| The development of the Protein A platform for monoclonal antibody purification was one of the most important advances in biotechnology. However, no analogous system exists for the purification of non-antibody proteins. The use of self-cleaving intein tag technology is a potential platform for non-antibody protein purification. In this dissertation, recent advances in self-cleaving intein tag technology will be discussed. First, the DeltaI-CM self-cleaving intein system was modified for use in high throughput protein purification applications. Second, the DeltaI-CM intein was paired with a novel affinity tag, the choline-binding domain tag, to allow for affinity purification of recombinant proteins using commercially available anion exchange resins. This new purification was further optimized using a design of experiments approach. Finally, an ongoing project for the development of improved inteins will be presented and discussed. Currently, self-cleaving intein tag technology is limited to bacterial expression systems due to non-ideal intein cleaving kinetics. To be a useful platform, intein technology must be compatible with all types of expression hosts, especially mammalian cell culture. To overcome this, a method to engineer inteins with improved properties using directed evolution and yeast surface display was developed. This method was used to enrich libraries of intein variants for variants that are suitable for use in mammalian cell culture. Initial characterization of these enriched libraries identified several candidates with improved properties. Overall, the work described here lowers the barrier for widespread adoption of self-cleaving intein tag technology in the biotechnology and pharmaceutical industries. |
