ACE2 (18-740) Human, Biotin

What is ACE2 (18-740) Human, Biotin and what are its structural characteristics?

ACE2 (18-740) Human, Biotin is a recombinant protein comprising amino acids 18-740 of the human Angiotensin-Converting Enzyme 2, expressed in HEK293 cells and enzymatically biotinylated. The protein contains the extracellular domain of ACE2 that retains both catalytic activity and binding capability for SARS-CoV-2 spike protein. Structurally, it features a C-terminal His-tag or His-AVI tag used for purification and detection, with biotinylation typically occurring at a specific lysine residue within the AVI tag sequence . The recombinant protein has a predicted molecular weight of approximately 87.2 kDa but typically migrates at 95-125 kDa under reducing conditions on SDS-PAGE due to glycosylation .

The protein maintains its native zinc-dependent metalloprotease activity domain containing the HEXXH zinc-binding motif, crucial for its carboxypeptidase function. This biotinylated version provides advantages for immobilization or detection in multiple experimental systems while preserving biological activity. The biotinylation level typically exceeds 90%, allowing efficient capture by streptavidin surfaces or detection reagents . The monomeric protein in solution can form functional dimers under certain experimental conditions, which may influence binding kinetics measurements.

What enzymatic activities does ACE2 (18-740) Human, Biotin retain compared to native ACE2?

ACE2 (18-740) Human, Biotin retains the essential carboxypeptidase activities of the native transmembrane protein despite lacking the membrane-spanning domain. It efficiently converts angiotensin I to angiotensin 1-9 and angiotensin II to angiotensin 1-7, maintaining its critical role in regulating the renin-angiotensin system . Enzymatic assays demonstrate that the biotinylated recombinant protein exhibits specific activity typically exceeding 1,000,000 U/mg, confirming its functionality .

This recombinant protein also maintains the ability to cleave multiple biologically relevant peptides beyond angiotensins. These include apelins (apelin-13, [Pyr1]apelin-13, apelin-17, apelin-36), casomorphins (beta-casomorphin-7, neocasomorphin), and dynorphin A with high efficiency . Additionally, it cleaves neurotensin, kinetensin, and des-Arg bradykinin, but shows no activity against bradykinin itself. The enzymatic activity can be measured using fluorogenic peptide substrates such as Mca-YVADAPK(Dnp)-OH, allowing researchers to verify functionality before binding experiments . Importantly, the biotinylation process preserves these catalytic functions when properly performed, with no significant activity differences observed between biotinylated and non-biotinylated variants.

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

ACE2 (18-740) Human, Biotin requires careful storage and handling to maintain its structural integrity and enzymatic activity. The protein is typically supplied as a sterile filtered clear solution in a buffer containing Tris (pH 8.0), NaCl, KCl, imidazole, and glycerol . For long-term storage, temperatures of -20°C or below are recommended, with -80°C preferred for periods exceeding 6 months. The protein should be stored in small aliquots to minimize freeze-thaw cycles, as repeated freezing and thawing significantly reduces activity .

When working with the protein, maintain it on ice and use within 8 hours after thawing. The addition of zinc (typically as ZnCl₂) in working buffers at concentrations of 10-50 μM helps maintain enzymatic activity since ACE2 is a zinc-dependent metalloprotease . Avoid buffers containing metal chelators such as EDTA or EGTA, which can strip the catalytic zinc and inactivate the enzyme. Optimal pH for stability ranges from 7.2-8.5, with activity decreasing significantly outside this range. For biotinylated preparations, minimize exposure to free biotin in experimental buffers, which can compete for streptavidin binding sites in downstream applications.

What methods can be used to validate ACE2 (18-740) Human, Biotin functionality before experimental use?

Validating the functionality of ACE2 (18-740) Human, Biotin before experimental use is critical for generating reliable research data. Multiple validation approaches are recommended, focusing on both enzymatic activity and binding capability. For enzymatic activity assessment, researchers can employ fluorogenic peptide substrates like Mca-YVADAPK(Dnp)-OH, where ACE2-mediated cleavage yields measurable fluorescence increases . Additionally, HPLC analysis of angiotensin II conversion to angiotensin 1-7 provides a direct measurement of physiologically relevant activity.

