Zhou Yu, Singh Bal Ram
Department of Chemistry and Biochemistry, and The School for Marine Science and Technology, University of Massachusetts-Dartmouth, North Dartmouth, MA 02747, USA.
Protein Expr Purif. 2004 Mar;34(1):8-16. doi: 10.1016/j.pep.2003.10.015.
Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses and associate with infant botulism. BoNT is a approximately 150kDa protein, consisting of a binding/translocating heavy chain (HC; 100kDa) and a toxifying light chain (LC; 50kDa) linked through a disulfide bond. C-terminal half of the heavy chain is binding domain, and N-terminal half of the heavy chain is translocation domain that includes transmembrane domain. A functional botulinum neurotoxin type B heavy chain transmembrane and binding domain (Ile 624-Glu 1291) has been cloned into a bacterial expression vector pET 15b and produced as an N-terminally six-histidine-tagged fusion protein (BoNT/B HC TBD). (His(6))-BoNT/B HC TBD was highly expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL and isolated from the E. coli inclusion bodies. After solubilizing the purified inclusion bodies with 6M guanidine-HCl in the presence of 10mM beta-mercaptoethanol, the protein was purified and refolded in a single step on Ni(2+) affinity column by removing beta-mercaptoethanol first, followed by the removal of urea. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. (His(6))-BoNT/B HC TBD retained binding to synaptotagmin II, the receptor of BoNT/B, which was confirmed by immunological dot blot assay, also to ganglioside, which was investigated using enzyme-linked immunosorbent assay.
肉毒杆菌神经毒素（BoNTs）是强效毒素，可抑制外周胆碱能突触释放神经递质，并与婴儿肉毒中毒有关。BoNT是一种约150kDa的蛋白质，由通过二硫键连接的结合/转运重链（HC；100kDa）和毒性轻链（LC；50kDa）组成。重链的C端一半是结合结构域，重链的N端一半是包括跨膜结构域的转运结构域。功能性B型肉毒杆菌神经毒素重链跨膜和结合结构域（Ile 624-Glu 1291）已被克隆到细菌表达载体pET 15b中，并作为N端带有六个组氨酸标签的融合蛋白（BoNT/B HC TBD）产生。（His(6)）-BoNT/B HC TBD在大肠杆菌BL21-CodonPlus（DE3）-RIL中高表达，并从大肠杆菌包涵体中分离出来。在用10mMβ-巯基乙醇存在下用6M盐酸胍溶解纯化的包涵体后，通过首先去除β-巯基乙醇，然后去除尿素，在Ni(2+)亲和柱上一步纯化并重折叠该蛋白质。通过SDS-聚丙烯酰胺凝胶评估，纯化的蛋白质纯度为98%。免疫斑点印迹分析证实（His(6)）-BoNT/B HC TBD保留了与BoNT/B的受体突触结合蛋白II的结合，酶联免疫吸附测定研究也证实了其与神经节苷脂的结合。
