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. 2026 Sep 7;49(9):e70524. doi: 10.1002/jssc.70524

Construction of α 1A‐Adrenoceptor‐Based Stationary Phase Triggered by the Ultrahigh Affinity Protein Pair Colicin E7 Dnase /IM7 for Targeted Bioactive Compound Screening in Herba leonuri

Xiaofang Qiao 1,2, Haosen Zhang 1, Yue Zhang 1, Qian Liu 1, Jia Guo 1, Jing Wang 1,3,✉
PMCID: PMC13548337  PMID: 42703902

ABSTRACT

Immobilization of functional proteins onto solid surfaces is pivotal to propel the advancement of biomaterials in both biomedical and industrial technologies. However, the process has been hindered when it comes to G protein‐coupled receptors due to the homogeneity of the seven transmembrane proteins. Herein, we exploited the efficient ultrahigh affinity of colicin E7 (CL7) and its cognate immunity protein IM7 for conjugation of functional protein onto macro‐porous silica gel and established a robust chromatographic system for separation of compounds from complex matrices. Benefiting from the ultrahigh affinity nature of the protein pair, conjugation of α1A‐adrenoceptor (α1A‐AR) was achieved at ambient conditions in less than 20 min with high specificity and stability. Feasibility of the platform was tested by determining the binding parameters between drugs and receptor under various conditions (different pHs and concentration of methanol). Under the desired condition, carvedilol gave the association constant of (11.56±0.7) × 105 M−1 indicating immobilization of α1A‐AR via this method still enhances its binding activity to drugs. Furthermore, stachydrine and leonurine were obtained as the bioactive compounds targeting the immobilized α1A‐AR. Our study is expected to offer a versatile platform to achieve effective and specific immobilization of other functional proteins, thus enabling easy separation of the target compound from complex systems.

Keywords: colicin E7 Dnase, inhibiting protein 7(IM7), targeted compound screening, ultra‐high affinity protein pair, α1A‐adrenoceptor

Benefiting from the ultrahigh affinity nature of the protein pair CL7/IM7, the α1A‐adrenoceptor was coated to the surface of the silica gel for construction of the chromatographic system. Stachydrine and leonurine were screened as the compounds in Herba leonuri that targeting the immobilized receptor by using this method.

graphic file with name JSSC-49-e70524-g003.webp

1. Introduction

The last decades have witnessed remarkable advancements in analytical methods which have revolutionized this field by significantly improving accuracy, sensitivity, and efficiency. Notably, with the progress in biotechnology, methodologies for the fabrication of functional protein‐based bio‐surfaces have gained increasing attention especially those approaches that enable controlled orientation, high specificity, and robust activity of proteins. These innovative methodologies are closely integrated with development of proteomics, interactomics, clinical diagnosis, and drug discovery [1, 2, 3, 4, 5]. That is because it facilitates a systematic understanding of biological phenomena at the molecular level. Apart from this, it has raised investigations of microarrays, biosensors, biochips, and continuous flow reactor systems into new heights where the target proteins are immobilized with the merits of improved stability, specificity, and reusability in continuous processes [6, 7, 8].

The literature has listed numerous strategies that are employed for protein immobilization, especially for G protein‐coupled receptors (GPCRs) [1, 9]. The non‐covalent protein adsorption method, relying on either passive adsorption or electrostatic interactions, was popularized in the earlier period for protein immobilization because it does not need additional coupling agents and modifications of the proteins. But the relatively weak interactions have hampered its wide applications. The covalent immobilization methods usually immobilize target proteins via covalent bonds which rely on the naturally present functional groups like the exposed amine groups or carboxyl groups. Among which, site‐specific covalent attachment is more attractable for its ability to offer precise control over the immobilized proteins. It enables a uniform conformation of the protein, thereby enhancing its robustness and minimizing steric hindrance between the protein and solid support [9, 10, 11]. However, the methods that are utilizing this strategy is challengeable due to the numerous functional groups on the side chain of the protein and the cysteine thiol groups in disulfide forms which complicates the process [12]. Consequently, the tertiary structure of the immobilized protein is compromised, and the protein's function is often lost [13, 14]. To address these limitations and establish a more stable protein conjugates, a method that relies on high selectivity of natural ligand‐receptor complexes like glutathione S‐transferases tag/glutathione, streptavidin/biotin has emerged [15, 16]. Yet, for any of the aforementioned affinity‐directed immobilization methods, sizable tags should be incorporated into the terminus of the target protein, which limits the density of the immobilized proteins.

