Hamorsky Krystal, Matoba Nobuyuki
Owensboro Cancer Research Program, University of Louisville James Graham Brown Cancer Center, 1020 Breckenridge Street Suite 201, Owensboro, KY, 42303, USA.
Department of Medicine, University of Louisville School of Medicine, Louisville, KY, USA.
Methods Mol Biol. 2016;1404:511-518. doi: 10.1007/978-1-4939-3389-1_33.
Herein, we report an Escherichia coli-based expression and purification method of recombinant cholera toxin B subunit (CTB). The CTB gene (E. coli codon optimized) is cloned into commercial pET-22b(+) vector using standard molecular biology techniques and the resulting vector is transformed into BL21(DE3) electrocompetent cells. The bacterial cells are grown and induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) results in accumulation of CTB in the culture medium. CTB is purified from the culture medium using a simple two-step chromatography process: immobilized metal affinity chromatography (IMAC) followed by ceramic hydroxyapatite (CHT). CTB is purified to >95 % homogeneity with a yield of over 10 mg per liter of culture. Depending on the application, endotoxin is removed using a commercially available endotoxin removal resin to <1 EU/mg.
在此，我们报道了一种基于大肠杆菌的重组霍乱毒素B亚基（CTB）的表达和纯化方法。使用标准分子生物学技术将CTB基因（经大肠杆菌密码子优化）克隆到商业pET-22b(+)载体中，并将所得载体转化到BL21(DE3)电感受态细胞中。培养细菌细胞，用异丙基β-D-1-硫代半乳糖苷（IPTG）诱导，导致CTB在培养基中积累。使用简单的两步色谱法从培养基中纯化CTB：固定化金属亲和色谱（IMAC），然后是陶瓷羟基磷灰石（CHT）。CTB纯化至>95%的纯度，每升培养物的产量超过10 mg。根据应用需求，使用市售的内毒素去除树脂将内毒素去除至<1 EU/mg。
