 spike proteins. The methodology requires careful preparation: proteins should be desalted on appropriate columns (e.g., Zeba Spin 7K MWCO), and binding assays conducted in HBS-N buffer containing EDTA and Tween 20, with flow rates of approximately 30 μL/min . Typical experimental parameters include 120 seconds for association and 180 seconds for dissociation, with reference-subtracted SPR binding curves analyzed using a 1:1 binding model to derive kinetic parameters and dissociation constants (KD) . The table below shows comparative binding affinities for different ACE2 constructs against various SARS-CoV-2 variants:

This systematic approach enables quantitative comparison of binding affinities across different viral variants and ACE2 constructs .

How can ACE2 (18-740) be incorporated into therapeutic development strategies?

ACE2 (18-740) can be incorporated into therapeutic development through several advanced strategies. One promising approach involves creating chimeric ACE2/Fc fusion proteins with enhanced binding properties. These chimeras combine an engineered ACE2 domain (containing mutations like T27L, H34V, and N90E) with modified antibody domains . Two key variants include an "Fc-silent" version (containing L234S/L235T/G236R mutations, termed "STR") that minimizes antibody-dependent enhancement (ADE) risk, and a "live longer" Fc-silent version with YTE mutations (M252Y/S254T/T256E) that increase FcRn receptor binding for extended biological half-life . These chimeric constructs have demonstrated ultrahigh binding affinities (in the picomolar to femtomolar range) to a wide variety of SARS-CoV-2 variants, including emerging Omicron sublineages . The therapeutic potential of these constructs should be validated through surrogate viral neutralization tests and live virus neutralization assays with physiologically relevant models like airway organoids, which better recapitulate human respiratory tissues than simple cell culture systems .

What control experiments are essential when using ACE2 (18-740) in viral binding studies?

When designing viral binding studies with ACE2 (18-740), several essential controls must be incorporated for result validation. First, researchers should include both positive and negative binding controls: a validated wild-type ACE2-Fc construct (such as those from GenScript or ACRO) serves as a positive control with established binding properties (KD ~3-16 nM for the Wuhan variant) , while an irrelevant protein of similar size and structure provides a negative control to assess non-specific binding. Second, concentration-dependent binding assays should include a range spanning at least three orders of magnitude (e.g., 0.0064-100 μg/ml as described in viral neutralization studies) . Third, multiple viral variants should be tested in parallel to assess binding specificity, particularly when evaluating engineered ACE2 variants designed for "variant-agnostic" binding. Finally, when conducting SPR or similar binding assays, researchers should perform reciprocal experimental setups (immobilizing either ACE2 or viral protein) to ensure binding parameters are consistent regardless of which partner is immobilized.

How should researchers design ACE2 (18-740) neutralization assays for SARS-CoV-2?

A comprehensive neutralization assay design for testing ACE2 (18-740) against SARS-CoV-2 should employ a multi-tiered approach. Initially, surrogate Viral Neutralization Tests (sVNT) provide efficient screening capability. The GenScript C-PASS system described in the literature uses an ELISA-based approach with HRP-conjugated recombinant SARS-CoV-2 RBD fragment and human ACE2 receptor protein . For definitive analysis, researchers should progress to live virus neutralization assays in appropriate biosafety level 3 facilities. A methodologically sound approach involves challenging human airway organoids (AOs) with virus (MOI = 0.1) in the presence of ACE2 proteins at concentrations ranging from 0.0064 to 100 μg/ml (n=3 replicates per concentration) . After 24 hours, media should be replaced (including fresh ACE2 proteins), followed by supernatant harvesting 24 hours later for viral load quantification . This design enables generation of complete dose-response curves and determination of IC50 values for neutralization activity, allowing direct comparison between different ACE2 constructs or against therapeutic antibodies.

What factors affect experimental reproducibility when working with ACE2 (18-740)?

