Frankel A, Tagge E, Chandler J, Burbage C, Hancock G, Vesely J, Willingham M
Department of Medicine, Medical University of South Carolina, Charleston 29425, USA.
Bioconjug Chem. 1995 Nov-Dec;6(6):666-72. doi: 10.1021/bc00036a002.
Fusion toxins consist of peptide ligands linked through amide bonds to polypeptide toxins. The ligand directs the molecule to the surface of target cells and the toxin enters the cytosol and induces cell death. Ricin toxin is an excellent candidate for use in fusion toxins because of its extreme potency, the extensive knowledge of its atomic structure, and the years of experience with RTA chemical conjugates in clinical trials. We synthesized a baculovirus transfer vector with the polyhedrin promoter followed sequentially from the 5' end with DNA encoding the gp67A leader sequence, the tripeptide ADP, IL2, another ADP tripeptide, and RTB. Recombinant baculovirus was generated in Sf9 insect cells and used to infect Sf9 cells. Recombinant IL2-RTB protein was recovered at high yields from day 5 insect cell supernatants, partially purified by affinity chromatography, and characterized. The recombinant product was soluble and immunoreactive with antibodies to RTB and IL2, bound asialofetuin and lactose, and reassociated with RTA. In the presence of lactose to block galactose-binding sites on RTB, the IL2-RTB-RTA heterodimer was selectively cytotoxic to IL2 receptor, bearing cells. Specific cytotoxicity could be blocked with IL2. Thus, we report a novel targeted plant toxin fusion protein with full biological activity.
融合毒素由通过酰胺键与多肽毒素相连的肽配体组成。配体将分子导向靶细胞表面，毒素进入细胞质并诱导细胞死亡。蓖麻毒素因其极高的毒性、对其原子结构的广泛了解以及在临床试验中对RTA化学偶联物的多年经验，是用于融合毒素的极佳候选物。我们合成了一种杆状病毒转移载体，其多角体蛋白启动子从5'端依次连接编码gp67A前导序列、三肽ADP、IL2、另一个ADP三肽和RTB的DNA。重组杆状病毒在Sf9昆虫细胞中产生并用于感染Sf9细胞。从第5天的昆虫细胞上清液中高产率回收重组IL2-RTB蛋白，通过亲和层析进行部分纯化并进行表征。重组产物可溶，与抗RTB和IL2的抗体发生免疫反应，结合去唾液酸胎球蛋白和乳糖，并与RTA重新结合。在乳糖存在以阻断RTB上半乳糖结合位点的情况下，IL2-RTB-RTA异二聚体对表达IL2受体的细胞具有选择性细胞毒性。特异性细胞毒性可用IL2阻断。因此，我们报道了一种具有完全生物活性的新型靶向植物毒素融合蛋白。