The high affinity protein pairs like Fab1/GA1 (Kd∼50 nM) [17], LD/LDI (Kd∼42 pM) [18], pyocin S2 DNase/ImS2(Kd∼10−14 M) [19], and Colicin E7 DNase(CL7)/Inhibiting protein 7(IM7)(Kd∼ 10−17 M) [20], are always used in the structure analysis of protein complexes, purification of target proteins, and detection proximity assay based on the β‐lactamase split enzyme system since they are reported in the literatures. Few of them have been used for construction of bio‐surfaces for analytical tests, especially for chromatographic investigations. CL7/IM7, a reported ultra‐high protein pair (with a dissociation constant of 10−17 M), has been extensively applied in purification of various proteins in a single step. Considering this, we believe that it preserves a high tendency that capable of overcoming the limitations in target protein immobilization with the priority of ultra‐high affinity, high efficiency, and high activity. Taking advantage of this protein pair, Hosse et al. have developed a ligand capture system suitable for accurate kinetic ranking of library clones [21, 22]. But, they have been seldom used in construction of the chromatographic systems, especially for GPCRs.

In a recent study, the CL7/IM7 has been reported for the establishment of a chromatographic system to investigate the binding interactions between compounds and the immobilized receptor [23]. Screening methods for bioactive compounds have received little attention, especially regarding the development and validation of the corresponding analytical process. Herein, we attempted to construct the chromatographic system using the immobilized α1A‐adrenoceptor(α1A‐AR) as the stationary phase to screen bioactive compounds in a complex system. The stationary phase was prepared via the ultra‐high affinity CL7/IM7 protein pair, a strategy that ensured the immobilized α1A‐AR retained high activity. After evaluation of the efficiency and the amount of the immobilized α1A‐AR, the morphology and bioactivity of the stationary phase were characterized by scanning electron microscopy (SEM), x‐ray photoelectron spectroscopy (XPS), and chromatographic analysis. More importantly, stachydrine and leonurine were identified as the targeted compounds screened from Herba leonuri that binding with the immobilized α1A‐AR. Our results demonstrated that the established method preserves the merits of robustness, specificity, and efficiency. Taken together, it has confirmed the great potential of this ultra‐high affinity protein pair in bio‐surface fabrication, especially for functional proteins.

2. Experimental

2.1. Preparetion of the Immobilized α1A‐AR ColumnTriggered by CL7/IM7 Protein Pair

We immobilized the expressed CL7‐tagged α1A‐AR onto the macro‐porous silica gel surface via CL7/IM7 protein pair in a covalent mode through the following steps.

2.1.1. Activation of the Silica Gel

1.5 g of the bare silica gel was weighed, dried, and incubated in 40 mL of 20% HCl at 110°C for 2 h by hot refluxing method at 60°C using nitrogen as the protection gas overnight. The gels were then incubated in anhydrous toluene, and activated with γ‐aminopropyl triethoxysilane (3.0 mL) for 12 h under the nitrogen atmosphere. With the removal of the unreacted section, we obtained the activated silica gel by washing it with toluene (5 mL × 3), dried it at 60°C overnight.

