利用球三糖神经酰胺偶联的辛基琼脂糖凝胶CL-4B通过受体介导亲和色谱法一步纯化志贺毒素-1 B亚基的方法。

Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramide-conjugated octyl sepharose CL-4B.

作者信息

Nakajima H, Katagiri Y U, Kiyokawa N, Taguchi T, Suzuki T, Sekino T, Mimori K, Saito M, Nakao H, Takeda T, Fujimoto J

机构信息

Department of Pathology, National Children's Medical Research Center, Taishido, Tokyo, Japan.

出版信息

Protein Expr Purif. 2001 Jul;22(2):267-75. doi: 10.1006/prep.2001.1449.

DOI:10.1006/prep.2001.1449

PMID:11437603

Abstract

A new single-step purification method for Shiga toxin (Stx) was developed using receptor-mediated affinity chromatography, in which Gb3Cer (globotriaosylceramide) was conjugated to octyl Sepharose CL-4B as a carrier. This method achieves high yield and high purity in a small column on which Gb3Cer has been immobilized at high density. Using this affinity column, the Stx1 B subunit was purified with homogeneity by a one-step procedure from a crude extract of recombinant Stx1 B subunit-producing Escherichia coli. The purified Stx1 B subunit conserved a natural pentamer structure confirmed by gel filtration and sedimentation equilibrium analysis. Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer expressed on Burkitt's lymphoma cells. This versatile purification method can be used to isolate various types of natural as well as recombinant Stx, facilitating fundamental studies of human diseases caused by this toxin.

摘要

利用受体介导的亲和色谱法开发了一种新的志贺毒素（Stx）单步纯化方法，其中将Gb3Cer（球三糖神经酰胺）与辛基琼脂糖凝胶CL-4B偶联作为载体。该方法在一个小柱上实现了高产率和高纯度，在该小柱上Gb3Cer已被高密度固定。使用该亲和柱，通过一步法从产生重组Stx1 B亚基的大肠杆菌粗提物中以均一性纯化了Stx1 B亚基。通过凝胶过滤和沉降平衡分析证实，纯化的Stx1 B亚基保留了天然五聚体结构。此外，纯化的Stx1 B亚基能够特异性结合在伯基特淋巴瘤细胞上表达的Gb3Cer。这种通用的纯化方法可用于分离各种类型的天然以及重组Stx，有助于对由该毒素引起的人类疾病进行基础研究。