Intein-catalyzed protein splicing: functional versatility and utility

This proposal continues our long-term study of intein-catalyzed protein splicing and its utilities. Inteins are found in nature as a novel class of enzymatic proteins that catalyzes a protein splicing reaction to excise the intein and ligate the flanking host proteins with a peptide bond. We recently focused on split-inteins that can catalyze a trans-splicing reaction to ligate two separate proteins. Inteins can generally work in non-native proteins of interest and have many uses in various fields of protein research, protein engineering and biotechnology. In past 20 years, my group has discovered numerous natural inteins, produced insights on intein evolution and structure-function, and engineered useful inteins for protein splicing and cleavage. In the last six years, we have made important breakthroughs in engineering intein-catalyzed protein and/or peptide ligations. We successfully used protein ligation in production and analysis of artificial spider silks. Over the next five years, our first goal is to further develop the functional versatility (efficiency and generality) of intein-catalyzed protein and/or peptide ligations, using advanced molecular biology methods including directed evolution. Protein and peptide ligations are highly desirable in various fields of protein research and industry. Our proposed research is the closest to achieving a successful engineering of intein-derived protein splicing ligase that can be generally useful for ligating proteins and/or synthetic peptides. This is expected to open up tremendously useful avenues of producing chemically synthesized proteins and semi-synthetic proteins, allowing site-specific additions of various desired chemical modifications and unnatural amino acids to proteins, which are expected to provide great values to many protein-related research and industrial fields including pharmaceutical protein drugs. These can benefit many Canadian researchers and biotech companies who often wish to add site-specific chemical modifications or unnatural amino acids to their proteins of interest. Our second goal is to apply protein ligations to the production of a large number of novel artificial spider silks, through tandem and combinatorial protein ligations. This will allow us to explore systematically various artificial spider silks formed from various forms of large and chimeric spider silk proteins. Artificial spider silks have been widely recognized as potentially useful biomaterials, because they are exceptionally strong, biocompatible, and biodegradable. Artificial spider silks from this proposal can benefit Canadian researchers and biotech industry, for example, these novel biomaterials may be used in making scaffoldings for tissue engineering.