Structure-Based Design and Synthesis of a New Phenylboronic-Modified Affinity Medium for Metalloprotease Purification

Abstract

Metalloproteases are emerging as useful agents in the treatment of many diseases including arthritis, cancer, cardiovascular diseases, and fibrosis. Studies that could shed light on the metalloprotease pharmaceutical applications require the pure enzyme. Here, we reported the structure-based design and synthesis of the affinity medium for the efficient purification of metalloprotease using the 4-aminophenylboronic acid (4-APBA) as affinity ligand, which was coupled with Sepharose 6B via cyanuric chloride as spacer. The molecular docking analysis showed that the boron atom was interacting with the hydroxyl group of Ser176 residue, whereas the hydroxyl group of the boronic moiety is oriented toward Leu175 and His177 residues. In addition to the covalent bond between the boron atom and hydroxyl group of Ser176, the spacer between boronic acid derivatives and medium beads contributes to the formation of an enzyme-medium complex. With this synthesized medium, we developed and optimized a one-step purification procedure and applied it for the affinity purification of metalloproteases from three commercial enzyme products. The native metalloproteases were purified to high homogeneity with more than 95% purity. The novel purification method developed in this work provides new opportunities for scientific, industrial and pharmaceutical projects.

Design and Synthesis of a Chitodisaccharide-Based Affinity Resin for Chitosanases Purification

“…Chitooligosaccharides (CHOS) are natural cationic saccharides, while the catalytic domain of chitosanases is rich in acidic amino acids [12,13]; the acid-base interactions between the two molecules can provide affinity force during affinity purification. Immobilization of a ligand onto epoxy-activated resin can be achieved via nucleophilic groups (often is primary amine) presented in the ligand [14,19,20,21,22]. Because CHOS contains an amine group at the C-2 position of the sugar ring, we thus focused our efforts on the design of CHOS-based affinity resin for purification of chitosanase.…”

“…CHDS-based affinity resins were synthesized according to our previous published method [20,21,27]. The synthesis scheme is shown in Figure 1.…”

“…To clear away free cyanuric chloride, 50% ( v/v ) acetone was utilized to wash the resins. The ninhydrin test was applied to examine the density of free amino groups and the linkage of cyanuric chloride to the amino groups, according to the previously described procedure [18,20,21]. Briefly, a small aliquot of gel was smeared on filter paper, sprayed with ninhydrin solution (0.2%, w/v , in acetone), and heated briefly with a hair dryer.…”