The long-term goals of this application are to better understand the biological importance of inteins as post-
translational regulatory elements, their emerging role in pathogen stress response, and to harness the power
of their unique chemistry to invent useful technologies for the biomedical research community. Inteins, or
intervening proteins, are self-catalytic, mobile genetic elements removed from host genes through protein
splicing. From the applied perspective, the ability of inteins to shuffle peptide bonds in highly specific ways has
proven exceptionally useful in protein engineering, leading to the development of numerous technologies.
While intein applications have dominated their investigation, and new intein-based technologies are developed
frequently, the biological importance of inteins in nature is poorly understood. Inteins are abundant mobile
genetic elements in the microbial world, found in approximately one-half of archaea and one-quarter of
bacteria. Contrary to long-standing assumption that inteins are molecular parasites, mounting recent evidence
suggests that some intein-containing proteins have evolved to couple splicing, and thus host protein activation,
to environmental signals. This represents a novel and potentially widespread form of post-translational
regulation. Further, given inteins are absent in humans and located within essential genes of several
pathogens, understanding the environmental factors that regulate protein splicing inform possible antimicrobial
We propose the following two aims, building upon recent discoveries, as well as expanding into new arenas.
Aim 1 will for the first time investigate the role inteins play as nascent chains, broadly determining whether
splicing is possible prior to release from the ribosome, as well as examining possible stress response
strategies of bacterial and fungal pathogens. Aim 2 seeks to develop two novel intein-based technologies.
First, a general strategy to improve expression of misfolding-prone proteins in bacteria and second, a zinc-
controlled self-removing protein purification tag for use in both bacterial and mammalian systems. Through
these Aims, this work will enhance our understanding of the roles these exciting and understudied elements
play in nature, their role in pathogen stress response, and will lead to new intein-based technologies for protein