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

尿鸟苷前体肽起分子内伴侣的作用，以正确折叠成熟多肽尿鸟苷。为了进一步研究分子内伴侣功能，制备了一系列分子内伴侣功能多肽，其中的三级结构被尿鸟苷前体肽动力学捕获，并对其生物学活性和折叠进行了研究。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原。使用Hampton Research的晶体筛选试剂盒进行晶体筛选。尿鸟苷原的晶体在8号春被用来收集反射，目前正在进行物相测定，前肽的分子内功能被应用于生物活性多肽的从头设计。为此，设计并化学合成了一系列尿鸟苷与耐热肠毒素(ST)的二硫键杂交肽。用大肠杆菌表达系统制备了尿鸟苷前体肽与杂交肽的融合蛋白。为了判断前肽是否能够以动力学方式捕获杂交肽，对杂交肽和融合蛋白的折叠反应进行了研究。利用该筛选系统，我们获得了一种新的生物活性多肽，该活性多肽被前肽动力学捕获。为了获得结构信息，进行了CD测量。杂交肽的圆二色谱与ST和尿鸟苷的CD谱相似，结果表明，唯一的生物活性多肽可以被前肽捕获，这表明前肽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.