Large amount production of recombinant peptides of the crustacean hyperglycemic hormone-family and their tertiary

甲壳动物高血糖激素家族及其三级重组肽的大量生产

基本信息

批准号：
11460054
负责人：
NAGASAWA Hiromichi
金额：
$ 8.96万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

In crustaceans, neuropeptides produced in the X-organ and stored in the sinus glands are responsible for many physiological phenomena such as glucose metabolism, molting, vitellogenesis, body color changes and so on. Among them, a group of peptides called crustacean hyperglycemic hormone (CHH) family involves hyperglycemic hormone, molt-inhibiting hormone (MIH) and vitellogenesis-inhibiting hormone. We previously isolated and characterized CHH-family peptides from the kuruma prawn, and then cloned their cDNAs, thereby clarifying the structures of hormone precursors. The aim of this study is to clarify structure-activity relationship of these peptides at a tertiary structural level. First, a recombinant MIH was produced in the Escherichia colt expression system using the cDNA. The recombinant peptide was not hormonally active just after expression, but a refolding reaction according to the conventional method afforded an active recombinant MIH. The recombinant MIH was as active as the natural MIH. The arrangement of the three disulfide bonds in the recombinant MIH was determined by analyzing its tryptic peptides and was found to be identical to that of the natural MIH. The CD spectrum of the recombinant MIH was very close to that of the natural MIH. Which suggested that they were rich in a-helix. Next, we prepared a double-labled recombinant MIH with ^<13>C and ^<15>N, and attempted to analyze its tertiary structure by nuclear magnetic rezonance techniques. Until now, we have finished to assign about 80 % of proton signals and have also obtained much NOE information. The analyzes are in progress and I hope the tertiary structure will be obtained in the near future. By the same method, we are producing one of CHHs and will analyze its tertiary structure and compare it with that of MIH. Such analyzes will give us important information on the molecular evolution of these peptides.

在甲壳类动物中，X器官产生并储存在窦腺中的神经肽负责许多生理现象，如葡萄糖代谢、蜕皮、卵黄生成、体色变化等。其中，甲壳动物高血糖激素（CHH）家族的一组肽涉及高血糖激素、蜕皮抑制激素（MIH）和卵黄生成抑制激素。我们之前从车虾中分离和表征了 CHH 家族肽，然后克隆了它们的 cDNA，从而阐明了激素前体的结构。本研究的目的是在三级结构水平上阐明这些肽的构效关系。首先，使用该cDNA在大肠杆菌表达系统中产生重组MIH。重组肽在表达后不具有激素活性，但根据常规方法的重折叠反应提供了活性重组MIH。重组 MIH 与天然 MIH 一样活跃。通过分析重组MIH的胰蛋白酶肽确定了重组MIH中三个二硫键的排列，发现其与天然MIH的排列相同。重组MIH的CD谱与天然MIH的CD谱非常接近。这表明它们富含α螺旋。接下来，我们制备了具有^ 13 C和^ 15 N的双标记重组MIH，并尝试通过核磁共振技术分析其三级结构。到目前为止，我们已经完成了大约80%的质子信号分配，也获得了大量的NOE信息。分析正在进行中，我希望在不久的将来能够获得三级结构。通过同样的方法，我们正在生产一种CHH，并将分析其三级结构并与MIH进行比较。此类分析将为我们提供有关这些肽的分子进化的重要信息。