Dialysis membrane

The induced GSTσ in E. coli BL-21 cells were harvested at 8000 x g for 8 min and pellet corresponding to 50 ml culture was resuspended in bacterial lysis solution (50 mM Tris-Cl, 300 mM NaCl, 0.1% Triton X-100, and 10% Glycerol) supplemented with 100 µg/ml lysozyme and 1 mM PMSF. The cells were incubated for 20 min at 4 °C and 20 min at room temperature before sonicating the sample on ice. After centrifugation at 20,000 rpm for 20 min at 4 °C, the supernatant was collected and used for purification of recombinant rParg-GSTσ using Ni⁺²-NTA affinity chromatography (Thermo-Fisher) according to manufacturer’s protocol. The elution of protein was performed with 100 mM, 200 mM, 300 mM, 400 mM and 500 mM imidazole gradient prepared in bacterial lysis buffer. The purified protein was analyzed on 12% SDS-PAGE and pure homogeneous fractions were pooled and dialyzed overnight at 4 °C in Phosphate buffer saline (pH-6.5) using dialysis membrane (Thermo-Fisher). The dialyzed protein was concentrated using 15 ml 10 MWK concentrator (Merck) and protein concentration was estimated spectrophotometrically at 595 nm using Bradford reagent (Sigma-Aldrich) following manufacturer’s protocol; BSA was used as protein standard. The concentrated protein was used for performing enzyme assays and polyclonal antibody generation.

There are different methods to generate silk hydrogels under controlled conditions. In the present study, we induced the hydrogel formation with methanol, as previously described (Numata et al., 2014 (link); Numata and Kaplan, 2010 (link)). Briefly, cocoons from the silkworm Bombyx mori were grounded and boiled for 30 min in a 20 mM Na₂CO₃ solution and then washed with distilled water to remove sericin proteins and wax. Extracted proteins were dried and dissolved in a warm 9 M LiB solution and kept for 2 h. The silk solution was dialyzed using distilled water for 2 days using a dialysis membrane with a molecular weight cut-off of 3,500 (Thermo Fisher Scientific, Waltham, MA, United States). The ilk solution was then concentrated. To produce the hydrogels, an aliquot of the concentrated silk solution was introduced into a silicon cylinder and the cylinder was immersed in methanol for 12 h at room temperature (25–27°C). The resulting silk hydrogel was mixed with 1 µM of the different molecular beacons used in this study.

The GF binding peptides, KGLPLGNSH [19 (link)] (Genscript, Piscataway, NJ, USA), targeting the human Transforming Growth Factor beta 1 (hTGFβ1), and DRVQRQTTTVVA [20 (link)] (Genscript), targeting the human Vascular Endothelial Growth Factor (hVEGF), and the cell adhesion peptide, RGDS (Genscript), were conjugated to Acrylate-PEG-Succinimidyl Valerate (ACRL-PEG-SVA 3.4 kDa, Laysan Bio, Arab, AL, USA). The peptides were dissolved in a 50 mM NaHCO3 pH 8.5 buffer and reacted with ACRL-PEG-SVA at a 1:1 molar ratio for 2 h. The products (ACRL-PEG-KGLPLGNSH, ACRL-PEG-DRVQRQTTTVVA and ACRL-PEG-RGDS) were purified through a dialysis membrane (3.5 kDa, Thermo Fisher Scientific, Waltham, MA, USA) for 24 h and then lyophilized. The products were stored at −20 °C until further use. Peptide conjugation to ACRL-PEG-SVA was confirmed using ATR-FTIR.