Hiprep 16 60 sephacryl s 200 hr column

DNA encoding the wild-type and various mutants of SsArd1 was amplified by PCR and subcloned into a pET28a vector (Novagen) for fusing the gene to a C-terminal His₆-tag. The final constructs were verified by DNA sequencing. All of the recombinant plasmids were transformed into Escherichia coli strain BL21 (DE3). Cells were grown in lysogeny broth (LB) at 37°C until OD₆₀₀ 0.6, and protein overexpression was induced for 4 h by the addition of 1.0 mM isopropyl 1-thio-β-D-galactopyranoside. The cultures were harvested at 6,000 rpm for 30 min. Cell pellets were resuspended in buffer A (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT), then lysed by sonication on ice, and insoluble pellets were removed by centrifugation at 17,418 g for 30 min at 4°C. The supernatant was filtered through a 0.45-μm filter membrane to remove cell debris before being applied to Ni-Sepharose columns. Columns were washed with buffer B (buffer A containing 50 mM imidazole) and target protein was eluted with buffer C (buffer A containing 300 mM imidazole). Further purification involved size-exclusion chromatography with a HiPrep 16/60 Sephacryl S-200 HR column (GE Healthcare) with buffer D (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM DTT). Peak fractions were examined by Coomassie blue-staining SDS-PAGE. Fractions containing the target protein were pooled and concentrated to 6 mg/ml.

Constituents of hydrolysed sericin were analysed via sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Equal amounts of protein mixed with loading dye were heated at 95 °C for 5 min and added onto 12% (w/v) gel of SDS-PAGE. The separated protein constituents were stained overnight with Coomassie brilliant blue R-250 solution. The molecular weight distribution of hydrolysed sericin was clearly observed after destaining the gel with isopropanol: acetic acid: water (10%: 10%: 80% v/v) solution (Laemmli and Favre 1973 (link)). Additionally, the size distribution profile of RSM-optimized sericin hydrolysates was also generated through fast protein liquid chromatography (FPLC) coupled with HiPrep 16/60 Sephacryl S-200 HR column (GE Healthcare, Stockholm, Sweden). Briefly, sericin hydrolysates at 4 mg/mL was prepared in Tris-HCl buffer (50 mM Tris–HCl, pH 8.0, 200 mM NaCl) for loading on the size exclusion chromatography column preequilibrated with Tris-HCl buffer. Then, the protein sample was eluted with Tris-HCl buffer at a flow rate of 1 mL/min. Absorbance of the eluent at 214 nm was determined to estimate protein concentration.

Size-exclusion chromatography separation was carried out in a fast protein liquid chromatography system (ÄKTA purifier FPLC system) (GE Healthcare UK Ltd., Amersham, UK). One hundred microliter of defatted and filtered pistachio protein extract was injected into a HiPrep 16/60 Sephacryl S-200 HR column (GE Healthcare UK Ltd., Amersham, UK) previously equilibrated with PBS. The flow rate was maintained at 0.5 mL min⁻¹. Eluted fractions were collected in 1.5 mL glass vials and stored at −20 °C until further use.