Division of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden.
Biotechnol J. 2013 Jan;8(1):139-45. doi: 10.1002/biot.201200228. Epub 2012 Oct 10.
We have previously generated an affibody molecule for the disease-associated amyloid beta (Aβ) peptide, which has been shown to inhibit the formation of various Aβ aggregates and revert the neurotoxicity of Aβ in a fruit fly model of Alzheimer's disease. In this study, we have investigated a new bacterial display system for combinatorial protein engineering of the Aβ-binder as a head-to-tail dimeric construct for future optimization efforts, e.g. affinity maturation. Using the bacterial display platform, we have: (i) demonstrated functional expression of the dimeric binder on the cell surface, (ii) determined the affinity and investigated the pH sensitivity of the interaction, (iii) demonstrated the importance of an intramolecular disulfide bond through selections from a cell-displayed combinatorial library, as well as (iv) investigated the effects from rational truncation of the N-terminal part of the affibody molecule on surface expression level and Aβ binding. Overall, the detailed engineering and characterization of this promising Aβ-specific affibody molecule have yielded valuable insights concerning its unusual binding mechanism. The results also demonstrated that our bacterial display system is a suitable technology for future protein engineering and characterization efforts of homo- or heterodimeric affinity proteins.
我们之前已经为与疾病相关的淀粉样β（Aβ）肽生成了一种亲和体分子，该分子已被证明可以抑制各种 Aβ 聚集体的形成，并在阿尔茨海默病的果蝇模型中逆转 Aβ 的神经毒性。在这项研究中，我们研究了一种新的细菌展示系统，用于对 Aβ 结合物进行组合蛋白工程，将其构建为头到尾的二聚体，以进行未来的优化工作，例如亲和力成熟。使用细菌展示平台，我们：（i）证明了二聚体结合物在细胞表面的功能性表达，（ii）确定了亲和力并研究了相互作用的 pH 敏感性，（iii）通过从细胞展示的组合文库中进行选择，证明了分子内二硫键的重要性，以及（iv）研究了合理截短亲和体分子的 N 末端部分对表面表达水平和 Aβ 结合的影响。总的来说，对这种有前途的 Aβ 特异性亲和体分子的详细工程和表征为其不寻常的结合机制提供了有价值的见解。结果还表明，我们的细菌展示系统是未来同型或异型亲和蛋白的同源或异源二聚体的蛋白质工程和表征的合适技术。