Multiple factors can impact experimental reproducibility when working with ACE2 (18-740), requiring careful experimental design and quality control. Batch-to-batch variation in protein production is a primary concern—researchers should verify >95% purity by SDS-PAGE and HPLC for each batch and perform comparative binding assays to confirm consistent functionality. Storage conditions significantly affect stability, with freeze-thaw cycles potentially degrading activity; researchers should use fresh aliquots whenever possible and validate protein activity before critical experiments. Buffer composition can alter protein conformation and activity, necessitating consistent preparation; for certain applications like SPR, desalting procedures using specific columns (e.g., Zeba Spin 7K MWCO) are recommended . Post-translational modifications, particularly glycosylation patterns, affect ACE2 functionality; the N90 glycosylation site specifically impacts SARS-CoV-2 binding, highlighting the importance of consistent expression systems . Finally, experimental conditions (temperature, incubation times, reagent concentrations) must be meticulously standardized and documented to enable meaningful comparisons across experiments and between laboratories.

What is the molecular basis for differential binding of ACE2 (18-740) to various SARS-CoV-2 variants?

The molecular basis for differential binding of ACE2 (18-740) to various SARS-CoV-2 variants lies in specific amino acid interactions at the binding interface. Molecular modeling studies have revealed that certain amino acid positions in both ACE2 and the viral receptor binding domain (RBD) are critical determinants of binding affinity . In wild-type ACE2, residues T27 and H34 interact with SARS-CoV-2 RBD amino acids at positions 455, 453, 473, and 456 . Mutations in these regions, either in the viral RBD (as occurs in variants like Alpha, Delta, and Omicron) or in engineered ACE2 constructs (such as the T27L and H34V substitutions), can significantly alter binding energetics. The T27L and H34V substitutions in engineered ACE2 variants optimize interactions with conserved SARS-CoV-2 RBD residues, creating a "variant-agnostic" binding profile . Additionally, the N90E substitution eliminates glycosylation-related steric hindrance at the binding interface . The effectiveness of these strategic substitutions is demonstrated by the dramatic improvement in binding affinities, from nanomolar KD values for wild-type ACE2 to picomolar or femtomolar values for engineered variants, representing up to several thousand-fold enhancement in binding strength .

How can ACE2 (18-740) be used to predict the binding properties of emerging SARS-CoV-2 variants?

ACE2 (18-740) can serve as a powerful tool for predicting the binding properties of emerging SARS-CoV-2 variants through a systematic analytical approach. Researchers can express and purify the spike protein RBD or S1 subunit from new variants, then perform quantitative binding studies using Surface Plasmon Resonance (SPR) . By comparing binding kinetics and affinities (KD values) to those of established variants, researchers can rapidly classify new variants according to their ACE2 binding properties. This approach is particularly valuable when combined with engineered "variant-agnostic" ACE2 constructs like the LVE variant, which maintains high affinity across multiple viral lineages . The methodology should follow established protocols: proteins desalted on appropriate columns, binding assays conducted in HBS-N buffer with EDTA and Tween 20, flow rates of 30 μL/min, and standard association/dissociation times . This approach allows for rapid assessment of new variants even before extensive clinical data becomes available, potentially identifying variants with enhanced transmission capabilities due to altered ACE2 binding properties.

What are the advantages of ACE2 (18-740)-based therapeutics compared to monoclonal antibodies for COVID-19?