For binding functionality, a functional ELISA remains the gold standard, where immobilized ACE2 (18-740) Human, Biotin should successfully bind to SARS-CoV-2 spike protein receptor binding domain (RBD) at concentrations of approximately 2 μg/ml . Surface plasmon resonance (SPR) offers a more quantitative assessment, with properly functional biotinylated ACE2 exhibiting binding affinity to SARS-CoV-2 spike RBD of approximately 15 nM . Biotinylation levels can be verified using gel shift assays with streptavidin, where complete migration shifts indicate high-quality biotinylation exceeding 90%. Lastly, SDS-PAGE analysis should show a predominant band at 95-125 kDa, with purity exceeding 95% for most research applications.

How can ACE2 (18-740) Human, Biotin be optimally utilized in SARS-CoV-2 binding studies?

ACE2 (18-740) Human, Biotin offers significant advantages in SARS-CoV-2 binding studies due to its high-affinity interaction with the viral spike protein and the versatility provided by biotinylation. For optimal experimental design, researchers should consider several methodological approaches. In biolayer interferometry (BLI) experiments, streptavidin sensors loaded with biotinylated ACE2 at concentrations of 5-10 μg/ml in PBS with 0.05% Tween-20 provide stable baselines and consistent binding data. Association and dissociation kinetics should be measured using spike protein concentrations ranging from 1-100 nM to accurately determine KD values .

For ELISA-based binding assays, optimal coating concentration of streptavidin is 5 μg/ml, followed by capture of biotinylated ACE2 at 2 μg/ml. When developing neutralization assays, pre-complexing ACE2 with spike protein for 30 minutes at room temperature before adding to target cells yields the most reproducible inhibition data. For flow cytometry applications, streptavidin-fluorophore conjugates at 1:500 dilution effectively detect ACE2-bound viral particles or expressing cells. Importantly, including controls with non-biotinylated ACE2 and irrelevant biotinylated proteins helps distinguish specific from non-specific interactions. Zinc supplementation (10 μM ZnCl₂) in binding buffers enhances structural stability without interfering with spike protein interactions .

What methodological considerations are important when using ACE2 (18-740) Human, Biotin in surface plasmon resonance experiments?

Surface plasmon resonance (SPR) experiments with ACE2 (18-740) Human, Biotin require careful optimization to generate reliable binding data. For sensor chip preparation, streptavidin-coated chips (e.g., SA chips) should be conditioned with three 1-minute injections of 1 M NaCl/50 mM NaOH before ACE2 immobilization. Optimal biotinylated ACE2 loading concentrations range from 0.5-5 μg/ml at flow rates of 5-10 μl/min, targeting a response level of 200-400 RU to minimize mass transport limitations while maintaining sufficient signal .

Running buffer composition significantly impacts data quality, with HBS-EP+ (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20) supplemented with 10 μM ZnCl₂ providing optimal stability for ACE2 while minimizing non-specific binding. Regeneration conditions must be carefully optimized; mild regeneration with 10 mM glycine pH 2.0 for 15-30 seconds typically removes bound analytes without damaging immobilized ACE2. For kinetic analyses, spike protein concentrations should span 0.5-50 nM, with an empty flow cell and irrelevant protein injections serving as references. Data analysis using a 1:1 Langmuir binding model typically yields KD values of 10-20 nM for wild-type spike protein . Temperature stability studies should be conducted at 4-37°C to understand physiological binding dynamics, with most consistent results obtained at 25°C.

How do glycosylation patterns of recombinant ACE2 (18-740) Human, Biotin impact binding affinity to SARS-CoV-2 variants?

The glycosylation pattern of ACE2 (18-740) Human, Biotin significantly influences its binding characteristics to SARS-CoV-2 spike protein variants. Human ACE2 contains seven potential N-linked glycosylation sites within the recombinant extracellular domain (18-740), and the specific glycan structures present depend on the expression system used. HEK293-derived ACE2 exhibits complex N-glycans that more closely resemble native human patterns compared to insect or yeast expression systems . These glycosylation differences can alter binding affinities to different SARS-CoV-2 variants by up to 2-4 fold.

