Evolutionary and functional versatility of inteins

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The objective of this proposed research is to study a novel protein element named intein. Inteins catalyze a unique reaction termed protein splicing, which is a protein maturation event that removes the intein from a precursor protein to form the mature and functional protein. Supported by a previous NSERC grant, we have discovered natural inteins of various forms and sizes, engineered artificial inteins capable of various desirable functions, and established a directed evolution system for improving intein functions under laboratory conditions. We propose to continue these intein studies to achieve the following goals. 1) To construct controllable inteins capable of efficient protein splicing through intein fragment complementation, which may be useful as a molecular switches to control the functions of other proteins of interest. 2) To construct functional intein-marker cassettes by inserting various desirable protein markers or tags into inteins, which can be generally useful tools for the production and purification of recombinant proteins. 3) To engineer specialty inteins capable of adding synthetic peptides onto recombinant proteins, through rational design and directed evolution, and use these inteins to develop novel methods of site-specific protein labeling and modifications, which can be valuable tools of protein research and production. 4) To produce super inteins that are both fast and general, through specially designed directed evolution, which can greatly expand the general utilities of intein-based technologies. These proposed researches have both academic and practical significances, because proteins are of central importance in life science research and in biotechnology.