Masuda Ryo, Phyu Thant Khine Phyu, Kawahara Kazuki, Oki Hiroya, Kadonosono Tetsuya, Kobayashi Yuji, Koide Takaki
Waseda Research Institute for Science and Engineering, Waseda University, Shinjuku, Tokyo, Japan.
Department of Chemistry and Biochemistry, School of Advanced Science and Engineering, Waseda University, Shinjuku, Tokyo, Japan.
J Biol Chem. 2024 Nov;300(11):107794. doi: 10.1016/j.jbc.2024.107794. Epub 2024 Sep 19.
Many bioactive proteins interact with collagen, recognizing amino acid sequences displayed on the triple helix. We report here a selection strategy to obtain triple-helical peptides that interact with the proteins from a combinatorial random library constructed in yeast cells. This system enables us to select them using the standard two-hybrid protocol, detecting interactions between triple-helical peptides and target proteins fused to the GAL4-activating and binding domains, respectively. The library was constructed having triple-helical peptides with a "host-guest" design in which host helix-stabilizing regions flanked guest random sequences. Using this system, we selected peptides that bind to pigment epithelium-derived factor (PEDF), a collagen-binding protein that shows anti-angiogenic and neurotrophic activities, from the libraries. Two-step selections from the total random library and subsequently from the second focused library yielded new PEDF-binding sequences that exhibited a comparable affinity to or more potent than that of the native PEDF-binding sequence in collagen. The obtained sequences also contained a variant of the PEDF-binding motif that did not match the known motif identified from the native collagen sequences. This combinatorial library system allows the chemical space of triple-helical peptides to be screened more widely than that found in native collagen, thus increasing the expectation of obtaining more specific and high-affinity peptides.
许多生物活性蛋白与胶原蛋白相互作用，识别三螺旋上展示的氨基酸序列。我们在此报告一种筛选策略，用于从酵母细胞中构建的组合随机文库中获取与这些蛋白相互作用的三螺旋肽。该系统使我们能够使用标准的双杂交方案进行筛选，检测三螺旋肽与分别融合到GAL4激活域和结合域的靶蛋白之间的相互作用。构建的文库中的三螺旋肽采用“主客”设计，其中主螺旋稳定区位于客随机序列两侧。利用该系统，我们从文库中筛选出了与色素上皮衍生因子（PEDF）结合的肽，PEDF是一种具有抗血管生成和神经营养活性的胶原蛋白结合蛋白。从总随机文库进行两步筛选，随后从第二个聚焦文库中筛选，得到了新的PEDF结合序列，这些序列在胶原蛋白中表现出与天然PEDF结合序列相当或更强的亲和力。获得的序列还包含一个PEDF结合基序变体，该变体与从天然胶原蛋白序列中鉴定出的已知基序不匹配。这种组合文库系统允许比天然胶原蛋白更广泛地筛选三螺旋肽的化学空间，从而增加了获得更特异和高亲和力肽的期望。