Comparative binding studies between fully glycosylated and enzymatically deglycosylated ACE2 reveal that N-glycans at positions N90 and N322 particularly influence the interaction with the receptor binding domain (RBD) of variant B.1.1.7 (Alpha), while having minimal effect on ancestral strain binding. The following table summarizes observed binding affinity differences across SARS-CoV-2 variants:

These findings highlight the importance of maintaining consistent ACE2 glycosylation patterns across experiments when comparing variant binding affinities. Researchers should characterize glycosylation profiles using lectin blotting or mass spectrometry when precise binding measurements are required .

What are the critical controls needed when using biotinylated ACE2 in viral neutralization assays?

Designing robust viral neutralization assays using ACE2 (18-740) Human, Biotin requires implementation of several critical controls to ensure data validity and reproducibility. First, include a biotinylation control using irrelevant biotinylated protein of similar size (e.g., biotinylated BSA) at equivalent concentrations to verify that observed neutralization effects are specific to ACE2 rather than resulting from the biotin moiety or general protein interference . Second, incorporate both non-biotinylated ACE2 and the soluble form of ACE2 (without tags) to distinguish potential functional differences caused by the biotinylation process.

Dose-response curves must include sufficient concentration ranges, typically spanning 0.01-100 μg/ml of ACE2, to accurately determine IC50 values. Time-course controls are essential, as pre-incubation time between virus and ACE2 significantly impacts neutralization efficiency, with 30-60 minutes at 37°C generally yielding optimal results. Cell-based assays should include ACE2-negative cell lines to confirm the specificity of the neutralization mechanism. For pseudovirus systems, VSV or lentivirus constructs lacking spike protein provide critical background controls. Finally, established neutralizing antibodies with known IC50 values should be included as benchmark controls to normalize results across different experimental batches. The following control checklist ensures comprehensive experimental validation:

• Irrelevant biotinylated protein control

• Non-biotinylated ACE2 control

• Soluble ACE2 without tags

• Full dose-response curve (0.01-100 μg/ml)

• Pre-incubation time course (15, 30, 60, 120 minutes)

• ACE2-negative cell line control

• Virus/pseudovirus lacking spike protein

• Reference neutralizing antibody

How can researchers develop a competitive binding assay using ACE2 (18-740) Human, Biotin to screen for potential therapeutic inhibitors?

Developing a competitive binding assay using ACE2 (18-740) Human, Biotin provides an efficient platform for screening potential therapeutic inhibitors of SARS-CoV-2 entry. The optimal assay design utilizes streptavidin-coated microplates to capture the biotinylated ACE2 at a concentration of 2 μg/ml, creating a uniform binding surface. After washing, a fixed concentration of labeled SARS-CoV-2 spike RBD (typically 10-20 nM, depending on the KD) is added in the presence or absence of test compounds . The RBD can be labeled with a fluorophore, HRP, or other detection tag depending on the desired readout system.

For assay optimization, researchers should determine the concentration of labeled RBD that gives 70-80% of maximum binding (EC70-EC80) for the competition studies. The Z' factor for a properly optimized assay should exceed 0.7, indicating excellent assay quality. Testing compounds should be pre-incubated with the labeled RBD for 30 minutes before adding to the immobilized ACE2. Including control inhibitors with known IC50 values, such as soluble ACE2 (IC50 ~50 nM) or characterized neutralizing antibodies, provides essential reference points for comparing compound potency .

The assay buffer composition significantly impacts screening results. Optimal conditions include PBS pH 7.4 with 0.05% Tween-20, 0.1% BSA, and 10 μM ZnCl₂. Higher detergent or salt concentrations may disrupt weak but potentially important inhibitor interactions. For high-throughput applications, a 384-well format with 50 μl reaction volumes provides sufficient sensitivity while conserving reagents. When analyzing results, plot percent inhibition versus inhibitor concentration and fit to a four-parameter logistic equation to determine IC50 values. Counter-screening against biotinylated irrelevant proteins helps identify false positives that interact with the biotin-streptavidin system rather than disrupting the ACE2-RBD interaction.

How can discrepancies in ACE2 enzymatic activity across different biotinylated preparations be reconciled?

Variations in enzymatic activity between different preparations of ACE2 (18-740) Human, Biotin present significant challenges for research reproducibility. These discrepancies typically stem from several factors that can be systematically addressed. First, the position and degree of biotinylation significantly impact activity, with site-specific enzymatic biotinylation via AVI-tag causing minimal interference compared to random chemical biotinylation methods . Researchers should verify biotinylation locations using mass spectrometry to ensure consistency between batches and vendors.

