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，我们对新发现的转氨酶底物（称为陷阱蛋白）进行了以下基础和生物技术研究。陷阱蛋白是一类在其氨基末端具有锚定序列的蛋白质，通过锚定序列，它们被共价捕获在其作用位点;锚定序列被称为粘固妥英部分。1)使用重组牙骨质妥英部分，我们表明，他们的序列中富含Gln，Lys和Pro具有无规卷曲结构，并作为一个很好的底物转氨酶，一个非常有用的特性，用于开发生物技术的蛋白质交联或蛋白质胶合。2)SPAI是一种循环血浆捕获蛋白，由小肠腺底附近的肠内分泌细胞合成和分泌。3)将其与绿色荧光蛋白融合制备荧光牙骨质妥英。在融合蛋白中保留了组分蛋白的有用性质，即我们成功地制备了在蛋白质工程中具有特殊意义和重要性的荧光牙骨质妥英部分。4)阐明了牙骨质妥因家族基因的进化。基因结构分析表明，陷阱蛋白基因是通过REST和SLPI基因之间的结构域改组而进化的，并且内含子序列已通过基因转换而均质化。

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.