2.1.2. Functionalization of the Gel

The resulting gels were treated by stirring for 2 h in 30 mL of anhydrous acetonitrile within 0.6 mM of carbonyldiimidazole. Following washing with anhydrous acetonitrile and tris‐HCl buffer (20 mM, pH = 7.40) for three times, the gels were suspended into the tris‐HCl buffer containing a certain content of the purified His‐tagged IM7 and stirred for 2 h. Then the gels were washed by the same buffer before the functionalized gels were well prepared.

2.1.3. Ligation of the Target Protein

We harvested α1A‐AR coated gels by suspended IM7 functionalized gels in 50 mL of the cell lysate containing CL7‐tagged α1A‐AR and stirred for 0.5 h. Following the washing steps, the gels were transferred to the solution containing glycine in a concentration of 1% for 30 min. Then, they were rinsed by 20 mM phosphate buffer (PB, pH = 7.40).

2.1.4. Packing of the Immobilized α1A‐AR Column

The immobilized α1A‐AR was packed into a steel stainless column (4.6 × 30 mm) by a slurry method using 20 mM PB as the displacement reagent.

2.2. Evaluation of the Immobilized α1A‐AR Column

2.2.1. Binding Interaction Analysis

We prepared series concentrations of drugs to investigate the binding interactions between the drugs and the immobilized α1A‐AR. They were 0.01, 0.08, 0.1, 0.2, 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mmol/L for phentolamine, 0.01, 0.05, 0.1, 0.2, 0.5, 0.8, 1.0, 1.5, 2.0, 3.0, and 4.0 mmol/L for carvedilol, 0.01, 0.05, 0.1, 0.2, 0.5, 0.75, 1.0, 1.5, and 2.0 mmol/L and labetolol. The mobile phases were 20 mM phosphate buffer (PB, pH = 7.40) for phentolamine and labetolol with detection wavelength of 278 nm and 230 nm for the two drugs, respectively. While for carvedilol, the mobile phase was 20 mM PB within isopropanol (5%), and the detection wavelength was 285 nm, respectively. The injection volume, the flow rate, and the temperature for the interaction analysis were 10 µL, 0.5 mL/min, and 25°C. Triplicate injections were used for these drugs.

2.2.2. Bioactive Compound Separation

We tested the immobilized α1A‐AR chromatography by utilizing it in screening bioactive compounds from a complex system which was performed using Herba leonuri as a real sample. A total of 10 µL Herba Leonuri extraction was injected into the column containing the immobilized α1A‐AR to screen the bioactive compounds that specifically bind to the receptor. The mobile phase used here was 20 mM ammonium acetate buffer (pH = 7.40). The flow rate and the detection wavelength were set as 0.5 mL/min and 280 nm. We collected the retained fractions in the column for further analysis.

3. Results and Discussion

3.1. Quantitative Production of the Target Protein

Over‐expression of the target protein with high activity raises fundamental concerns for the fabrication of the bio‐surface. For such purpose, we selected a conventional method that uses a native expression system (E. coli cells) for quantitative production of the target proteins. This is because the E. coli system is demonstrated to be well‐characterized and rapidly growing, which are easily to be cultured and selected for protein production. Second, a large variety of expression plasmids exist due to the genetic tractability nature of the cells. Third, the report has confirmed that it is easy to conduct isotopic labeling experiments by using this system [24]. As such, in this work, we used E. coli as the expression system and used CL7 as the fusion partner to direct the correct insertion of the receptor into the membrane whilst increasing the overall stability. As illustrated in Figure 1A–C, we achieved successful soluble expression of CL7‐tagged α1A‐AR in the supernatant of the cell lysate at the molecular weight of about 67.0 kDa. Such a result was reasonable since the theoretical molecular weight of CL7‐tag and α1A‐AR were 14.73 and 51.49 kDa, respectively. Meanwhile, a large amount of His‐tagged IM7 was expressed at the proper condition and purified for further immobilization of α1A‐AR (Figures 1D–F and Figure S1). The high concentrations of the target proteins are more than sufficient for further immobilization of α1A‐AR. Furthermore, Western Blot analysis had validated the successful expression of His‐tagged IM7 and CL7‐tagged α1A‐AR. After evaluation and semi‐quantification of the bands, the amount of the purified IM7 and α1A‐AR were calculated as 103 µg/mL and 80 µg/mL. Yet, we found that the band of the purified His‐tagged IM7 migrated slight slower than the theoretical band (29.87 kDa). This may be because: (1) poly alkaline amino acid like 6×histidine at the C‐terminus of the target protein will binds to SDS, which may lower the migration rate of the protein. (2) Interaction of the hydrophilic proteins to water may lead to an increased size of the protein complex which will slow down the migration rate (> 60% of the hydrophilic amino acid were included in the His‐tagged IM7).

