Abe Koichi, Yoshida Wataru, Terada Kotaro, Yagi-Ishii Yukiko, Ferri Stefano, Ikebukuro Kazunori, Sode Koji
Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture & Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Int J Mol Sci. 2013 Nov 25;14(12):23244-56. doi: 10.3390/ijms141223244.
We have developed a novel method, antagonistic template-based biopanning, for screening peptide ligands specifically recognizing local tertiary protein structures. We chose water-soluble pyrroloquinoline quinone (PQQ) glucose dehydrogenase (GDH-B) as a model enzyme for this screening. Two GDH-B mutants were constructed as antagonistic templates; these have some point mutations to induce disruption of local tertiary structures within the loop regions that are located at near glucose-binding pocket. Using phage display, we selected 12-mer peptides that specifically bound to wild-type GDH-B but not to the antagonistic templates. Consequently, a peptide ligand showing inhibitory activity against GDH-B was obtained. These results demonstrate that the antagonistic template-based biopanning is useful for screening peptide ligands recognizing the specific local tertiary structure of proteins.
我们开发了一种新方法——基于拮抗模板的生物淘选，用于筛选能特异性识别局部蛋白质三级结构的肽配体。我们选择水溶性吡咯并喹啉醌（PQQ）葡萄糖脱氢酶（GDH-B）作为该筛选的模型酶。构建了两个GDH-B突变体作为拮抗模板；这些突变体有一些点突变，可诱导位于葡萄糖结合口袋附近的环区域内的局部三级结构破坏。利用噬菌体展示技术，我们筛选出了能特异性结合野生型GDH-B但不与拮抗模板结合的12肽。因此，获得了一种对GDH-B具有抑制活性的肽配体。这些结果表明，基于拮抗模板的生物淘选可用于筛选识别蛋白质特定局部三级结构的肽配体。
