Rapid and efficient one‐step purification of a serralysin family protease by using a p‐aminobenzamidine‐modified affinity medium

Abstract

The metalloproteinase MP belongs to the serralysin family, which is involved in important functions such as nutrient acquisition and infection pathogenesis. Serralysin proteases in highly purified form are commonly used at the industrial level with several purposes. In this study, we set up an efficient and rapid purification protocol for MP using a p-aminobenzamidine-modified affinity chromatography. The affinity medium was synthesized by using p-aminobenzamidine as affinity ligand immobilized via cyanuric chloride spacer to Sepharose 6B sorbent carrier. According to the adsorption analysis, the dissociation constant K and theoretical maximum adsorption Q of this medium were 24.2 μg/mL and 24.1 mg/g wet sorbent, respectively. The purity of MP was assessed by a high-performance liquid chromatography on a TSK3000SW column and sodium dodecyl sulfate polyacrylamide gel electrophoresis, revealing values of 98.7 and ∼98%, respectively. The specific activity of purified MP was 95.6 U/mg, which is similar to values obtained through traditional purification protocols. In conclusion, our protocol could be easily employed for the rapid isolation of MP with high purity, and could be implemented for other serralysin family proteases.

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

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

“…The purity of the purified chitosanase was estimated based on the intensity of the protein band using Gelpro Analyzer 3.2, a commonly used gel imaging analysis system. HPLC analysis was conducted using Agilent 1260, equipped with a TSK3000SW column (Tosoh Co., Tokyo, Japan), wherein protein was monitored by absorbance at 280 nm [27]. The mobile phase was 0.1 M PBS, pH 6.7, 0.1 M Na 2 SO 4 , 0.05% NaN 3 .…”