Strategies for Functionalization of Recombinant Spider Silk

The use of silk-based materials for medical and biotechnological applications has been investigated for many years, with particular progress the last fifteen years. Extensive research has been conducted on silk derived from the silkworm Bombyx mori, but lately the evolvement in recombinant production has made mimics of spider silk proteins increasingly available. Revealed characteristics of silk such as biocompatibility, biodegradability and mechanical strength are features highly desirable in materials for medical purpose. This, in combination with techniques for functionalization (addition of new functions), incentivize further development of silk into highly sophisticated materials useful for advanced applications. The main objective of this thesis has been to investigate novel strategies for functionalization of the recombinant spider silk protein 4RepCT. Two distinct approaches were used, coating and genetic fusion. We showed that coating of silk with polyelectrolytes could be employed to make the fibers electrically conductive as well as fluorescent. Parameters affecting the coating efficiency were investigated, and pH was shown to play an important role. Genetic engineering was employed to fuse 4RepCT with moderately sized protein domains with inherent binding affinities for IgG, albumin and biotin, respectively. We found that the designed silk fusion proteins could self-assemble into silk-like fibers with preserved affinity of the added domains to bind their intended targets. Moreover, we could also demonstrate a general principle for presentation of biomolecules on IgGbinding spider silk by applying a two-step procedure, exemplified by presentation of VEGF via an anti-VEGF antibody bound to IgG-binding silk. We also showed a proofof-principle for 4RepCT materials with catalytic activity by genetic linkage of an enzyme. To investigate potential use of other heterologous hosts for increased production of functionalized silk fusion proteins, the methylotrophic yeast Pichia pastoris was evaluated. We demonstrated expression and secretion of an IgG-binding silk fusion protein in P. pastoris, although the protein was subjected to degradation as well as glycosylation.