'Clickable' functionalised recombinant spider silk and its applications

Introduction We have recently reported1 the preparation of antibiotic and fluorophore functionalized silk fibres self-assembled from the miniaturized major spidroin protein 4RepCT. This is derived from the dragline silk of... [ view full abstract ]

Introduction

We have recently reported¹ the preparation of antibiotic and fluorophore functionalized silk fibres self-assembled from the miniaturized major spidroin protein 4RepCT. This is derived from the dragline silk of the South African nursery web spider, Euprosthenops australis.

Methods

The functionalized fibres were prepared by incorporating the bio-orthogonal amino acid L-azidohomoalanine (Aha) in place of the three methionines in the 4RepCT protein. This was achieved by expressing 4RepCT in an E. coli methionine auxotroph grown in a medium rich in Aha. Each 4RepCT protein was expressed as a fusion with the protein thioredoxin at its N-terminal in order to keep the silk protein soluble. The thioredoxin was removed by treatment with thrombin to leave 4RepCT^3Aha which then spontaneously self-assembles into fibres up to 1 m in length. The Aha residues can be selectively and efficiently modified with ligands bearing alkyne groups using a copper (I) catalysed azide alkyne cycloaddition (CuAAC) ‘click’ reaction.

Results and Discussion

We have demonstrated that we can functionalize the silk proteins with fluorophores (Figure 1) and with the broad spectrum antibiotic levofloxacin (Figure 2) using the CuAAC reaction. The antibiotic has been attached via a glycerol ester that is cleaved either through a drop in pH or by esterases released by E. coli as they grow. The 4RepCT_3Aha proteins can be modified either prior to or after silk fibre assembly and the tensile strength of the resulting fibres is unaffected. This has allowed fibres decorated with two or more different ligands to be prepared by mixing differently functionalized batches of fibres together prior to fibre assembly. The different ligands are found to be evenly distributed throughout the fibres as can be seen in the image of silk fibres labelled with both red and green fluorophores which produces a fibre that appears yellow when excited (Figure 2). Antibiotic functionalized silk fibres have been shown to prevent E. coli growth for >5 days giving potential uses in dressings for slow healing wounds such as diabetic ulcers.

1. Antibiotic Spider Silk: Site-Specific Functionalization of Recombinant Spider Silk Using "Click" Chemistry. Adv. Mater., 2017, 29, 1604245 (http://dx.doi.org/10.1002/adma...)