新型蛋白质水凝胶的制备及其应用

The Michael addition of the sulfhydryl group on the protein to the vinyl group on the polymer was often used for the formation of protein-based hydrogel. However, the introduction of cysteine into the protein frequently resulted in the changes of protein conformation, precipitation, and low yield of purification. The activity of the endogenous cysteine of protein itself had lost due to the formation of disulfide bond or the endogenous cysteine was unable to react with double bond because it was inside the protein domain. These disadvantages finally hindered the development of the research on protein-based hydrogels. Our group discovered a tetramer of ubiqitin-like domain (ULD) containing four endogenous cysteines which can be used to react with double bond of the maleimide on the end of four-armed poly(ethylene glycol), leading to the formation of protein-based hydrogel quickly. The active peptides and functional proteins are easy to be fused to ULD by genetic engineering and the resulting fusion proteins can be used for the formation of protein-based hydrogels. In this project, we plan to fuse active peptides with the ability of helping cells' adhesion and proliferation to ULD to obtain protein-based hydrogels which is suitable for the 3D cell culture. We also plan to fabricate the fusion protein of ULD-TIP-1 of which TIP-1 has the different affinity with different hexapeptides. We will use the ability of TIP-1 to reach the goal of the controlled release of the derivative of anti-cancer drug and hexapeptide from our protein-based hydrogel. Because the fusion proteins we designed are easy to be expressed and purified with high yield and the time for the formation of protein-based hydrogel is short, our protein-based hydrogel has big potential for the application in tissue engineering, controlled drug release, regenerative medicine, etc.

巯基和双键的迈克尔加成反应经常被应用于制备蛋白质水凝胶。然而，非内源性半胱氨酸的引入往往造成蛋白构象变化、易于沉淀和提纯产率低，而蛋白的内源性半胱氨酸往往已形成了二硫键或在蛋白内部，无法与双键反应。这些不足限制和阻碍了蛋白质水凝胶研究领域的发展。课题组发现了含有四个内源性半胱氨酸的ULD蛋白四聚体，可用于与马来酰亚胺结尾的四臂PEG发生迈克尔加成反应，迅速地形成水凝胶。通过基因工程的方法可将活性多肽和功能蛋白与ULD融合，获得融合蛋白，用于制备功能性水凝胶。项目拟将可帮助细胞粘附和增殖的活性多肽与ULD融合后用于制备适合细胞三维培养的水凝胶。同时拟构建ULD-TIP-1融合蛋白，通过TIP-1与六肽的不同结合力调控"抗癌药物分子-六肽衍生物"从水凝胶中的释放行为。由于各种融合蛋白具有表达与提纯简单、产量高、快速成胶的优点，本项目设计的新型蛋白质水凝胶体系有望应用于组织工程、药物控释等领域。

本研究计划拟通过基因工程的方法得到和ULD蛋白偶联新型融合蛋白，利用ULD蛋白具有内源性的半胱氨酸（cysteine）来构建蛋白质水凝胶，考察蛋白质水凝胶在三维细胞培养和作为药物载体的效果；同时探索表面诱导多肽自组装在细菌检测、组织工程等领域的应用。在2014-2017年间，项目负责人带领研究团队按照原定计划逐步实现了新型蛋白质水凝胶的构建，并考察了蛋白质水凝胶在三维细胞培养和作为药物控释载体的效果。同时，研究了细菌膜和表面诱导的多肽自组装，并将该策略应用到了细菌、酶等物质的检测等方面。我们的科研工作为新型蛋白质水凝胶的设计和制备提供新的思路和策略。项目执行期间申请了中国发明专利4项，一共发表了标注该基金项目的SCI收录论文13篇，其中包括影响因子大于10的Angew Chem Int Ed 文章1篇；过去4年时间里培养了自组装多肽研究方面的多名优秀人才，包括5名博士毕业生(其中4名加入国内高校或者科研院所继续从事科学研究，1名继续从事博士后) 和3名硕士生。2014年获得天津市自然科学一等奖（第三完成人）。