Sehnke P C, Ferl R J
Department of Horticultural Sciences, University of Florida, Gainesville, Florida 32611, USA.
Protein Expr Purif. 1999 Mar;15(2):188-95. doi: 10.1006/prep.1998.0993.
The plant protein toxin ricin has found widespread application as a potential therapeutic agent for many human diseases and in disease-model systems such as those involving apoptosis. Genetic engineering and expression of the complete two-polypeptide chain toxin have only been possible in plants, specifically in transgenic tobacco carrying the preproricin gene under the control the cauliflower mosaic virus 35S promoter. Production of modified ricin for altered controllable activity and/or fusion therapeutics to target delivery requires knowledge of the heterologous processing that occurs when preproricin is expressed in tobacco. Here, recombinant ricin from transgenic tobacco was purified using lectin affinity chromatography and characterized using various biochemical and biophysical techniques. Coomassie blue staining of an SDS-PAGE gel of lactose-agarose purified material identified predominant proteins of 30 and 35 kDa molecular weight. Western analysis using anti-ricin a- and b-chain antibodies confirmed the expression and purification of recombinant ricin, with identical protein banding profiles to that of authentic castor-bean-derived ricin. High-resolution gel filtration chromatography characterized the lactose binding complex as a 66-kDa native molecular weight protein which could be separated into 30- and 35-kDa proteins upon incubation with the reducing agent dithiothreitol. N-terminal sequencing of the recombinant ricin a-chain revealed that an equimolar ratio of two alternately processed peptides was present, which varied by an additional amino acid derived from the signal peptide. Similar analysis of ricin b-chain again identified two forms of this polypeptide as well; however, full-length ricin b-chain and b-chain missing the first alanine residue were present at 11:1 molar ratios. Transgenic tobacco plants expressing ricin were used to develop a stable cell suspension culture system from callus induced with the growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Double sandwich enzyme-linked immunosorbent assay using anti-ricin b-chain antibodies and Western analysis identified soluble ricin in the media of the cultures, indicating that cell cultures provide a safe and simple means to produce properly processed recombinant ricin.
植物蛋白毒素蓖麻毒素已作为一种潜在的治疗剂在许多人类疾病以及疾病模型系统（如涉及细胞凋亡的系统）中得到广泛应用。完整的双多肽链毒素的基因工程和表达仅在植物中可行，具体而言是在携带前蓖麻毒素基因且受花椰菜花叶病毒35S启动子控制的转基因烟草中。为了改变可控活性和/或用于靶向递送的融合治疗而生产修饰的蓖麻毒素，需要了解前蓖麻毒素在烟草中表达时发生的异源加工情况。在此，使用凝集素亲和层析法从转基因烟草中纯化重组蓖麻毒素，并使用各种生化和生物物理技术对其进行表征。乳糖 - 琼脂糖纯化材料的SDS - PAGE凝胶的考马斯亮蓝染色鉴定出分子量为30和35 kDa的主要蛋白质。使用抗蓖麻毒素a链和b链抗体的Western分析证实了重组蓖麻毒素的表达和纯化，其蛋白条带图谱与源自蓖麻籽的天然蓖麻毒素相同。高分辨率凝胶过滤层析将乳糖结合复合物表征为分子量为66 kDa的天然蛋白质，在用还原剂二硫苏糖醇孵育后可分离为30 kDa和35 kDa的蛋白质。重组蓖麻毒素a链的N端测序显示存在两种交替加工的肽的等摩尔比，它们因来自信号肽的一个额外氨基酸而有所不同。对蓖麻毒素b链的类似分析也再次鉴定出该多肽的两种形式；然而，全长蓖麻毒素b链和缺失第一个丙氨酸残基的b链以11:1的摩尔比存在。表达蓖麻毒素的转基因烟草植株被用于从用生长调节剂2,4 - 二氯苯氧乙酸和6 - 苄基腺嘌呤诱导的愈伤组织中建立稳定的细胞悬浮培养系统。使用抗蓖麻毒素b链抗体的双夹心酶联免疫吸附测定和Western分析鉴定出培养物培养基中的可溶性蓖麻毒素，表明细胞培养提供了一种安全且简单的方法来生产经过适当加工的重组蓖麻毒素。
