Antibiotic Spider Silk: Site-Specific Functionalization of Recombinant Spider Silk Using “Click” Chemistry

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.


Methods
Reagents were purchased from Sigma Aldrich unless stated otherwise.

Transformation of methionine auxotroph DL41
E. coli DL41 (Yale Coli Genetic Stock Center) were transformed with pJExpress401 plasmid encoding Hexa-His tag/sol tag/ Thioredoxin/ Enterokinase site/ Lys-C site/ Thrombin site/4RepCT (synthesized by Life Technologies).Transformed bacteria were streaked onto LB agar plates containing kanamycin and incubated at 37˚C.

Expression and purification of 4RepCT and 4RepCT 3Aha
For 4RepCT containing only canonical amino acids, selected DL41 colonies were grown at 37˚C in Luria-Bertani media containing kanamycin (LB KAN ) to an OD 600 of 1.0 -1.4, induced with 1 mM IPTG and incubated for a further 4 hours at 37˚C. For 4RepCT 3Aha , DL41 cells were grown at 37˚C in M9 minimal media containing kanamycin and all amino acids including methionine to an OD 600 of 1.0 -1.4. Cells were transferred to M9 minimal media with 50 mg/L L-azidohomoalanine (L-Aha)(synthesised in-house following references 27 and 28 in main text) in place of methionine, induced with 0.5 mM IPTG and incubated overnight at 25˚C. Cells were harvested by centrifugation for 12 minutes at 3500 x g, resuspended in Buffer A (20 mM Tris pH 7.5, 400 mM NaCl, 15 mM imidazole) and lysed by sonication. The lysate was centrifuged for 25 minutes at 40 000 x g and loaded onto a Ni-IMAC column (GE Life Sciences). Loosely bound proteins were eluted with Buffer A. Tightly bound 4RepCT/ 3Aha was eluted from the column with ~250 mM imidazole in buffer containing 20 mM Tris pH 7.5 and 400 mM NaCl. Purified 4RepCT/ 3Aha was dialysed against 20 mM Tris pH 8.0 overnight.

Fibre formation
Fibres were made by incubation with thrombin (5 units), Lys-C (Promega) (2 µg) or Enterokinase (5 units). Cleavage was achieved at a fusion protein concentration of 1-2 mg/ml, 1 mM potassium phosphate pH 7.5 and a total volume of 1-3 ml. The reaction was contained in sealed tubes, incubated at 30˚C with gentle rocking for 4 hours.
Silks were observed under 60 X (Nikon Apo TIRF NA 1.49 objective) and 4 X (Nikon Plan Fluor NA 0.13 objective) magnification.

Conjugation 4RepCT Aha with levofloxacin
4RepCT 3Aha fibres of ~1 cm in length were reacted with 9.27 mM of alkyne bearing linker (glycidyl propargyl ether)as described in 'conjugation with fluorophores' (in the case of non-functionalised and levofloxacin control fibres, linker was replaced with water). After incubation, fibres were washed with 3 ml of buffer containing 20 mM Tris pH 7.5, 1 mM EDTA (wash buffer) four times.
After washing, linker-labelled and levofloxacin control fibres were refluxed at 65˚C for 18 hours in methanol containing 50 mM levofloxacin (non-functionalised fibre was refluxed in methanol only).
All fibres were washed with 2 ml of methanol four times, 2 ml of wash buffer twice and stored in methanol.

Tensile strength measurements
A total of 3 4RepCT 3Aha fibres and 3 FAM modified 4RepCT 3Aha fibres were removed from solution and mounted onto cardboard windows. Once straightened, the fibres were glued into place using cyanoacrylate super glue (LOCTITE, Henkel Corporation). Glue was allowed to dry and the fibremounted cardboard window fixed into the grips of the tensile tester (Mecmesin MultiTest 2.5 i) equipped with a 2N load cell. The sides of the cardboard window were carefully cut, and tensile test initiated. Fibres were pulled vertically at a speed of 5 mm/min until the fibre broke (failed) and the load at failure recorded.

Scanning electron microscopy (SEM) and Energy-Dispersive X-ray spectroscopy (EDX)
Adhesive carbon pads were fixed atop aluminium SEM stubs, and were used to secure fibre samples on top of the stub. Once secure, fibre samples were coated with a fine layer of platinum and transferred into the sample carousel of the SEM (FEI Quanta 650). Images were taken at 5.00 kV. For EDX analysis uncoated fibre samples were placed into the carousel of the SEM (FEI Quanta 650 ) and analysed using an Oxford instruments Xmax 150 detector.
Supplementary Figure 1: EDX spectra of functionalised 4RepCT 3Aha fibres. (A) An unwashed FAM conjugated 4RepCT 3Aha fibre contains a negligible amount of copper -0.1% of the specimen weight, shown in the map sum spectrum. The area in which copper is found (between 8 -10 keV) is magnified in the inset picture and shows that the copper signal is noisy, indicating that copper content is low (B) A FAM conjugated fibre after one 20 mM Tris buffer pH 7.5 wash does not contain any detectable copper. (C) A FAM conjugated fibre after identical washing steps to those used on levofloxacin conjugated fibres does. The fibre does not contain any detectable copper.