Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography

Abstract

In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases.

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

“…In a previous work, we have developed an affinity purification protocol based on the immobilized metal affinity chromatography (IMAC). In particular, we used Cu‐iminodiacetic acid as the affinity ligand coupled with Sepharose 6B beads by a 14‐atom length spacer . Although with this approach we obtained a high activity recovery (74.6%), the use of elevated concentrations of toxic imidazole in the elution buffer required additional dialysis or desalting step.…”

“…Moreover, the IMAC procedure applied to MP purification is discouraged, because MP is a metalloprotein, and the metal ion from the resin can easily exchange with the metal ion from the metalloprotein. This metal transfer can cause a strong decrease in the stability of purified MP, which needs additional calcium ions to recover its stability and activity . Therefore, the development of a new affinity medium to obtain a simple and efficient purification protocol for MP is an important task.…”

“…To assay the protease activity, 0.1 mL of enzyme solution was mixed with 4.9 mL of casein solution (0.6% w/v in 25 mM borate buffer, pH 10.0) and incubated at 25°C for 10 min. The relative protease activity was measured using Folin–Ciocalteu's method . One unit (U) was defined as the amount of enzyme causing the release of 1 μg of tyrosine per min under the above conditions.…”

“…The obtained MP was highly purified using HPLC (Agilent 1260, USA) with a TSK3000SW gel filtration column (Tosoh, Japan) monitored at 280 nm . The purity was calculated based on the software EuroChrom 2000.…”

“…Interestingly, the purified MP has shown an excellent anti‐biofilm activity and represents a potential tool for the treatment of lung fibrosis. However, the protocol proposed to purify MP is challenging and time‐consuming, and a simple and effective protocol to obtain MP with high purity is recommended .…”