Buffer composition plays a crucial role in maintaining enzymatic function. The presence of stabilizing agents such as 10-50 μM ZnCl₂ and 10% glycerol significantly improves activity retention during storage. When comparing different preparations, standardizing the assay conditions becomes essential—use identical substrate concentrations, buffer compositions, and incubation times. A reference standard curve using a well-characterized ACE2 preparation allows normalization of activity across batches . The following standardization protocol ensures comparable activity measurements:

• Prepare master mix of fluorogenic substrate Mca-YVADAPK(Dnp)-OH at 10 μM in reaction buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10 μM ZnCl₂, 0.01% Brij-35)

• Add 50 μl substrate to 50 μl of serially diluted ACE2 preparations (0.1-100 ng/ml)

• Incubate at 37°C for precisely 30 minutes

• Stop reaction with 25 μl of 1M acetic acid

• Measure fluorescence (excitation 320 nm, emission 405 nm)

• Calculate specific activity as units per mg protein

This standardized approach typically reduces inter-batch variability from >50% to <15%, allowing more meaningful comparisons between different ACE2 preparations .

What are the best approaches for quantifying the degree of biotinylation in ACE2 preparations?

Mass spectrometry provides the most definitive biotinylation analysis. MALDI-TOF or ESI-MS can determine the exact number of biotin molecules per ACE2 protein by measuring the mass shift (~226 Da per biotin). Tryptic digestion followed by LC-MS/MS enables identification of specific biotinylated residues, particularly valuable when assessing site-specific enzymatic biotinylation via AVI-tag. For routine quality control, the streptavidin gel-shift assay offers a practical approach—incubating the biotinylated ACE2 with excess streptavidin causes a substantial molecular weight increase detectable by native PAGE, with the fraction of shifted protein corresponding to the biotinylated fraction .

A biotin quantification fluorescence assay using fluorophore-labeled streptavidin provides another sensitive option. The following protocol yields reliable biotinylation measurements:

• Prepare standards using non-biotinylated ACE2 spiked with known ratios of fully biotinylated ACE2

• Capture 100 ng protein samples on anti-ACE2 antibody-coated microplate wells

• Probe with streptavidin-Cy3 (1:2000 dilution)

• Measure fluorescence (excitation 550 nm, emission 570 nm)

• Calculate percent biotinylation using standard curve

This approach detects biotinylation levels between 5-100% with high accuracy (~±3%) and provides a reliable quality control method for batch consistency .

What factors influence the binding kinetics between ACE2 (18-740) Human, Biotin and SARS-CoV-2 spike protein variants?

Multiple factors influence the binding kinetics between ACE2 (18-740) Human, Biotin and SARS-CoV-2 spike protein variants, requiring careful experimental design and interpretation. Temperature significantly affects both association (ka) and dissociation (kd) rates, with measurements at 37°C typically showing 2-3 fold faster association but also increased dissociation compared to room temperature (25°C) experiments. This temperature dependency must be standardized when comparing variants or inhibitors .

The glycosylation state of both ACE2 and spike protein substantially affects binding parameters. Comparative analysis of kinetic values for different experimental conditions reveals:

These findings emphasize the importance of maintaining consistent experimental conditions when comparing spike variants or evaluating potential inhibitors .

How might engineered ACE2 (18-740) Human, Biotin variants be utilized to study emerging SARS-CoV-2 variants?

Engineered variants of ACE2 (18-740) Human, Biotin represent powerful tools for investigating emerging SARS-CoV-2 variants and developing improved therapeutics. Strategic amino acid substitutions based on structural analysis can enhance affinity for specific viral variants or broaden binding across multiple variants. For example, mutations like N90Q (removing a glycosylation site) and T92Q increase binding affinity to the Delta variant by approximately 3-fold without significantly altering binding to other variants . Systematic alanine scanning mutagenesis of the ACE2 binding interface identifies critical contact residues that could be modified to create variant-specific detection reagents.

Biotinylated ACE2 engineered with increased thermal stability (through disulfide bond introduction or consensus sequence design) provides more robust detection platforms for field applications. Creating domain-swapped chimeric proteins incorporating elements from ACE2 homologs found in resistant species (e.g., mice have low spike binding despite 82% ACE2 sequence identity) offers insights into species-specific viral susceptibility mechanisms . For multiplexed detection systems, differentially tagged ACE2 variants enable simultaneous profiling of multiple viral variants in a single sample.

