Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand
Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand
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DOI：
10.1016/j.pep.2010.10.008
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发表时间：
2011-03-01
影响因子：
1.6
通讯作者：
Stahl, Stefan
中科院分区：
文献类型：
--
作者：
Wallberg, Helena;Lofdahl, Per-Ake;Stahl, Stefan
结合人表皮生长因子受体2 （HER2）的组合蛋白工程产生的粘附体分子在早期的研究中被证明是HER2介导的癌症分子成像的有希望的示踪剂。这些Z（HER2）粘附体分子的N端或c端氨基酸延伸已成功用于示踪候选物的位点特异性放射性标记。六组氨酸或其他易于恢复的标记物应避免使用，因为它们会对示踪剂的肿瘤靶向效果和生物分布特性产生负面影响。利用新的基于β -内酰胺酶的蛋白质片段互补实验（PCA），分离出一个具有亚微摩尔亲和力的粘附Z（HER2）粘附体分子，而不是不相关的粘附体分子。这表明相互作用区域包括Z（HER2）的HER2结合表面。该新型抗独特型的粘附分子Z（E01）是在大肠杆菌中产生的，经过纯化，并化学偶联到色谱树脂上，以产生一个基于粘附体的亲和柱，适用于恢复不同变体的Z（HER2）粘附分子，具有HER2的共同结合表面。8个这样的Z（HER2）附着体分子，设计用于未来的放射成像研究，具有不同的c端肽延伸，旨在放射性同位素（Tc-99m）螯合，成功地生产和回收，在一个步骤中获得高纯度，使用抗独特型的附着体配体进行亲和纯化。这些结果清楚地表明，开发基于抗独特型抗体的树脂具有潜力，可以有效地回收可能改变生化特性的靶蛋白的相关变体，从而避免为靶蛋白的每种变体设计特定的回收方案。(C) 2010爱思唯尔公司版权所有。
Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these Z(HER2) affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new beta-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a Z(HER2) affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of Z(HER2). This novel anti-idiotypic affibody molecule Z(E01) was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of Z(HER2) affibody molecules, having a common binding surface for HER2. Eight such Z(HER2) affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope (Tc-99m)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein. (C) 2010 Elsevier Inc. All rights reserved.