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.

Keywords: adsorption analysis; affinity purification; aminophenylboronic acid; metalloprotease; molecular docking.

Figure 1

Synthesis protocol and scheme of the 4-APBA ligand coupled with actived Sepharose 6B via cyanuric chloride spacer. Reagents and conditions: (a) epichlorohydrin, DMSO, NaOH aqueous solution, 2.5 h; (b) 35% saturated ammonia, overnight; (c) cyanuric chloride, 50% acetone, pH 7–8; (d) 4-APBA, sodium carbonate, 24 h.

Figure 2

The scheme of four different affinity media. (A) 3-APBA ligand coupled with activated Sepharose 6B via cyanuric chloride spacer; (B) Aniline ligand coupled with activated Sepharose 6B via cyanuric chloride spacer; (C) 4-APBA ligand coupled with activated Sepharose 6B via 5-atom spacer arm; (D) 4-APBA ligand coupled with activated Sepharose 6B via 10-atom spacer arm.

Figure 3

Adsorption analyses of different affinity media. (A) Adsorption analysis of affinity media with three different ligands via the same spacer arm (cyanuric chloride); (B) Adsorption analysis of affinity media with the same ligand (4-APBA) via three different spacer arms. (1) Equilibrium adsorption of metalloprotease (MP) on the affinity medium in a batch system (50 mM Gly-NaOH buffer, pH 8.6, 25 °C), (2) Plot describing the equilibrium of the absorption on the medium and the enzyme concentration in the liquid phase.

Figure 4

The binding mode of MP and the 4-APBA-modified medium. The atom force field maps were generated using Autogrid4 software for AutoDock4 (Zn); binding conformation was analyzed by Lamarckian Genetic Algorithm-Local Search combined algorithm with default searching parameter.

Figure 5

Purity analysis of three purified enzyme products. (A) SDS-PAGE (10.0%) analysis showed that the enzymes were purified to an apparent homogeneous population with a molecular mass of 48 kDa and the purity was more than 95%. Lane M, molecular mass standard protein marker; Lane 1, the purified MP; Lane 2, the purified DENIE-B LPS-P; Lane 3, the purified ViscozymeL; (B) HPLC analysis using the size exclusion by gel filtration of the purified MP (1), DENIE-B LPS-P (2) and ViscozymeL (3) on a TSK 3000SW column.