Intein-catalyzed protein splicing: functional versatility and utility

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.