公开(公告)号：US20160312203A1

公开(公告)日：2016-10-27

申请号：US15203638

申请日：2016-07-06

Applicant: I-SHOU UNIVERSITY

Inventor： Chih-Hsin Hung ,  Huei-Ru Luo ,  Jen-Yi Wang

IPC: C12N9/36

CPC classification number: C12N9/2462 ,  C07K2319/00 ,  C07K2319/74 ,  C12N9/88 ,  C12N2795/00022 ,  C12Y302/01017 ,  C12Y402/02002

Abstract: The invention discloses a method of manufacturing a lysin protein capable of directly lysing Acinetobacter baumannii without pretreatment processing using chloroform or EDTA, comprising: transforming an expression plasmid into E. coli, wherein the expression plasmid is deposited at DSMA-Deutsche Sammlung von Mikroorganismen and Zellkulturen with deposit number DSM32023; expressing the expressing plasmid by the E. coli to form lysin protein having an amino acid sequence as set forth in SEQ ID NO: 6; lysing the E. coli containing the lysing protein to obtain a supernatant by centrifugating the E. coli lysate; mixing the supernatant and Ni2+ resins, washing the unbound protein by a binding buffer containing 50 mM Tris-HCl (pH 8.2), 15 mM MgCl2, 20% (v/v) glycerol, 0.05% β-ME and 0.1 mM PMSF; and eluting the purified lysing protein by an elution buffer containing 50 mM Tris-HCl (pH 8.2), 15 mM MgCl2, 20% (v/v) glycerol, 0.05% β-ME, 0.1 mM PMSF and 250 mM imidazole.

Abstract translation: 本发明公开了一种能够直接溶解鲍氏不动杆菌而不用氯仿或EDTA进行预处理的赖氨酸蛋白质的制造方法，其特征在于：将表达质粒转化到大肠杆菌中，其中所述表达质粒沉积在DSMA-Deutsche Sammlung von Mikroorganismen和Zellkulturen 存款编号DSM32023; 用大肠杆菌表达表达质粒以形成具有SEQ ID NO：6所示氨基酸序列的赖氨酸蛋白; 裂解含有裂解蛋白的大肠杆菌，通过离心大肠杆菌裂解液得到上清液; 混合上清液和Ni2 +树脂，通过含有50mM Tris-HCl（pH 8.2），15mM MgCl 2，20％（v / v）甘油，0.05％β-ME和0.1mM PMSF的结合缓冲液洗涤未结合的蛋白质; 并通过含有50mM Tris-HCl（pH 8.2），15mM MgCl 2，20％（v / v）甘油，0.05％β-ME，0.1mM PMSF和250mM咪唑的洗脱缓冲液洗脱纯化的裂解蛋白。