Functionalization of spider silk with affinity and bioactive domains via genetic engineering for in vitro disease diagnosis and tissue engineering

Abstract: In the recent past, spider silk has drawn significant attention from researchers mainly due to its distinguished mechanical strength, elasticity, biocompatibility and biodegradability. Technological advancements in genetic engineering have resulted in methods for creation of partial spider silk proteins. The main objective of this thesis has been to functionalize a partial spider silk protein, 4RepCT, with different affinity and bioactive domains via genetic engineering. Furthermore, the applicability of materials based on functionalized/bioactivated partial spider silk proteins for in vitro disease diagnosis and tissue engineering applications has been investigated.In Paper I, four affinity domains of different sizes and folds were genetically attached to 4RepCT. All four silk fusion proteins could self-assemble to silk-like fibers. The retained ability of each added affinity domain to bind its respective target while in silk format was also verified. A construct where a monomeric streptavidin domain was genetically fused to 4RepCT was used to allow non-covalent presentation of biotinylated growth factors. Such materials have potential for applications where capture of growth factors could be advantageous, for example in vitro cell culture studies.In Paper II, as a proof-of-concept, two recombinant antibody fragments (scFvs), previously shown to contribute to the candidate protein signature for diagnosing Systemic Lupus Erythematosus (SLE), were covalently attached to either ends of two types of partial spider silk proteins, 4RepCT and NTCT. All of the generated silk fusion proteins were shown able to self-assemble into fibres as well as defined spots in an array. Significantly higher target detection signal was reported from scFv-silk fusion proteins when compared to the same added amount of scFvs alone in micro- and nanoarrays. Thus, scFv-silk fusion proteins can be used as capture probes in the generation of sensitive diagnostic immunoassays for effective disease diagnosis.In Paper III, bioactivation of 4RepCT with a pleiotropic growth factor, basic fibroblast growth factor (bFGF) was investigated. The generated silk-bFGF fusion protein retained the propensity to self-assemble into surface coatings and silk-like fibers. Maintained functionality of the silk-bFGF coating to bind FGFR receptor was confirmed using surface plasmon resonance studies. Moreover, with the aim to create an artificial ECM, silk-bFGF protein was combined with FN-silk, an engineered spider silk protein previously reported to support cell adhesion. Retained bioactivity of the bFGF was confirmed by culture of primary human endothelial cells on combined silk coatings and within combined silk fibers, even when cultured in medium containing low serum and no supplemented soluble growth factors. These findings highlight the use of combined silk coatings for in vitro cell culture, and combined silk fibers as a potential scaffold for tissue engineering applications.In Paper IV, the possibility to genetically fuse two affibody-based VEGFR2 binders, Zdimer and Ztetramer, to 4RepCT was investigated. Maintained activity of added engineered affibodies was confirmed by receptor phosphorylation and cell proliferation studies. Furthermore, the possibility to create vessel-like structures within a FN-silk based cell scaffold containing Ztetramer-silk fibrils was reported. These findings highlight the future potential of herein developed silk based cell scaffolds in regenerative medicine.