Development of protein crosslinking technology using the newly found family transglutaminase substrates termed cementoin

为了开发一种高效的蛋白质交联方法，这是设计高度组织化的多功能蛋白质复合物所必需的，我们对新发现的谷氨酰胺转肽类底物进行了以下基础和生物技术研究。诱捕蛋白是一类在其氨基末端具有锚定序列的蛋白质，通过这些锚定序列，它们在其作用位点被共价捕获；锚定序列称为水泥蛋白片段。1)利用重组胶凝蛋白片段，我们发现它们富含Gin、Lys和Pro的序列具有随机线圈结构，可以作为谷氨酰胺转胺酶的良好底物，这一特性对开发蛋白质交联或蛋白质粘合的生物技术非常有用。2) SPAI是一种循环血浆诱捕素，由位于小肠隐窝基部附近的肠内分泌细胞合成和分泌。3)与绿色荧光蛋白融合制备荧光胶凝蛋白。在融合蛋白中保持了组分蛋白的有用性质，即我们成功地制备了一个荧光胶凝蛋白片段，这将在蛋白质工程中具有特殊的兴趣和重要性。4)明确了水泥蛋白家族基因的进化。基因结构分析表明，trappins基因是通过REST和SLPI基因之间的结构域改组而进化的，内含子序列是通过基因转换而均匀化的。

To develop an efficient method for protein crosslinking that is necessary for the design of highly organized, multifunctional protein complexes, we carried out the following basic and biotechnological studies on the newly found transglutaminase substrates termed trappins. Trappins are a family of proteins that have anchoring sequences at their amino termini through which they become covalently trapped at their sites of action ; the anchoring sequence is called cementoin moiety. 1) Using recombinant cementoin moieties, we showed that their sequences rich in Gin, Lys, and Pro have a random coil structure and serve as a good substrate for transglutaminase, a characteristics very useful for developing the biotechnology of protein crosslinking or protein gluing. 2) SPAI, a circulating plasma trappin, was demonstrated to be synthesized and secreted by the enteroendocrine cells located near the crypt base in small intestine. 3) Fluorescent cementoin was prepared by fusing it with green fluorescent protein. The useful properties of the component proteins are maintained in the fusion protein, namely we succeeded to prepare a fluorescent cementoin moiety that will be of special interest and importance in protein engineering. 4) Evolution of cementoin family genes was clarified. Analysis of gene structure revealed that genes for trappins have evolved by the shuffling of domains between the REST and SLPI genes and that the intron sequences have been homogenized by gene conversion.