title: 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.
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China.|Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Qingdao, China.|Ministry of Agriculture, Qingdao Key Laboratory of Marine Enzyme Engineering, Qingdao, China.
authors.forename: Shangyong
authors.initials: S
authors.lastname: Li
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China.|Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Qingdao, China.|Ministry of Agriculture, Qingdao Key Laboratory of Marine Enzyme Engineering, Qingdao, China.
authors.forename: Linna
authors.initials: L
authors.lastname: Wang
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China.|Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Qingdao, China.|Ministry of Agriculture, Qingdao Key Laboratory of Marine Enzyme Engineering, Qingdao, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Yang
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China.|Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Qingdao, China.|Ministry of Agriculture, Qingdao Key Laboratory of Marine Enzyme Engineering, Qingdao, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Bao
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China.|Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Qingdao, China.|Ministry of Agriculture, Qingdao Key Laboratory of Marine Enzyme Engineering, Qingdao, China.
authors.forename: Junzhong
authors.initials: J
authors.lastname: Liu
authors.affiliation: Laboratory of Oncology and Molecular Endocrinology, CHUL Research Center (CHUQ) and Laval University, Quebec, Canada.
authors.forename: Shengxiang
authors.initials: S
authors.lastname: Lin
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China.|Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Qingdao, China.|Ministry of Agriculture, Qingdao Key Laboratory of Marine Enzyme Engineering, Qingdao, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Hao
authors.affiliation: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China.|Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Qingdao, China.|Ministry of Agriculture, Qingdao Key Laboratory of Marine Enzyme Engineering, Qingdao, China.
authors.forename: Mi
authors.initials: M
authors.lastname: Sun
mesh_terms: D002846:Chromatography, Affinity; D005417:Flavobacterium / Q000201:enzymology*; D045726:Metalloproteases / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D045726:Metalloproteases
keywords: Adsorption analysis; Affinity purification; Iminmodiacetic acid; Marine bacterium; Metalloprotease