(17a) Viscoelastic Properties of Crosslinked Protein Polymer Hydrogels

We have used genetic engineering and bacterial expression to create high-molecular weight protein polymers that can be either enzymatically or chemically crosslinked into hydrogels with viscoelastic properties. The synthesis of protein polymers by genetic engineering allows for precisely controlled protein length (monodispersity) and specifically tailored amino acid sequence (controlled reactivity). Controlled cloning¹ was used to produce highly repetitive DNA templates, encoding the amino acid sequences(GKGSGKGA)_x, (GKGTGA)_n, and (GKAGTGSA)_m with lengths x = 15, 30, n = 20, 40 and m = 30, 60, 120 respectively. The proteins were expressed in E. coli with a 10 X Histidine tag to facilitate nickel affinity chromatography purification. Purified proteins are chemically crosslinked into a hydrogel using amine reactive crosslinking agents. For enzymatic crosslinking, the protein polymers can be grafted with synthetic peptides that serve as substrates for tissue transglutaminase crosslinking. In this designed family of protein polymers, the number of potential crosslinking sites is controlled by lysine spacing and molecular weight. The resulting hydrogel properties also depend on the degree of crosslinking, which is modified by the crosslinking agent and reaction conditions. Rheological studies are planned to quantitively determine the loss and storage moduli of the resulting hydrogels, as well as in vivo tests of hydrogel biocompatibility. Preliminary in vitro test with NIH 3T3 fibroblasts confirmed that the protein polymer hydrogels were non-toxic. This class of protein polymers can be used to create hydrogels with material properties specific for particular site implementation requirements for tissue engineering applications. Additionally, the amine groups in the protein polymers can be grafted with bioactive peptides that can be chosen to create hydrogels with customizable bioactivity. 1) J. Won, A. E. Barron, Macromolecules 2002, 35, 8281.