(477c) Construction of Intein-Mediated Bioactive Protein Hydrogel

Hydrogels made entirely of proteins/peptides or hybrid proteins/polymers have found applications as tissue engineering scaffolds, drug delivery carriers, controlled release depots and as key components of biosensing and bioanalytical devices. Hydrogels formed through a self-assembly process have the potential to produce precisely defined, hierarchical 3D structures – a particularly desirable trait when assembling enzymes/catalysts for cascade reactions. Taking advantage of intein-mediated protein ligation, we have constructed a protein hydrogel with the potential to self-assemble in situ for therapeutic applications. The building blocks of our protein hydrogels are two protein triblock copolymers that are expressed separately in E.coli. Each copolymer contains a bioactive protein flanked by one half of a naturally split intein and a crosslinker comprising multimeric protein subunit. Upon mixing of these two protein copolymers, the split intein in each copolymer covalently links the crosslinker/bioactive protein from the two triblock copolymers, resulting in the formation of a protein polymer molecule capable of self-assembling into a hydrogel. The gelation process is rapid upon mixing of the protein copolymers. We characterized the stability and mechanical properties of intein-mediated hydrogels comprising various cross-linking agents such as DsRed and CutA as the bioactive protein module. The described protein hydrogel technology enables precise molecular control of the constituent protein elements through manipulation of the genetic code and could provide scaffolds for industrial biocatalysis and tissue engineering applications.