ACE2 (18-740)-based therapeutics offer several significant advantages over monoclonal antibodies for COVID-19 treatment or prevention. First, engineered ACE2 variants demonstrate "variant-agnostic" binding properties, maintaining high affinity across multiple SARS-CoV-2 variants including those that escape monoclonal antibody neutralization . This is because ACE2-based therapeutics target the conserved, functionally critical receptor-binding interface that viruses cannot easily mutate without compromising infectivity. Second, chimeric ACE2/Fc fusion proteins can be designed with "Fc-silent" domains (containing L234S/L235T/G236R mutations) that minimize the risk of antibody-dependent enhancement (ADE) of infection . Third, these constructs can be further engineered with YTE mutations (M252Y/S254T/T256E) in the Fc domain to increase FcRn receptor binding, extending biological half-life 3-4 fold, particularly beneficial for nasal or aerosol administration . Fourth, ACE2-based therapeutics potentially offer dual benefits by not only neutralizing the virus but also replacing ACE2 enzymatic activity that may be disrupted during infection. Finally, the ultrahigh binding affinities achieved with engineered ACE2 variants (reaching femtomolar KD values for some variants) suggest potential for higher potency at lower doses compared to many monoclonal antibodies .

How should researchers design experiments to compare multiple engineered ACE2 (18-740) variants?

When designing experiments to compare multiple engineered ACE2 (18-740) variants, researchers should implement a comprehensive, multi-parameter assessment approach. First, binding affinity characterization should employ Surface Plasmon Resonance (SPR) as described in the literature, with consistent experimental conditions across all variants and multiple viral targets representing diverse SARS-CoV-2 lineages . The resulting kinetic parameters and KD values provide quantitative metrics for direct comparison. Second, researchers should assess neutralization potency using both surrogate assays (sVNT) and live virus neutralization with physiologically relevant models like airway organoids, testing each variant across a standardized concentration range (e.g., 0.0064-100 μg/ml) to generate complete dose-response curves and determine IC50 values . Third, stability testing should evaluate thermal stability, resistance to proteolytic degradation, and shelf-life under various storage conditions. Fourth, for variants intended as therapeutics, pharmacokinetic studies should assess half-life in circulation and in target tissues, particularly for constructs with modifications designed to extend biological persistence . Finally, manufacturability assessment should evaluate expression yield, purification efficiency, and consistency across batches. This multi-parameter approach enables comprehensive comparison between variants to identify optimal candidates for specific research or therapeutic applications.

How can researchers optimize ACE2 (18-740) expression for maximum yield and activity?

Optimizing ACE2 (18-740) expression requires systematic refinement of multiple parameters to achieve maximum yield without compromising activity. For expression system selection, HEK 293 cells have proven most effective for producing properly folded and glycosylated ACE2 . Vector design should incorporate strong promoters (such as CMV), optimized Kozak sequences, and efficient secretion signals, along with suitable purification tags (typically His-tag) . For transient transfection approaches, researchers should optimize transfection reagent type and DNA:reagent ratios through small-scale pilot experiments before scaling up. Cell culture conditions significantly impact yield—researchers should evaluate different media formulations (including those supplemented with protein expression enhancers), feeding strategies, and harvest timing. Temperature optimization can enhance proper folding; many researchers find that lowering culture temperature to 30-32°C during the expression phase improves the yield of correctly folded protein. For purification, a multi-step approach typically yields best results: IMAC using Ni-NTA for initial capture of His-tagged protein, followed by size exclusion chromatography to separate monomeric ACE2 from aggregates, and possibly ion exchange chromatography for final polishing. Throughout optimization, researchers should assess not only total protein yield but also specific activity (enzymatic function or viral binding capacity) to ensure that increased production doesn't come at the cost of reduced functionality.

What analytical techniques can verify the structural integrity of purified ACE2 (18-740)?

Verifying the structural integrity of purified ACE2 (18-740) requires a multi-technique analytical approach addressing different aspects of protein quality. SDS-PAGE and Western blotting provide basic assessments of size, purity (which should exceed 95%) , and immunoreactivity. Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) can determine the oligomeric state and detect aggregation. Circular dichroism (CD) spectroscopy offers information about secondary structure content, while differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC) assess thermal stability and can identify stabilizing buffer conditions. Mass spectrometry provides precise molecular weight determination and can confirm post-translational modifications, particularly glycosylation patterns which are critical for ACE2 function. Native mass spectrometry can further verify the intact structure and complex formation. Functional assays are equally important: enzymatic activity assays measuring conversion of angiotensin I to Ang1-9 or angiotensin II to Ang1-7 confirm catalytic function, while binding assays (SPR or ELISA-based) with SARS-CoV-2 RBD verify receptor functionality. For comprehensive validation, researchers should compare their purified ACE2 against commercial standards with established quality metrics. These combined approaches provide a thorough assessment of ACE2 structural integrity, essential for reliable experimental outcomes.