FIGURE 1.

FIGURE 1

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Expression of CL7‐tagged α1A‐AR and His‐tagged IM7. (A) and (D) were the SDS‐PAGE analysis of the two proteins. (B) and (E) displayed the western blot analysis of the two proteins. (C) and (F) were the corresponding semi‐quantification analysis of the proteins. Lane M: the standard protein marker. Lane U: un‐induced cells from the LB media. Lane I: induced cells. Lane S: supernatant of the cell lysate. Lane P: precipitant of the cell lysate. Lane P1 and P2: the purified protein loaded with 10 and 20 µL. The loaded volumes of the other lanes were 10 µL.

3.2. Immobilized α1A‐AR Retains Strong Responsiveness to Its Specific Ligand

The immobilization diagram was illustrated in Figure 2. To confirm the successful immobilization of target receptor, we firstly analyzed the efficiency of the protein immobilized onto the surface of the gel using the bicinchoninic acid (BCA) method. We observed that the adsorption capacity of purified IM7 increased markedly within less than 20 min (Figure S2). Once the incubation time exceeded 20 min, the adsorption of IM7 remained stable, indicating that 20 min is the optimal incubation time for column fabrication. While for CL7‐tagged α1A‐AR, we further quantified the total protein content of cell lysates before and after immobilization. A total protein reduction of 300 nmol was detected, which confirmed the successful immobilization of α1A‐AR (Figure S3). The immunofluorescence analysis also demonstrated the effectiveness of using the CL7/IM7 protein pair in immobilizing a target protein (Figure 3A–D). Specifically, the analysis showed a significant increase in fluorescence intensity when the receptor was coated onto the gel surface. The enhancement in fluorescence suggests high activity of the immobilized α1A‐AR for its antibody. No contaminants or aggregates of other proteins were detected during the immobilization process, thereby confirming the successful application of the protein pair in immobilizing the target protein.

FIGURE 2.

FIGURE 2

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Diagram for construction of α1A‐AR‐based chromatographic bio‐surface on silica gel. The silica gel was presented as blue circle. The receptor was displayed as helix. The gels were modified with IM7 using N, N′‐carbonyldiimidazole as the linker. Following immersion in the cell lysate for a 20‐min incubation period, the CL7‐tagged α1A‐AR were coated to the gel surface to create an α1A‐AR‐based chromatographic bio‐surface.

FIGURE 3.

FIGURE 3

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Characterization of the immobilized α1A‐AR. (A–D) were the Immunofluorescence analysis of the bare silica gel, aminopropyl silica gel, IM7 modified gel, and α1A‐AR coated gel. (E–H) were the corresponding SEM analysis of the gels. (I–L) were the XPS analysis of the gels.

SEM images of the gels (Figure3E–H) showed a spherical morphology with a relatively macro porous surface at a diameter of 6.0 ± 0.002 µm of the bare silica gel. The aminopropyl silica gel and IM7 coated gel did not show obvious changes in both the diameters and the surfaces. While lager particle size (∼6.022 ± 0.005 µm) with a rougher surface was observed on the α1A‐AR coated gels. Considering the diameters of GPCRs like beta2 adrenoceptor is about 10 nm [25, 26], which is sufficient to recognize in size changes on the gel surface evenly in comparison to the bare silica gels. As such, we reasoned that the receptor has been successfully immobilized onto the gel surface.

