**2. Materials and Methods**

## *2.1. Material*

Thai *Bombyx mori* silk cocoons were received from Queen Sirikit Sericulture Center, Nakhon Ratchasima province, Thailand. Recombinant protein eADF4(C16) based on dragline silk protein ADF-4 of *Araneus diadematus* was produced according to the published protocol [13]. Briefly, a bacterial expression plasmid containing gene corresponding to 16 repeats of module C of ADF4 protein (sequence: GSSAAAAAAAASGPGGYG PENQGPSGPGGYGPGGP) was induced in *E. coli* strain BLR(DE3) using isopropyl β-D-1-thiogalactopyranoside (IPTG). Approximately 3–4 h after the induction, cells were harvested, lysed and eADF4(C16) was isolated by precipitation using 30% ammonium sulfate, before redissolution in 6 M guanidine thiocyanate (GdnSCN) and lyophilization. All reagents used in this study were of analytical grade and supplied from Sigma-Aldrich, MO, USA, unless otherwise stated.

To extract SF from silk cocoons, 40 g of the cocoons were boiled in 1 L of 0.02 M Na2CO3 for 20 min twice to remove sericin, before leaving to dry. Four gram of dried silk fiber was dissolved in 16 mL of 9.3 M LiBr (1:4 weight-to-volume ratio), and incubated at 60 ◦C for 4 h. After that, LiBr was eliminated by dialyzing the SF solution against deionized water for 48 h using a dialysis tube with molecular weight cut-off (MWCO) of 12–16 kDa [14]. The concentration of the protein solution was determined from dry solid weight. To prepare the sterile SF solution, the solution was autoclaved at 121 ◦C for 20 min and stored in a refrigerator until usage.

eADF4(C16) powder was dissolved in 6 M GdnSCN at a concentration of 4 mg/mL and incubated at 37 ◦C for 1 h. The solution was then dialyzed against 10 mM Tris-HCl buffer (pH 7.4) using a dialysis tube with MWCO of 6–9 kDa. Subsequently, the protein solution was concentrated using dialysis against 20% *w*/*v* polyethylene glycol (PEG; Mn = 35 kDa), and the concentration was determined from the absorbance at 280 nm [10]. The eADF4(C16) was UV-irradiated for 20 min for sterilization.
