Enhancing Chromatographic Performance of Immobilized Angiotensin II Type 1 Receptor by Strain-Promoted Alkyne Azide Cycloaddition through Genetically Encoded Unnatural Amino Acid

Abstract

During integration to the solid surface, the effects of tags introduced for bioorthogonal reactions on protein activity have received far less investigation. This represents the major challenge of improving the performance of the immobilized protein-based assays. Herein, the relationship between the fusion tags and their reaction efficiency in mediating the assay performance was realized by determining the chromatographic performance using genetically encoded azide−alkyne cycloaddition, and Halo-and SNAP-tagged bioorthogonal reactions for synthesizing immobilized angiotensin II type 1 receptor (AT 1 R). We demonstrated that immobilization with the incorporation of unnatural amino acid in the receptor minimizes the peak tailings and broadenings of irbesartan, fimasartan, losartan, and tasosartan, while attachment via large tags (SNAP and Halo) leads to serious asymmetry peaks. Upon the first immobilization, the association constants of the four drugs to AT 1 R appeared to be 1 order of magnitude greater than the other two attachments. Such enhancement is likely reasoned by the improved association rate constants and the relatively identical dissociation rates due to the small tag. While demonstrating improved chromatographic performance, the immobilized AT 1 R prepared by the genetically encoded azide−alkyne reaction was applied in analyzing Uncaria Schreber nom. cons. extract, which identified hynchophylline as a specific ligand binding to the receptor. As immobilized proteins move toward diverse assays, our findings provide an unprecedented insight into the relation between fusion tags and their reaction efficiency in mediating the assay performance, which is thus dedicated to the creation of a protein-functionalized surface for precisely determining the drug−protein interaction and discovering the specific partner of the protein.

Affinity Chromatographic Method for Determining Drug–Protein Interaction with Enhanced Speed Than Typical Frontal Analysis

“…Since coined by Ellen M. Sletten and Carolyn R. Bertozzi in 2009, bioorthogonal chemistry/reaction has been successfully applied in different fields including in vivo imaging and protein conjugation, owing to the mild reaction conditions, high specificity, and minimized interfering with the native biochemical process. Taking inspiration from these advantages, our group was dedicated to create a one-step method for immobilizing a series of proteins like adrenoceptors, endothelin receptors, and angiotensin receptors from cell lysates using bioorthogonal reactions. , Upon these cases, this work extended the halo-tag mediating bioorthogonal dehalogenation reaction to the immobilization of PARP 1 . As illustrated in Figure a,b, the reaction occurred between the fusion protein in the cell lysates and the chloropropyl-group on the gel surface since demonstrable loss of band densities was observed in the gel.…”

“…The way to synthesize immobilized protein is a debated and critical factor needing to be considered when HPAC is applied in drug–protein binding analysis using immobilized protein as the stationary phase. , This raises the question as to what extent the stationary phase models the behavior of the original protein since immobilization proves to have an influence on the protein activity. In most cases, it induces loss of protein functionality by disordered orientation, denaturation, and changes of solubility .…”

“…Taking inspiration from these advantages, our group was dedicated to create a one-step method for immobilizing a series of proteins like adrenoceptors, endothelin receptors, and angiotensin receptors from cell lysates using bioorthogonal reactions. 35,38 Upon these cases, this work extended the halotag mediating bioorthogonal dehalogenation reaction to the immobilization of PARP 1 . As illustrated in Figure 1a,b, the reaction occurred between the fusion protein in the cell lysates and the chloropropyl-group on the gel surface since demonstrable loss of band densities was observed in the gel.…”

Site-Specific Covalent Immobilization of SMA-Stabilized ACE2 for SARS-CoV-2 Recognition and Drug Screening

“…The histidine tag (His-tag) contains six consecutive histidines with a molecular weight of only 0.84 kDa, which has minimal interference with protein expression and folding. , The imidazole part of His-tag can interact with immobilized metal-NTA complexes via noncovalently binding ( K D ≈ 1–10 μM), so they are widely used in the separation and purification of recombinant proteins . However, the reversible binding of His-tag to NTA leads to its slow and continuous dissociation, which is not suitable for protein immobilization.…”

Rapid separation and binding configuration prediction of the components in Danshen decoction to endothelin A receptor using affinity chromatography and molecular dynamics simulation

“…Since the first synthesization by Wainer's group for drug-receptor interaction analysis [15], the receptor stationary phase has been dramatically improved and optimized for drug discovery purposes. i) The receptors were immobilized via site-directed covalent methods rather than physical adsorption/weak affinity/random covalent methods to improve the stability of the receptor [16]; ii) The receptors were immobilized via bioorthogonal reactions to avoid the tedious purification steps [17][18][19]; iii) The receptors were immobilized via small tags like unnatural amino acid to improve the activity of the receptors [20]; iv) A series of methods (e.g., nonlinear chromatography, adsorption energy distribution, and injection amount dependent methods) [21][22][23] and parameters (binding efficiency index and surface efficiency index) [24] were introduced to evaluate the drug-like property of natural products rapidly. As an important quantitative metric for drug-like property evaluation, the kinetic data could reflect the time-dependent changes of ligand-receptor complex, thus, highly related to the druggability of a ligand [25].…”