Intein Engineering for Protien Hydrogel Synthesis and Protein Purification

Abstract

Inteins are proteins encoded within a precursor gene that excise themselves after translation and ligate the surrounding proteins with a peptide bond. Since their discovery two decades ago, many inteins have been engineered for various biotechnology applications. This dissertation focuses on the use and development of intein-based technologies for applications in protein purification and immobilization. The highly efficient naturally split DnaE intein from Nostoc punctiforme (Npu DnaE) was incorporated into synthetic protein building blocks for the synthesis of protein hydrogels, and engineered to catalyze rapid C-terminal cleavage reaction and used in the rapid purification of tag-less protein.

In the first application, we developed protein hydrogels as general scaffolds for protein immobilization. Immobilization has been shown to increase protein stability and facilitate enzyme recovery-and-recycle tasks. These hydrogels are composed of artificial protein building-blocks expressed in bacterial hosts. Hydrogel gelation is catalyzed by intein-mediated protein trans-splicing reactions or disulfide bond formation between different protein building blocks. The resulting artificial protein hydrogels possess high solution stability at a wide range of pHs and temperatures, undergo shear-thinning, and are compatible with organic solvents. These self-assembled protein hydrogels can protect immobilized enzymes from organic solvent denaturation during biosynthesis, be used in enzymatic biofuel cells, and are suitable for the immobilization of multiple enzymes.

In the second application, we engineered the Npu DnaE intein to catalyze rapid thio-induced C-terminal cleavage reaction and subsequently developed a split intein mediated technology for recombinant protein purification (SIRP). SIRP enables efficient purification of tag-less recombinant protein from E. coli lysate in less than 1 hour – the hitherto fastest reported intein technology for protein purification.