Abstract:

Proteins play a prominent role in biotechnology, and a number of new tools have been developed to facilitate their study. Inteins, which are self-splicing protein segments that can also be engineered to self-cleave, offer a number of attractive methods for the study and purification of proteins. In this work for example, the utility of existing intein-based biosensors has been demonstrated in several new applications. These include the identification of estrogenic compounds in crude botanical extracts, as well as in the screening of computationally designed drug-like ligands. Further, the first reported sugar-controlled splicing intein has been engineered, which could be adapted to form new biosensors or to controllably modulate protein activity. Intein-based protein purification has also been investigated in this work, with new technologies developed and current techniques advanced. In one case, an engineered cleaving intein has been incorporated into a vector system that permits rapid cloning and purification of proteins by self-cleaving affinity tags, which could facilitate process optimization as well as high-throughput screening of protein libraries. Additionally, a previously developed non-chromatographic method has been used to co-purify a multi-subunit enzyme, thus significantly enhancing the demonstrated utility of this method. Finally, a mathematical model that can be used to precisely control the concentration profile exiting a column has been refined and synthesized. The ability to predictably generate a desired concentration profile in the effluent of a chromatographic column could greatly improve the efficiency of protein purifications. The newly developed tools and novel applications of existing tools presented in this work significantly enhance the utility of inteins in the study and purification of proteins.