What are the most promising future research directions for ACE2 (18-740) Human?

The most promising future research directions for ACE2 (18-740) Human span both basic science and translational applications. In therapeutics development, the creation of multivalent ACE2-based molecules could further enhance binding avidity and neutralization potency beyond the already impressive femtomolar affinities achieved with engineered variants . Next-generation ACE2 chimeras combining the viral-binding domain with novel functional domains—such as immunomodulators or tissue-targeting moieties—could expand therapeutic applications beyond direct viral neutralization. For drug delivery, ACE2-based targeting strategies could enable selective delivery of therapeutic payloads to tissues expressing SARS-CoV-2 spike protein. In diagnostics, engineered high-affinity ACE2 variants could serve as superior capture agents for ultrasensitive viral detection assays. For basic virology research, ACE2 (18-740) provides a powerful tool for studying viral evolution and predicting the emergence of variants with altered transmissibility. In structural biology, continued refinement of our understanding of the ACE2-viral interface could reveal new opportunities for therapeutic intervention. Finally, broader application of the protein engineering strategies that yielded high-affinity ACE2 variants could inform the development of engineered receptors for other emerging pathogens, creating a platform technology for rapid response to future pandemic threats.

What key lessons have been learned from engineering high-affinity ACE2 (18-740) variants?

The engineering of high-affinity ACE2 (18-740) variants has yielded several key lessons applicable to both COVID-19 research and broader protein engineering efforts. First, the remarkable success of the "LVE" (T27L, H34V, N90E) variant demonstrates that strategic mutations targeted to the protein-protein interface can dramatically enhance binding affinity, achieving improvements of several thousand-fold (from nanomolar to femtomolar KD values) . Second, the N90E mutation specifically highlights how glycosylation can create steric hindrance at binding interfaces; eliminating glycosylation sites can significantly improve protein-protein interactions when the glycan impedes binding . Third, the "variant-agnostic" binding profile of engineered ACE2 variants emphasizes the value of targeting conserved viral regions that cannot easily mutate without compromising viral fitness . Fourth, the creation of chimeric ACE2/Fc fusion proteins with modified Fc domains (STR or YTE variants) demonstrates how combining optimized binding domains with engineered functional domains can create multifunctional therapeutic candidates with enhanced properties like extended half-life . Finally, the systematic integration of computational modeling (using tools like Protean 3D with DFIRE scoring) with experimental validation (SPR and viral neutralization assays) provides a powerful template for rational protein engineering across diverse applications . These lessons extend beyond COVID-19 research to inform protein engineering strategies for other receptor-ligand systems and therapeutic targets.

How might ACE2 (18-740) research contribute to preparedness for future coronavirus outbreaks?

ACE2 (18-740) research contributes significantly to preparedness for future coronavirus outbreaks through multiple pathways. First, the development of "variant-agnostic" high-affinity ACE2 variants provides a versatile platform for rapidly responding to novel coronaviruses that utilize ACE2 as an entry receptor . Unlike antibodies that might lose efficacy against new viral strains, engineered ACE2-based molecules target the conserved receptor-binding interface that all ACE2-utilizing coronaviruses must maintain. Second, the methodologies established for characterizing ACE2-viral interactions—including SPR binding assays, surrogate neutralization tests, and airway organoid infection models—create a standardized toolkit for rapidly assessing new viral threats . Third, the chimeric ACE2/Fc fusion protein designs, particularly those with Fc-silent domains to prevent antibody-dependent enhancement, offer templates for developing prophylactic agents that could be deployed early in an outbreak . Fourth, the molecular understanding gained from ACE2-SARS-CoV-2 structural studies informs predictions about potential mutations that might emerge in future outbreaks. Finally, the "LiVE-Longer YTE" chimera specifically designed for nasal administration represents an approach to blocking viral entry at the primary site of infection, potentially providing a model for rapid-response prophylactics against future respiratory pathogens . Collectively, these advances create both technological platforms and conceptual frameworks that can significantly accelerate response times to future coronavirus outbreaks.

