Abstract

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 by ab initio computer 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.

摘要

为了寻找能让我们有效地将融合蛋白束缚在聚合物表面上的肽基序，我们设计了一种 KLKLKLKLKL (KL5) 十肽，其中碱性氨基酸和疏水性氨基酸交替连接。通过基因工程技术和细菌表达系统，表皮生长因子（EGF）、碱性成纤维细胞生长因子和基质细胞衍生因子-1α KL5 融合物与其对照对应物一起制备，但不含 KL5。通过表面等离子体共振分析对这些融合蛋白在聚苯乙烯、亲水化聚苯乙烯和聚己内酯表面上进行吸附实验。为了理解结合分析的结果，融合蛋白的结构是通过从头预测的计算机模拟并通过圆二色光谱进行经验分析。结构分析结果表明，KL5 肽暴露于外部，对蛋白质伴侣的结构影响可忽略不计。然而，发现KL5作为肽基序的效率在很大程度上取决于蛋白质伴侣。我们的结果表明，KL5 在与酸性蛋白质（如 EGF）融合时最有效地发挥其作为肽基序的功能。事实上，在通过 KL5 肽系留 EGF 的聚苯乙烯和聚己内酯表面上，7 天培养后测定的活人间充质干细胞数量比对照表面更大。根据本研究中获得的结果，我们得出结论，KL5 可用作肽基序，用于连接特定类别的蛋白质伴侣。