The immobilization of proteins on solid surfaces plays a pivotal role in various fields such as biomedical and bioanalytical techniques which facilitates a systemic understanding of biological phenomena at the molecular level [1], [2], [3]. Many modern bio-based applications, including biochips, biosensors, drug screening, and drug delivery systems often involve protein-protein interaction or protein-drug interaction analysis [4], [5], [6], [7]. These broad applications mainly rely on the activity of the immobilized functional proteins because, in such situations, the biomedical properties of the functional proteins are transferred to the material surface, thereby allowing selective interaction [8]. Despite the promising achievement in linking functional proteins onto solid surfaces in physical adsorption, crosslinking, capsulation, and covalent attachment, the unmet challenge in such a field emerges when the immobilized proteins are required for more homogeneous in orientation, more stable, and easy separation from the reaction mixture. Unfortunately, the classical method like physical adsorption offers an easier and simple way for protein immobilization, it generally leads to disordered orientation which influences the protein activity or reduces the accessibility to its functional sites. Generally, the amines, thiols, or carboxylic acid groups present in the side chain of the amino acid are exploited as the active binding site for robust covalent immobilization of the proteins [9], [10], [11]. Considering the multiple copies of the amino acids, the homogenous orientation of the immobilized proteins needs to be improved. Therefore, increasing efforts are needed for developing new methods to immobilize proteins in a controllable and homogeneous orientation, which is advantageous for its applications, hence the sensitivity and reproducibility are promoted.

G protein-coupled receptors (GPCRs) represent the largest trans-membrane protein family encoded by more than 800 genes in human genome [12]. Generally, GPCRs undergo conformational changes when binding to their specific ligand, thus causing the activation of the signaling networks involved in the key physiological effects. Given the tremendous diversity of GPCRs, there remains enormous potential for the development of new drugs for ameliorating diseases such as cardiovascular diseases, inflammatory diseases, respiratory system diseases, cancer, neurological disorders, as well as metabolic imbalances [13], [14], [15]. Considering the significant role of GPCRs, a variety of modern methods have been developed for pursuing drug candidates to fight against these diseases by using immobilized GPCRs. But these methods are challenged because of the consumption of time and labor when it meets the purification needs of the receptors. Meanwhile, the activity loss of the purified GPCRs ascribed to the misfolding of the receptors, which may be the core factor in the immobilized method.

VirD2 is a protein from the plant parasite A. tumefaciens with the ability to establish covalent bond at a specific sequence location of an unmodified nucleic acid molecule [16,17]. The covalent phosphodiester linkage formed by VirD2 is located within the first 200 amino acids of the protein. The main amino acid involved in the covalent bond is Y29, which bridges the protein to the 5’ phosphate of a nucleotide just downstream of its recognition sequence in a self-enzymatic mode. Importantly, the DNA-protein conjugate can be formed in vitro in the presence of other proteins or biomolecules. Based on this mechanism, the small size and efficacy of VirD2, conjugating to DNA molecules, has been exploited for some other bioanalytical investigations, such as DNA nanotechnology and biosensors.

Herein, we propose a one-step immobilization method for capturing Cysteinyl leukotriene receptor 1(CysLTR1) from the cell lysate by the enzymatic effect of VirD2 fused as a tag at the C terminus of the receptor (Fig. 1). Meanwhile, we modified the specific DNA sequence onto the surface of the macro-porous silica gel by condensation reaction between the carboxyl group from the modified silica gel and the amino group at the 3’ end of the DNA. After a fast, site-specific conjugation process in physiological conditions, the receptor was covalently linked to the silica gel surface. We utilized the immobilized CysLTR1 column to investigate the binding interactions of three drugs to the receptor by chromatographic methods like non-linear chromatography analysis. Utilized the established platform, we selectively screened and identified the CysLTR1-targeted bioactive compounds from traditional Chinese medicine. Our results demonstrated that the established chromatographic method meets the requirements of high specificity and stability in obtaining bioactive compounds from complex matrices due to its minimal loss of the receptor. We believe that the method is promising in conjugating other functional proteins onto the solid surface for further investigations.