Recombinant derivatives of botulinum neurotoxin A engineered for trafficking studies and neuronal delivery
Recombinant derivatives of botulinum neurotoxin A engineered for trafficking studies and neuronal delivery
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DOI：
10.1016/j.pep.2009.12.013
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发表时间：
2010-05-01
影响因子：
1.6
通讯作者：
Ichtchenko, Konstantin
中科院分区：
文献类型：
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作者：
Band, Philip A.;Blais, Steven;Ichtchenko, Konstantin
多个实验室的工作已经阐明了肉毒杆菌神经毒素 A (BoNT/A) 的结构域如何使神经元胞吐作用失效，但重要的问题仍未得到解答。由于 BoNT/A 中毒会使其自身无法吸收，因此轻链 (LC) 在神经元中不会以可检测的水平积累。因此，我们设计、表达和纯化了一系列 BoNT/A 无毒衍生物 (ad)，它们保留了天然运输所需的野生型特征。 BoNT/A1ad(ek) 和 BoNT/Alad(tev) 是全长衍生物，通过 LC 蛋白酶的双点突变（E-224 > A；Y-366 > A）而无毒。 Delta LC-肽-BoNT/A(tev)和Delta LC-GFP-BoNT/A(tev)是衍生物，其中LC的催化部分被短肽或用GFP加肽替代。在所有四种衍生物中，我们将 S6 肽序列 GDSLSWLLRLLN 融合到蛋白质的 N 末端，以便使用 Sfp 磷酸泛酰乙胺基转移酶实现 Cargo 的位点特异性附着。货物可以以提供均质衍生物群体的方式附着，而不是单标记和多标记分子种类的多分散混合物。所有四种衍生物均含有引入的裂解位点，用于转化为二硫键异二聚体。这些构建体在杆状病毒系统中表达，并且蛋白质被分泌到培养基中并纯化至均质，产量范围为每升1至30毫克。这些衍生物提供了独特的工具来研究体内毒素运输，并评估与重链 (HQ) 连接的货物结构如何影响神经元细胞质的递送。此外，它们还创造了设计基于 BoNT 的分子载体的潜力，该分子载体可以将治疗剂靶向神经元细胞质。(C) 2009 Elsevier Inc. 保留所有权利。
Work from multiple laboratories has clarified how the structural domains of botulinum neurotoxin A (BoNT/A) disable neuronal exocytosis, but important questions remain unanswered. Because BoNT/A intoxication disables its own uptake, light chain (LC) does not accumulate in neurons at detectable levels. We have therefore designed, expressed and purified a series of BoNT/A atoxic derivatives (ad) that retain the wild type features required for native trafficking. BoNT/A1ad(ek) and BoNT/Alad(tev) are full length derivatives rendered atoxic through double point Mutations in the LC protease (E-224 > A; Y-366 > A). Delta LC-peptide-BoNT/A(tev) and Delta LC-GFP-BoNT/A(tev) are derivatives wherein the catalytic portion of the LC is replaced with a short peptide or with GFP plus the peptide. In all four derivatives, we have fused the S6 peptide sequence GDSLSWLLRLLN to the N-terminus of the proteins to enable site-specific attachment Of Cargo using Sfp phosphopantetheinyl transferase. Cargo can be attached in a manner that provides a homogeneous derivative population rather than a polydisperse mixture of singly and multiply-labeled molecular species. All four derivatives contain an introduced cleavage site for conversion into disulfide-bonded heterodimers. These Constructs were expressed in a baculovirus system and the proteins were secreted into culture medium and purified to homogeneity in yields ranging from 1 to 30 mg per liter. These derivatives provide unique tools to study toxin trafficking in vivo, and to assess how the structure of cargo linked to the heavy chain (HQ influences delivery to the neuronal cytosol. Moreover, they create the potential to engineer BoNT-based molecular vehicles that can target therapeutic agents to the neuronal cytoplasm. (C) 2009 Elsevier Inc. All rights reserved.