Intein-catalyzed protein splicing: functional versatility and utility

这项研究的长期目标是了解内部催化的蛋白质剪接。在自然界中，肠内蛋白广泛存在于各种宿主蛋白中，其中肠内蛋白催化蛋白质剪接反应去除肠内蛋白，并伴随肽键连接其侧翼序列。目前尚不清楚蛋白质是如何起源、进化和获得任何生物学功能的。我们特别感兴趣的是由位于两个单独的前体蛋白上的两个互补片段组成的分裂-内部蛋白，其中内部蛋白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，因为没有已知的蛋白质连接酶。蛋白是自剪接的，因此它们可以通过重组DNA放置在非天然宿主蛋白中，实现蛋白剪接或反式剪接。内部催化的蛋白质剪接在蛋白质研究和生物技术的各个领域都有许多已经实现和潜在的应用。我课课组对内含蛋白进行了超过15年的研究，在此期间，我们发现了大量新颖的内含蛋白，对内含蛋白的进化和传播产生了重要的见解，揭示了内含蛋白有趣的结构-功能关系，并对大量的分裂内含蛋白进行了基因工程改造，实现了可控的蛋白剪接和裂解。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. 当插入到非天然宿主蛋白中时，蛋白是如何适应新环境的？当用于细胞内外任何感兴趣的蛋白时，我们能否（通过顺序定向进化）产生能够高效、快速拼接的超级蛋白？2. 如何将内含蛋白（通过结构断裂和改变）转化为能够将蛋白质与合成肽拼接在一起的新型分裂内含蛋白？我们能否设计一种基于内含蛋白的“蛋白质连接酶”，用于一般蛋白质和肽的连接？3. 多重分裂蛋白如何在溶液和活体哺乳动物细胞表面的混合系统中避免交叉反应？解决这些问题将加深我们对蛋白质结构-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.