Evaluation of a peptide motif designed for protein tethering to polymer surfaces
Evaluation of a peptide motif designed for protein tethering to polymer surfaces
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评估用于将蛋白质束缚到聚合物表面的肽基序
DOI：
10.1080/09205063.2020.1816870
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Koichi Kato
中科院分区：
文献类型：
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作者：
Ayana Nakano;Isao Hirata;Binh Vinh Pham;Ajay Shakya;Kotaro Tanimoto;Koichi Kato
为了寻找能够有效地将融合蛋白连接到聚合物表面的多肽基序，我们设计了一种KLKLKLKLL(KL5)十肽，其中碱性氨基酸和疏水氨基酸交替连接。利用基因工程技术和细菌表达系统，制备了表皮生长因子、碱性成纤维细胞生长因子和基质细胞衍生因子-1α的KL5融合基因，并与不含KL5的对照基因进行了比较。通过表面等离子体共振分析，研究了这些融合蛋白在聚苯乙烯、亲水性聚苯乙烯和聚己内酯表面的吸附实验。为了理解结合分析的结果，用从头算计算机模拟预测了融合蛋白的结构，并用圆二色谱进行了实证分析。结构分析的结果表明，KL5肽暴露在外界，对蛋白质伴侣的结构影响可以忽略不计。然而，人们发现KL5作为多肽基序的效率很大程度上依赖于蛋白质伙伴。我们的结果表明，当KL5与EGF等酸性蛋白融合时，KL5最有效地发挥了其多肽基序的功能。事实上，培养7天后，在通过KL5肽连接EGF的聚苯乙烯和聚己内酯表面上，检测到的活的人类间充质干细胞数量比对照表面多。根据这项研究的结果，我们得出结论，KL5是一个有用的多肽基序，用于拴系一类特定的蛋白质伙伴。
In search for peptide motifs that allow us to efficiently tether fusion proteins onto polymer surfaces, we designed a KLKLKLKLKL (KL5) decapeptide in which basic and hydrophobic amino acids were alternately linked. By means of genetic engineering technology together with a bacterial expression system, the KL5 fusions of epidermal growth factor (EGF), basic fibroblast growth factor, and stromal cell-derived factor-1α were prepared together with their control counterparts without KL5. The adsorption experiments were performed for these fusion proteins on the surface of polystyrene, hydrophilized polystyrene, and polycaprolactone by surface plasmon resonance analysis. To understand the results of the binding assays, the structure of the fusion proteins was predicted byab initiocomputer simulation and analyzed empirically by circular dichroism spectroscopy. The result of structural analyses suggested that the KL5 peptide is exposed to the outside and has a negligible effect on the structure of the protein partners. However, it was found that the efficiency of KL5 as a peptide motif greatly depends on protein partners. Our results showed that KL5 exerts most effectively its function as a peptide motif when fused to acidic proteins such as EGF. Indeed, the number of living human mesenchymal stem cells determined after 7-day culture was larger on the polystyrene and polycaprolactone surfaces with EGF tethered through the KL5 peptide than control surfaces. According to the results obtained in this study, we conclude that KL5 is useful as a peptide motif for tethering a specific class of protein partners.