XPS analysis also confirmed the successful immobilization of α1A‐AR (Figure 3I–L). The content of C1s increased from 33.02% in the bare silica gel to 45.32% in the α1A‐AR coated gel, while the content of N1s, absent in the bare gel, rose to 4.92% in the coated gel. Given a complete reaction between IM7 and the gel, the amount of α1A‐AR on the gel surface should be 300 nmol/g gel according to the density of IM7.

Furthermore, taking sodium nitrite, tamsulosin, labetolol, phentolamine, and carvedilol as probes, we tested the specificity of the chromatographic system (Figure S4A). We observed that the four drugs (tamsulosin, labetolol, phentolamine, and carvedilol) showed significant difference in retention times but the similar tailed peaks on the immobilized α1A‐AR column, suggesting immobilized α1A‐AR retained its bioactivity. Different from these drugs, sodium nitrite showed a shorter retention time and non‐tailed peak. This is reasonable since it was a non‐retained compound of α1A‐AR. Thus, we regarded retention time of sodium nitrite as the void time of the chromatographic system. Meanwhile, the retention behaviors of these drugs were also examined in the control column without the immobilized α1A‐AR, yielding results that were comparable to the retention of sodium nitrite on the immobilized α1A‐AR column (Table S1). The stability of the chromatographic system was tested by injection of tamsulosin under the desired chromatographic condition (Figure S4B). After 30 days of continuous flushing with the mobile phase, no demonstrable changes were observed for tamsulosin on the immobilized α1A‐AR column, either in retention time or in chromatographic profile, confirming the stability of the immobilized receptor throughout the investigation period. Overall, we demonstrated that CL7/IM7 is capable of anchoring target protein onto the silica gel surface for fabrication of bio‐surface via ultra‐high affinity.

3.3. The Immobilized Stationary Phase Exhibits Enhanced Stability and Selectivity

We tested the stability of α1A‐AR pro‐ and after‐ immobilization to evaluate whether immobilization of the receptor via the CL7/IM7 protein pair will improve the stability of the target protein. We found that after immobilization, α1A‐AR showed minimal loss of ligand binding capacities during the freeze (−80°C)–thaw (25°C) cycles when phentolamine was taken as an example (Figure S5). While for the free soluble α1A‐AR, the binding capacity lost an average of 14.8% during the first two freeze–thaw cycles, and 60.6% for the third and the fourth cycles. These results are predictable since G‐protein coupled receptors are sensitive to the existing environment, especially the membrane architecture. When the soluble receptor is treated to lose the membrane, it will easily lose the activity for binding to its specific ligand. In contrast, when the receptor was immobilized onto the gel surface through a covalent bond via the CL7/IM7 protein pair, the flexibility of the receptor was hindered because the solid space is applicable for the receptor to avoid aggregation.

Then, we checked the effect of pH and the solvent environment on the stationary phase. We measured the binding constants of the immobilized α1A‐AR at a specific pH range of 4.0–9.0. And the solvent effect was checked by adding a series of concentrations of methanol in the mobile phase. We defined the relative activity as the affinity of the immobilized α1A‐AR to phentolamine, relative to the binding constants of the same ligands at initial application under the desirable conditions (20 mM phosphate buffer solution, pH = 7.4). We found that the relative activity of the immobilized α1A‐AR increased where the pH value was in the range of 4.0–7.0 (from 5.3% to 79.6%, Figure S6A), and it decreased to 10% when the pH value increased to 9.0. Clear loss was found when the pH value was lower than 6.0 and higher than 8.0. It was reasonable since the extreme pH value of the solution induced denature of the protein. As such, the affinity of the drug to the immobilized α1A‐AR decreased. While with the increase of the concentration of methanol in the mobile phase, a decreased relative activity of the immobilized α1A‐AR appeared (Figure S6B). The highest concentration of methanol was determined to be 40%. When higher than this value, the activity decreased to 5%. Meanwhile, when the concentration of methanol decreased more than 10%, the activity increased to 80%.