Future development directions include creating ACE2 fusion proteins with reporter enzymes directly attached instead of biotin, eliminating the streptavidin capture step for more streamlined assays. Site-specific introduction of photo-crosslinking amino acids at the binding interface would allow covalent capture of transient spike protein conformations for structural studies of fusion intermediates. These engineered ACE2 variants provide essential research tools for monitoring SARS-CoV-2 evolution and developing next-generation therapeutics as the virus continues to adapt .

What are the emerging applications of ACE2 (18-740) Human, Biotin in therapeutics development?

Biotinylated ACE2 (18-740) Human is emerging as a versatile platform for developing novel therapeutic approaches beyond its conventional research applications. As a tool for drug discovery, immobilized biotinylated ACE2 enables high-throughput screening of small molecule libraries to identify compounds that disrupt the ACE2-spike interaction. The precise orientation provided by streptavidin-biotin capture ensures consistent exposure of the binding interface for more reliable screening results compared to randomly oriented protein .

In antibody development, biotinylated ACE2 serves as both a selection tool and a competition standard for evaluating therapeutic antibodies. By designing competitive binding assays with labeled ACE2, researchers can rapidly identify antibodies that block the ACE2-spike interaction without requiring live virus neutralization assays, accelerating the screening process. The well-defined biotinylation site provides consistent presentation of ACE2, minimizing batch variation in screening campaigns .

Several innovative therapeutic approaches utilize ACE2 (18-740) Human, Biotin directly:

• Decoy Receptor Strategy: Biotinylated ACE2 conjugated to nanoparticles creates multivalent viral traps with enhanced avidity compared to soluble ACE2.

• Bispecific Constructs: Fusion of biotinylated ACE2 with Fc domains or other binding moieties creates molecules that both neutralize virus and recruit immune effectors.

• Targeted Delivery: Conjugation with cell-type specific antibodies allows directed delivery of ACE2 decoys to tissues with high viral loads.

• Diagnostic Applications: Integration into lateral flow assays provides rapid detection of spike protein with physiologically relevant binding.

The ability to precisely control orientation through the biotin-streptavidin interaction makes these applications more reproducible and manufacturable than approaches using random chemical conjugation methods .

How can ACE2 (18-740) Human, Biotin be utilized to investigate the dual enzymatic and viral receptor functions in disease models?

The dual functionality of ACE2 as both an essential enzyme in the renin-angiotensin system and the primary receptor for SARS-CoV-2 creates unique research opportunities using biotinylated ACE2 (18-740) Human. This bifunctional protein enables investigation of how viral binding affects endogenous enzymatic activity—a potentially crucial mechanism in COVID-19 pathophysiology. By using site-specifically biotinylated ACE2, researchers can immobilize the protein in defined orientations that preserve both functions for mechanistic studies .

Experimental approaches to study this dual functionality include real-time monitoring systems where enzymatic activity is measured before, during, and after spike protein binding. Using fluorogenic substrates like Mca-YVADAPK(Dnp)-OH, researchers can quantify how spike binding modulates catalytic efficiency (kcat/KM). Current data indicate that spike binding reduces angiotensin II conversion by 40-60%, potentially contributing to angiotensin II accumulation and associated vascular pathologies in COVID-19 patients .

Advanced tissue culture models incorporating biotinylated ACE2 enable investigation of organ-specific effects. For pulmonary research, ALI (air-liquid interface) cultures of primary bronchial epithelial cells can be supplemented with biotinylated ACE2 to study how viral binding alters local angiotensin processing. Similar approaches using kidney organoids examine potential mechanisms of renal injury in COVID-19. The biotinylation allows precise tracking of exogenous ACE2 distribution and fate using streptavidin-conjugated fluorophores or quantum dots .

Mouse models with tissue-specific expression of human ACE2, combined with administered biotinylated ACE2, allow in vivo investigation of therapeutic potential while monitoring both viral neutralization and enzymatic restoration. The biotinylated protein's extended serum half-life (compared to non-biotinylated versions) provides a pharmacokinetic advantage for such studies while allowing precise quantification through biotin-specific detection methods .