Selection of Peptides that Associate with Dye-Conjugated Solid Surfaces in a pH-Dependent Manner Using cDNA Display
Selection of Peptides that Associate with Dye-Conjugated Solid Surfaces in a pH-Dependent Manner Using cDNA Display
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DOI：
10.1021/acsomega.9b00631
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发表时间：
2019-04
期刊：
影响因子：
4.1
通讯作者：
Takuya Terai;Hiroki Anzai;N. Nemoto
中科院分区：
文献类型：
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作者：
Takuya Terai;Hiroki Anzai;N. Nemoto
识别包括合成聚合物和小分子的人工材料的肽在生物技术和化学生物学领域引起了关注。特别地，仅在特定条件下与靶分子缔合的可逆肽适体是令人感兴趣的。在这项工作中，肽适体识别的酚酞衍生物（PhP：pH敏感的有机染料）固定在固体表面上的pH依赖性的方式被选择使用在体外展示方法（cDNA展示）。考虑到PhP的疏水性和芳香性，我们制备了编码比标准随机密码子更多芳香氨基酸的偏向DNA文库（3A文库），并从>1010个肽种类中进行了七轮选择。选择的肽包括LVFLIWWM（LV59）与PhP-修饰的固体支持物（琼脂糖树脂和磁珠）在中性缓冲液中，但容易解离在碱性条件下，PhP经历大的结构变化，从内酯醌，这是伴随着增加的亲水性和阴离子电荷。对照实验表明，LV59识别苯酚和内酯部分，并且在中性pH下的缔合主要由肽与PhP之间的π堆积和疏水相互作用驱动。然而，值得注意的是，总疏水性和芳香环的数量并不能完全解释亲和力，并且在一定程度上观察到序列特异性。经过进一步优化，这种相互作用对将实际用于蛋白质纯化。
Peptides that recognize artificial materials including synthetic polymers and small molecules are drawing attention in the fields of biotechnology and chemical biology. In particular, reversible peptide aptamers that associate with the target molecules only under specific conditions are interesting. In this work, peptide aptamers that recognize a phenolphthalein derivative (PhP: a pH-sensitive organic dye) immobilized on a solid surface in a pH-dependent manner were selected using an in vitro display method (cDNA display). Considering the hydrophobic and aromatic nature of PhP, we prepared a biased DNA library (3A library) that encodes more aromatic amino acids than the standard random codon and performed seven rounds of selection from >1010 peptide species. The selected peptides including LVFLIWWM (LV59) associated with PhP-modified solid support (sepharose resin and magnetic beads) in neutral buffer but readily dissociated under basic conditions where PhP undergoes large structural change from lactone to quinoid, which is accompanied by increase of hydrophilicity and anionic charge. Control experiments suggested that LV59 recognized both phenol and lactone moieties, and the association under neutral pH is mainly driven by π-stacking and hydrophobic interaction between the peptide and PhP. Notably, however, total hydrophobicity and number of aromatic rings did not completely explain the affinity, and sequence specificity was observed to some extent. After further optimization, this interaction pair would be practically useful for protein purification.