Abstract
In a new, versatile approach to fun-ction-alizing recombinant spider silk, L-azidohomoalanine is introduced residue-specifically in the minispidroin protein 4RepCT through expression in an E. coli methionine auxotroph. Both fluorophores and the antibiotic levofloxacin are attached to this bio-orthogonal amino acid using copper-catalyzed click chemistry, either before or after the silk fibers are self-assembled.
Highlights
We report the incorporation of L-Aha into the methionine sites of a previously characterised recombinant silk protein 4RepCT, to give 4RepCT3Aha and allowing for downstream site-specific chemical modification of the protein using click chemistry
Unlike previous attempts to incorporate azide groups into silk proteins that involve multi-step processes that are non-specific or only allow the introduction of only a single azide group at the N-terminal, our method achieves this in just one step and allows complete control over the number of azides per protein subunit, based on the number of methionine codons it’s gene contains.[23,24]
By washing functionalised fibres with buffers containing EDTA and by utilising THPTA which binds and stabilises the Cu (I) ion in the click reaction, the traces of copper (
Summary
[25,26,27,28] The azide side chains of L-Aha allow highly specific and efficient site-specific conjugation to a large and varied repertoire of functional molecules via Staudinger ligation with phosphine reagents, Copper (I)-catalysed azide-alkyne cycloaddition (CuAAC) or Strain promoted azidealkyne cycloaddition (SPAAC) in so called ‘click’ reactions.[25,29,30] This paper demonstrates the CuAAC mediated conjugation of 4RepCT3Aha with two different fluorophores and the antibiotic levofloxacin, showcasing the potential of covalently functionalised recombinant spider silk proteins as biomaterials with enhanced properties.
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