Application of the intra-molecular chaperon function of propeptide of peptide hormone for de novo design of bioactive peptide

尿鸟苷蛋白前肽作为分子内伴侣，促进成熟肽尿鸟苷蛋白的正确折叠。为了进一步研究分子内伴侣功能，制备了一系列从头设计的肽，其三级结构被尿鸟苷蛋白原肽动力学捕获，并研究其生物活性和折叠。为了研究伴侣功能的结构基础，利用大肠杆菌表达系统制备了尿鸟苷蛋白原。使用汉普顿研究的晶体筛选试剂盒进行晶体筛选。 8月份利用尿鸟苷蛋白原晶体进行反射收集，目前正在进行相位测定。利用前肽的分子内功能从头设计生物活性肽。为此，设计并化学合成了一系列尿鸟苷蛋白和热稳定肠毒素（ST）的二硫键杂合肽。还利用大肠杆菌表达系统制备了尿鸟苷蛋白前肽与杂合肽的融合蛋白。为了估计前肽是否能够在动力学上捕获杂合肽，检查了杂合肽和融合蛋白的折叠反应。我们使用该筛选系统获得了一种新型生物活性肽，它被前肽动力学捕获。为了获得结构信息，进行了CD测量。杂合肽显示出与ST和尿鸟苷蛋白相似的CD谱。结果表明，唯一的生物活性肽可以被前肽捕获，这表明前肽中相互作用空间的三级结构为生物活性结构提供了模板。

A pro-peptide of uroguanylin functions as an intra-molecular chaperone for the correct folding of the mature peptide, uroguanylin. In order to further study the intra-molecular chaperone function, a series of de novo designed peptides, of which tertiary structures were kinetically trapped by the pro-peptide of uroguanylin, was prepared and its biological activity and folding.In order to investigate the structural basis of the chaperone function, pro-uroguanylin was prepared by E. coli expression system. Crystal screening was performed using the crystal screen kit of Hampton research. Crystals of pro-uroguanylin was applied for the collection of reflections in Spring 8. The phase determination is now in progress.The intra-molecular function of propeptide was applied for the de novo design of bioactive peptide. For this purpose, a series of disulfide hybrid peptides of uroguanylin and heat-stable enterotoxin (ST) was designed and chemically synthesized. The fusion proteins of propeptide of uroguanylin and the hybrid peptides were also prepared by E. coli expression system. In order to estimate whether the propeptide is able to kinetically trap the hybrid peptides, folding reactions of hybrid peptides and fusion proteins were examined. We obtained a novel bioactive peptide, which was kinetically trapped by the propeptide, using this screening system. In order to obtain the structural information, CD measurements were performed. The hybrid peptide showed a CD spectrum similar to that of ST and uroguanylin.The results showed that the only bioactive peptide can be trapped by the propeptide, suggesting that the tertiary structure of the interacting space in the propeptide provides the template for the bioactive structure.