Afffinity tag excision strategies for Car9-tagged proteins

Thesis (Master's)--University of Washington, 2016-06

The demand for cheap methods to evaluate and purify of proteins is growing especially in the emerging sectors of the biologics industries and in research laboratories. In accommodating this demand for growth, we explore the possibility that the Car9 affinity tag, a silica-binding dodecamer, may provide an inexpensive alternative to the current gold-standard, the hexahistidine tag. Specifically, we show that the Car9 extension, which was previously shown to be functional as a C-terminal affinity tag, supports rapid purification when appended to the N-terminus of proteins, yielding a >85% purity in a manner of minutes. In addition, we demonstrate two novel methods of Car9 tag removal using the outer-membrane protease, OmpT. The first involves the use of OmpT-overproducing E. coli cells crosslinked to chitosan microspheres using glutaraldehyde. The second employs a mutated version of purified Ompt that is itself modified with a Car9 tag. We show that the immobilized cells support complete excision of a C-terminal Car9 tag in 24h of incubation with no contaminant formation. These catalytic microspheres are reusable, storable, and show promise for larger scale experiments. An OmpT-Car9 fusion protein refolded from inclusion bodies was also shown to support Car9 tag cleavage, and removal with silica. However, further experimentation will be required to determine binding affinity of OmpT-Car9 for future purification and retrieval optimization experiments.