Functionalisation of spider silk protein 4RepCT using un-natural amino acid mutagenesis and click chemistry
Abstract
Interest in spider silk as a biomaterial has recently increased owing to its strength, biocompatibility, biodegradability, lack of immunogenicity and pyrogenicity. Unfortunately large scale production from spiders is highly impractical, as their cannibalistic nature prevents them from being farmed. As a consequence of this, scalable production of recombinant spider silk sequences in E.coli has emerged. There is a demand for functionalised silk materials tailored for specific novel applications. Previously this has been achieved through genetic fusion, which can prove difficult when using highly GC rich silk sequences, or classical chemical conjugation methods that are non-residue or non-site specific. The aim of the research described in this thesis was to produce a chemically modifiable self-assembling spider silk protein through the incorporation of a non-canonical amino acid into a miniature spidroin construct (4RepCT). To achieve this, a methionine auxotrophic strain of E.coli has been used to incorporate a methionine analogue (L-azidohomoalanine (L-Aha)) in place of all three methionine residues in the mature 4RepCT sequence to give 4RepCT3Aha. I have demonstrated that this did not adversely affect the normal self-assembly process of the protein into fibres. Subsequently the silk protein was functionalised using either copper-catalysed azide-alkyne cycloaddition (CuAAC) or strain-promoted azide-alkyne cycloaddition (SPAAC) in so called click chemistry reactions for a…
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