Generation of ubiquitin-based binder with an inserted active peptide
Generation of ubiquitin-based binder with an inserted active peptide
复制标题
生成带有插入活性肽的基于泛素的结合物
DOI：
10.1016/j.bbrc.2018.08.110
复制
发表时间：
2018
影响因子：
3.1
通讯作者：
Honda Shinya
中科院分区：
文献类型：
--
作者：
Miyafusa Takamitsu;Hirota Kiyonori;Honda Shinya
将活性肽接枝到结构稳定的支架蛋白上对于产生功能蛋白是有效的。在这项研究中，我们的目的是开发一种使用泛素作为支架蛋白的接枝方法。泛素是由76个氨基酸残基组成的小蛋白质，其对热和pH应激高度稳定，这是支架蛋白的期望特征。此外，即使它被分裂或插入额外的残基，其结构也保持不变。因此，我们假设将活性肽接枝到泛素中将产生功能蛋白。作为概念验证，我们开发了基于泛素的结合剂（UbB），其中p53（17-28）肽插入Ile 36和Pro37之间。在本工作中用作模型活性肽的p53（17-28）肽已知与小鼠双微体2同源物（Mdm 2）结合。尺寸排阻色谱和圆二色性表明，UbB保持了类似的结构的泛素。通过表面等离子体共振测量的对Mdm 2的亲和力比p53（17-28）肽的亲和力大292倍。这些观察结果表明，泛素是一个强大的支架肽移植。我们希望这项研究将有助于进一步发展的泛素为基础的蛋白质工程。
The grafting of active peptides onto structurally stable scaffold proteins is effective for the generation of functional proteins. In this study, we aimed to develop a grafting method using ubiquitin as a scaffold protein. Ubiquitin is a small protein consisting of 76 amino acid residues that is highly stable against heat and pH stress, which are desirable characteristics for a scaffold protein. Moreover, its structure is maintained even if it is split or additional residues are inserted. Therefore, we assumed that grafting of an active peptide into ubiquitin would result in a functional protein. As a proof of concept, we developed the ubiquitin-based binder (UbB), into which the p53 (17–28) peptide was inserted between Ile36 and Pro37. The p53 (17–28) peptide, utilized as a model active peptide in this work, is known to bind to mouse double minute 2 homolog (Mdm2). Size exclusion chromatography and circular dichroism indicated that UbB maintained a similar structure to that of ubiquitin. The affinity for Mdm2 measured by surface plasmon resonance was 292 times greater than that of the p53 (17–28) peptide. These observations indicate that ubiquitin is a robust scaffold for peptide grafting. We hope that this study will aid further development of ubiquitin-based protein engineering.