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.
Highlights
Proteases are enzymes that catalyze the hydrolysis of peptide bonds
Some reports refer to the high-yield purification of metalloproteases in one-step procedure, these protocols were based on immobilized metal affinity chromatography (IMAC) that has its disadvantages [21,22]
To confirm that boronic acid derivatives (BADs) could inhibit MP, ten BADs were purchased or synthesized, and their inhibitory effect was tested on MP
Summary
Proteases are enzymes that catalyze the hydrolysis of peptide bonds. Based on the mechanism of catalysis, proteases can be classified into six classes, including metallo, serine, aspartic, cysteine, glutamic, and threonine proteases [1]. Among all of the six classes of proteases, only untagged serine proteases can be purified in one step using p-aminobenzamidine-modified affinity medium [8,9] This simple procedure of affinity purification significantly accelerated the pharmaceutical application of many serine proteases [10,11,12,13,14,15]. Some reports refer to the high-yield purification of metalloproteases (more than 90% purity) in one-step procedure, these protocols were based on immobilized metal affinity chromatography (IMAC) that has its disadvantages [21,22]. YS-80-122, has been previously isolated in our laboratory [3] This enzyme is a typical Zn-containing metalloprotease with antioxidant activity, and it has been commercially used as a detergent additive. Our research provides new opportunities for the development of industrial methods of metalloprotease purification
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