3.4. Immobilized α1A‐AR Exhibits Improved Accuracy in Drug–Receptor Interaction Investigations

Three canonical drugs, phentolamine, carvedilol, and labetolol, were used to test the feasibility of the chromatographic system (Figure S7). Figure 4 illustrated the chromatograms of these drugs on the immobilized α1A‐AR column. We found asymmetric peaks appeared for these drugs at various concentrations. The observed tailing peaks were reasonable since the dissociation rate constant is always lower than the association rate constant. As such, we can calculate the binding parameters between the drugs and the immobilized α1A‐AR based on the correlation between the peaks and the kinetic constants [27] (Supporting Information S1). Meanwhile, the tendency of the corresponding capacity factors decreased when the injected concentration of the drug increased.

FIGURE 4.

FIGURE 4

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Nonlinear chromatography investigations between the immobilized α1A‐AR and phentolamine(A1), carvedilol(A2), and labetolol(A3), respectively. (B1–B3) were the trend of the corresponding capacity factor changing as the drug concentrations increased. (C1–C3) displayed the fitted curves (the black line) of each drug at appropriate concentration. The grey lines were the fitted curves. The association constants were calculated and recorded.

We then pre‐processed the raw data of these drugs at each concentration and normalized them as y = Y/C 0, and x = t/t 0, where C 0 is the concentration of solute injected multiplied by 5.22 × 10−2 (the width of the injection pulse calculated from the void volume of the system and a 20.0 µL loop), and t 0 is the void time of the chromatographic system. The representative experimental peak profiles and the fitted curves generated using non‐linear chromatography functions were displayed in Figure 4C1–C3 and compared using the “goodness of fit” parameters r 2. The results showed that the experimentally obtained peaks were in consistent with the fitted ones with corresponding coefficient parameters greater than 0.97. The corresponding association constants of these drugs were also calculated. It is worthwhile to compare the binding parameters investigated in this work and in the previous report. We found that our results are four orders of magnitude lower than the results from the literature using radioligand binding assay, but at the same order of magnitude when chromatographic method is applied in the binding constant investigations [28]. This is rational since the radioligand binding assay using 3H‐ligand represents the gold standard and most classical approach for determining the affinity of ligands for the receptor in an aqueous system. When the system changed to the solid‐liquid interface, the counts of the immobilized receptor strongly decreased, thus resulting a decreased tendency in the orders of magnitude in the binding constant. However, the results obtained from the current work were slightly higher than those from the previous report via chromatographic analysis. We attributed this to the ultrahigh affinity between the protein pair CL7/IM7, which brought an improvement of the binding constant in comparison to that from the literature. Furthermore, the detecting concentration of the drug using the current method was kept lower than that in the literature [29]. Taken together, the receptor showed an improved sensitivity and activity, providing evidence for recognizing its specific ligands. These results, taken together, have confirmed the possibility of immobilization of target proteins triggered by CL7/IM7 protein pair.

