功能性六聚组氨酸标记的蓖麻毒素B在烟草中的表达

Expression of functional hexahistidine-tagged ricin B in tobacco.

作者信息

Reed Deborah G, Nopo-Olazabal Luis H, Funk Vanessa, Woffenden Bonnie J, Reidy Michael J, Dolan Maureen C, Cramer Carole L, Medina-Bolivar Fabricio

机构信息

Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0331, USA.

出版信息

Plant Cell Rep. 2005 Apr;24(1):15-24. doi: 10.1007/s00299-004-0901-0. Epub 2004 Dec 15.

DOI:10.1007/s00299-004-0901-0

PMID:15599751

Abstract

Ricin B (RTB), the lectin subunit of ricin, shows promise as an effective mucosal adjuvant and carrier for use in humans. In order to obtain a recombinant plant source of RTB that is devoid of the toxic ricin A subunit, we expressed RTB in Nicotiana tabacum. RTB was engineered with an N-terminal hexahistidine tag (His-RTB), which may affect protein stability. Lactose-affinity purification of His-RTB from leaves yielded three major glycosylated products of 32, 33.5 and 35 kDa. Their identity as RTB was verified by mass spectrometry and immunoblotting with anti-ricin antibodies. Functionality of His-RTB was confirmed by binding to asialofetuin, lactose and galactose.

摘要

蓖麻毒素B（RTB）是蓖麻毒素的凝集素亚基，有望成为一种有效的用于人类的黏膜佐剂和载体。为了获得不含毒性蓖麻毒素A亚基的重组植物源RTB，我们在烟草中表达了RTB。RTB被设计带有一个N端六聚组氨酸标签（His-RTB），这可能会影响蛋白质稳定性。从叶片中通过乳糖亲和纯化His-RTB得到了三种主要的糖基化产物，分子量分别为32 kDa、33.5 kDa和35 kDa。通过质谱分析以及用抗蓖麻毒素抗体进行免疫印迹验证了它们作为RTB的身份。His-RTB与去唾液酸胎球蛋白、乳糖和半乳糖的结合证实了其功能。