How should researchers interpret differences in binding affinities between ACE2 variants and viral proteins?

Second, researchers should compare both kinetic parameters (kon and koff rates)—affinity improvements driven by faster association rates may have different functional implications than those driven by slower dissociation rates. Third, the relationship between affinity and neutralization potency is not always linear; beyond certain affinity thresholds, other factors like steric accessibility or concentration at sites of viral entry may become limiting. Fourth, variant-specific differences can provide insights into viral evolution—consistent binding by engineered ACE2 variants across multiple viral variants demonstrates successful targeting of conserved viral features . Finally, researchers should consider physiological context—femtomolar affinities observed in purified protein systems may translate differently in complex biological environments with competing interactions and diffusion limitations.

What statistical approaches are appropriate for analyzing ACE2 binding and neutralization data?

Appropriate statistical analysis of ACE2 binding and neutralization data requires careful consideration of experimental design and data characteristics. For binding kinetics determined by SPR, non-linear regression using a 1:1 binding model is typically applied to derive association rate (kon), dissociation rate (koff), and equilibrium dissociation constant (KD) parameters . Goodness-of-fit should be evaluated using residual plots and chi-square values. When comparing multiple ACE2 variants or viral strains, researchers should perform at least three independent experiments and report mean values with standard deviations or standard errors. For neutralization assays, dose-response curves should be analyzed using four-parameter logistic regression to determine IC50 values (concentration providing 50% inhibition) . Statistical significance of differences between ACE2 variants can be assessed using parametric tests (ANOVA with appropriate post-hoc comparisons) for normally distributed data or non-parametric alternatives (Kruskal-Wallis) when normality assumptions are violated. For correlation analyses between binding affinity and neutralization potency, Spearman's rank correlation is often more appropriate than Pearson's correlation due to the non-linear relationship between these parameters. Power analysis should guide sample size determination, particularly for resource-intensive experiments like live virus neutralization assays with airway organoids . Finally, researchers should clearly report both statistical significance (p-values) and effect sizes to communicate both the reliability and magnitude of observed differences.

How can computational modeling enhance understanding of ACE2-viral interactions?

Computational modeling provides powerful insights into ACE2-viral interactions that complement experimental approaches. Molecular dynamics simulations can reveal dynamic aspects of the binding interface not captured in static crystal structures, identifying transient interactions and conformational changes that influence binding energetics. Molecular docking with knowledge-based scoring functions (like DFIRE described in the research) enables rapid screening of potential ACE2 mutations and prediction of their effects on binding affinity . More sophisticated approaches like free energy perturbation calculations can provide quantitative estimates of binding energy changes caused by specific mutations. Sequence-based evolutionary analysis can identify conserved regions in viral spike proteins that are constrained due to functional requirements, representing ideal targets for engineered ACE2 variants. Network analysis of amino acid interactions at the binding interface can identify key residues that contribute disproportionately to binding stability, as demonstrated by the identification of T27L, H34V, and N90E substitutions in engineered ACE2 variants . Glycan modeling can predict steric effects of glycosylation on binding interactions, explaining observations like the improved binding seen with the N90E mutation that eliminates a glycosylation site . By integrating these computational approaches with experimental validation, researchers can develop more accurate predictive models and accelerate the rational design of improved ACE2 variants for both research and therapeutic applications.