3.5. Rapid Discovery of the Selective α1A‐AR Ligand by the Immobilized Receptor Column

Over 300 prescriptions have been prescribed with Herba leonuri in clinical practice to confirm its diverse pharmacological effects including cardioprotection, antioxidant properties, anticancer activity, and anti‐inflammatory capabilities [30, 31]. These are closely related to the material basis and the underlying mechanism of the drug, especially for heart disease. Prior studies have established the significance of α1A‐AR ligands in the treatment of cardiovascular and cerebrovascular diseases, prompting our assumption that Herba leonuri contains compounds specifically interact with α1A‐AR. Figure 5A displayed the chromatogram of the extract from Herba leonuri, obtained using the immobilized α1A‐AR column. Two intensive peaks, annotated as Peak1 and Peak 2, appeared with retention times of 1.0 and 2.65 min, respectively. We hypothesized that Peak 1 represented compounds in Herba leonuri that were not retained on the column, as its retention time was close to the void time of the chromatographic system. Conversely, Peak 2 exhibited a retention time significantly longer than the void time of the chromatographic system, leading us to conclude that it contained compounds that specifically bind to α1A‐AR. Subsequently, Peak 2 was collected and further analyzed using LC‐MS. The results revealed that two compounds were included in Peak 2 (Figure 5B). The father ion at m/z 181.05 [M+H]+ yielded a daughter ion at m/z 132.11 [M+H]+, while the father ion at m/z 144.10 [M+H]+ generated a daughter ion at m/z 84.09 [M+H]+. Focusing on identifying these two compounds by the reference standards and the reported mass information, we identified the compounds that specifically bind to the immobilized α1A‐AR as leonurine and stachydrine (Figure 5C,D).

FIGURE 5.

FIGURE 5

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Screening of the bioactive compounds in Herba leonuri. (A) The chromatogram of the extract of Herba leonuri; (B–D) were identification of the compounds; and (E–J) were binding interaction analysis of the screened compounds by nonlinear chromatography.

Figure 5E,H illustrated the chromatograms of the two drugs on the immobilized α1A‐AR column. We found that their retention times decreased along with an increased concentration. This observation suggests that the immobilized α1A‐AR column became saturated and the immobilized receptor interacted with the drug. As such, we analyzed the binding interactions of the two drugs by non‐linear chromatography (Figure 5G,J). The corresponding parameters (a0, a1, a2, and a3) were recorded in Table S2–S4. The association constants for the two drugs were calculated as (1.45 ± 0.21) × 105 and (1.24 ± 0.18) × 104 M−1, respectively. We found that in comparison with stachydrine, leonurine had a stronger binding interaction with α1A‐AR. The binding interactions of the two compounds to the receptor were mainly attributed to van der Waals' force and hydrogen bonding, as illustrated in Figure S8. This underlying mechanism helps explain the pharmacological effects exerted by these two compounds. Taken together, these results confirm the successful application of the immobilized α1A‐AR column in screening potential compounds within complex systems. The ability to differentiate binding interactions and calculate association constants provides valuable insights into the pharmacological properties of the drugs and their interactions with the α1A‐AR receptor.

4. Conclusion

In this study, we presented a strategy for immobilizing α1A‐AR using the CL7/IM7 affinity protein pair, which offers significant advantages in terms of high affinity, high efficiency, and specificity. Leveraging these benefits, we successfully prepared the immobilized α1A‐AR column in a rapid and robust manner. Through systematic investigations, we demonstrated the feasibility of using this column to reveal binding interactions between drugs and the immobilized receptor, as well as to screen bioactive compounds from complex systems. Our findings suggest that the immobilized receptor has the potential to emerge as a viable alternative in drug‐receptor interaction analysis and lead screening. The advantages of our approach, including the high affinity and specificity of the CL7/IM7 affinity protein pair, and the robust preparation of the immobilized α1A‐AR column, make it a promising tool for advancing research in this area. We believe that our method will contribute to the development of new drugs and the understanding of drug‐receptor interactions.

Author Contributions

Xiaofang Qiao: investigation, methodology, formal analysis, writing – original draft. Haosen Zhang: formal analysis, data curation. Yue Zhang: data curation, investigation. Qian Liu: writing – original draft, investigation. Jia Guo: investigation, formal analysis. Jing Wang: conceptualization, investigation, project administration, writing – review and editing, funding acquisition.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: jssc70524‐sup‐0001‐SuppMat.docx.

JSSC-49-e70524-s001.docx (736KB, docx)

Acknowledgments

We thank for the financial support from the National Natural Science Foundation of China (22004097), and the Open Research Fund of Yunnan Characteristic Plant Extraction Laboratory (YKKF2023010).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting File: jssc70524‐sup‐0001‐SuppMat.docx.

JSSC-49-e70524-s001.docx (736KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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