Functionalization of partial spider silk with affinity domains and its use for diverse applications

Abstract: Over the past years, spider silk has drawn considerable attention from researchers because of its renowned mechanical strength (force needed to break), elasticity, biocompatibility and biodegradability. The advancements in genetic engineering have led to the production of artificial mimics of spider silk proteins. The main objective of this thesis is to functionalize two variants of partial spider silk, 4RepCT (RC), 23 kDa and NTCT (NC), 27 kDa, by covalent attachment to affinity domains at gene level. Retained functional properties of the silk part and added affinity domains were studied in the resulting silk fusion proteins. In Paper I, four affinity domains of different sizes (5-17 kDa) with different folds were genetically attached to the RC silk variant. We confirmed that all four RC silk fusion proteins could self-assemble to silk-like fibers. The ability of each added affinity domain to bind its respective target while in RC silk fusion protein was also confirmed. A non-covalent way of presenting biotinylated growth factors was achieved by one of the constructs; M4-4RepCT silk. In a similar way, presentation of an active enzyme was verified by the activity measurement of the silk-fusion material with bound enzyme. Thus, these findings highlight the use of such materials in for example cell culture and tissue engineering applications. In Paper II, as a proof-of-concept, two recombinant antibody fragments (scFvs) each of 30 kDa, previously shown to contribute to the candidate protein signature for diagnosing systemic lupus erythematosus (SLE), were covalently attached to either ends of RC and NC silk variants. All of the generated scFv-RC and scFv-NC silk fusion proteins were shown able to self-assemble to fibres. The retained functionalities of scFv domains in scFv-RC/NC silk fusion proteins were confirmed in micro- and nanoarrays, respectively. Significantly higher target detection signal was reported by scFv-silk fusion proteins when compared to the same added amount of scFvs alone in the immunoassays. Thereby, suggesting the use of scFv-silk fusion proteins as capture probes in generation of sensitive diagnostic immunoassays. The overall results from this thesis thus highlight the diverse possible applications of partial spider silk proteins after being functionalized with various affinity domains.