Design of an Optimized Scaffold for Affibody Molecules
Design of an Optimized Scaffold for Affibody Molecules
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DOI：
10.1016/j.jmb.2010.03.002
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发表时间：
2010-04-30
影响因子：
5.6
通讯作者：
Abrahmsen, Lars
中科院分区：
文献类型：
--
作者：
Feldwisch, Joachim;Tolmachev, Vladimir;Abrahmsen, Lars
亲和体分子是基于三螺旋束蛋白结构域的非免疫球蛋白衍生的亲和蛋白。在这里，我们描述了优化的亲和体分子支架的设计过程，其具有改进的特性和与亲本支架明显不同的表面。通过在人表皮生长因子受体2（HER 2）特异性亲和体分子Z（HER 2：342）的背景下应用氨基酸取代的迭代过程来实现该改进。通过使用亲本Z结构域和亲和体分子的可用结构的广泛结构建模来指导N-末端区域、环1、螺旋2和螺旋3中的置换。分析几个单一取代的效果，然后分析多达11个不同取代的组合。两个氨基酸取代N23 T和S33 K占最显着的改进，包括增加的热稳定性与高达+12摄氏度的熔融温度。优化的支架在非结合表面含有11个氨基酸取代，其特征在于改善的热稳定性和化学稳定性，以及增加的亲水性，并且能够通过化学肽合成和重组细菌表达产生相同的亲和体分子。HER 2特异性亲和体示踪剂[MMA-DOTA-Cys 61]-Z（HER 2：2891）-Cys（ABY-025）是通过将MMA-DOTA（马来酰亚胺单酰胺-1，4，7，10-四氮杂环十二烷-1，4，7，10-四乙酸）与化学产生的肽或大肠杆菌中产生的肽缀合而产生的。ABY-025显示出对HER 2的高亲和力和特异性（平衡解离常数K-D为76 pM），并在SKOV-3异种移植物和人乳腺肿瘤的组织切片中检测到HER 2。在加热至90 ° C然后冷却至室温的三个循环后，HER 2结合能力完全保留。此外，靶向其他蛋白质（肿瘤坏死因子α，胰岛素，Tag聚合酶，表皮生长因子受体或血小板衍生生长因子受体β）的五种亲和体分子的结合表面被移植到优化的支架上，从而产生具有改善的热稳定性和更亲水的非结合表面的分子。(C)2010爱思唯尔有限公司版权所有。
Affibody molecules are non-immunoglobulin-derived affinity proteins based on a three-helical bundle protein domain. Here, we describe the design process of an optimized Affibody molecule scaffold with improved properties and a surface distinctly different from that of the parental scaffold. The improvement was achieved by applying an iterative process of amino acid substitutions in the context of the human epidermal growth factor receptor 2 (HER2)-specific Affibody molecule Z(HER2:342). Replacements in the N-terminal region, loop 1, helix 2 and helix 3 were guided by extensive structural modeling using the available structures of the parent Z domain and Affibody molecules. The effect of several single substitutions was analyzed followed by combination of up to 11 different substitutions. The two amino acid substitutions N23T and S33K accounted for the most dramatic improvements, including increased thermal stability with elevated melting temperatures of up to +12 degrees C. The optimized scaffold contains 11 amino acid substitutions in the nonbinding surface and is characterized by improved thermal and chemical stability, as well as increased hydrophilicity, and enables generation of identical Affibody molecules both by chemical peptide synthesis and by recombinant bacterial expression. A HER2-specific Affibody tracer, [MMA-DOTA-Cys61]-Z(HER2:2891)-Cys (ABY-025), was produced by conjugating MMA-DOTA (maleimide-monoamide-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the peptide produced either chemically or in Escherichia coli. ABY-025 showed high affinity and specificity for HER2 (equilibrium dissociation constant, K-D, of 76 pM) and detected HER2 in tissue sections of SKOV-3 xenograft and human breast tumors. The HER2-binding capacity was fully retained after three cycles of heating to 90 degrees C followed by cooling to room temperature. Furthermore, the binding surfaces of five Affibody molecules targeting other proteins (tumor necrosis factor alpha, insulin, Tag polymerase, epidermal growth factor receptor or platelet-derived growth factor receptor beta) were grafted onto the optimized scaffold, resulting in molecules with improved thermal stability and a more hydrophilic nonbinding surface. (C) 2010 Elsevier Ltd. All rights reserved.