Intein-catalyzed protein splicing: functional versatility and utility

本研究的长期目标是了解内含肽催化的蛋白质剪接。内含肽在自然界中广泛存在，作为嵌入各种宿主蛋白质中的内部蛋白质，其中内含肽催化蛋白质剪接反应以切除内含肽并伴随地将其侧翼序列与肽键连接。目前尚不清楚内含肽是如何起源、进化和获得任何生物学功能的。我们特别感兴趣的是分裂内含肽，它由位于两个单独的前体蛋白上的两个互补片段组成，内含肽片段可以聚集在一起催化前体蛋白之间的反式剪接反应，这可能是最接近的蛋白质连接形式，因为没有蛋白质连接酶是已知的。内含肽具有自我剪接的特性，因此可以通过重组DNA将其置于非天然宿主的目的蛋白中，实现蛋白质的剪接或反式剪接。内含肽催化的蛋白质剪接在蛋白质研究和生物技术的各个领域有着广泛的应用前景。本课题组对内含肽的研究已经超过15年，在此期间，我们发现了大量新的内含肽，对内含肽的进化和分布产生了重要的见解，揭示了内含肽有趣的结构-功能关系，并通过基因工程改造了大量的断裂内含肽，用于蛋白质的可控剪接和切割。在目前的建议中，我们希望解决以下三个基本问题和相关目标。1.内含肽插入非天然宿主蛋白质时如何适应新的环境，我们能否（通过顺序定向进化）产生超级内含肽，当用于细胞内外的任何感兴趣的蛋白质时，它们可以有效快速地剪接？2.内含肽如何转化（通过结构断裂和改变）为新的分裂内含肽，可以将蛋白质与合成肽拼接在一起，我们能否设计一种基于内含肽的“蛋白质连接酶”用于一般的蛋白质和肽连接？3.多个分裂内含肽如何避免在溶液和活哺乳动物细胞表面的混合系统中的交叉反应？解决这些问题将加深我们对内含肽结构-功能和进化的某些基本方面的理解，也将为蛋白质研究和生物技术的一般领域提供有价值的工具。

The long term objective of this study is to understand intein-catalyzed protein splicing. Inteins are widespread in nature as internal proteins embedded in various host proteins, where the intein catalyzes a protein splicing reaction to excise the intein and concomitantly ligate its flanking sequences with a peptide bond. It is not clear how intein have originated, evolved, and acquired any biological functions. We are particularly interested in split-inteins that consist of two complementary fragments located on two separate precursor proteins, where the intein fragments can come together to catalyze a trans-splicing reaction between the precursor proteins, which is probably the closest form of protein ligation, because no protein ligase has been known. Inteins are self-splicing, therefore they can be placed in non-native host proteins of interest, through recombinant DNA, to achieve protein splicing or trans-splicing. Intein-catalyzed protein splicing has many realized and potential uses in various fields of protein research and biotechnology. My research group has studied inteins for over 15 years, during which we discovered a large number of novel inteins, produced important insights on the evolution and spread of inteins, revealed interesting structure-function relationship of inteins, and genetically engineered a large number of split-inteins for controllable protein splicing and cleavages. In the current proposal, we want to address the following three fundamental questions and related goals. 1. How do inteins adapt to new environments when inserted in non-native host proteins, and can we produce (through sequential directed evolution) super inteins that can splice efficiently and rapidly when used in any protein of interest inside and outside cells? 2. How can inteins be converted (through structural fragmentation and alteration) to novel split-inteins that can splice together proteins with synthetic peptides, and can we engineer an intein-based “protein ligase” for general protein and peptide ligation? 3. How can multiple split-inteins avoid cross-reactions in a mixed system both in solution and on the surface of live mammalian cells? Addressing these questions will deepen our understandings of certain fundamental aspects of intein structure-function and evolution, and it will also produce valuable tools for the general field of protein research and biotechnology.