Sephadex g 75 column

The expression and purification of recombinant VP6 (reVP6) of BTV-10 by use of baculovirus in the Sf9 cell line have been described previously (14 (link)). Additionally, His-tagged wild-type (WT) and mutant reVP6 proteins were expressed in E. coli strain BL21(DE3) pLysS. The His-tagged reVP6 proteins were purified using Ni-NTA affinity purification, eluting the purified protein with a buffer comprising 20 mM Tris-HCl, 200 mM NaCl (pH 7.4), and 250 mM imidazole. Imidazole was then removed by buffer exchange with Sephadex G-75 columns (GE Healthcare).

C. elegans SAS-6 (Uniprot ID 062479) fragments were prepared as described earlier^{18 (link),25 (link)}; briefly, fragments comprising the protein N-terminal domain (CeSAS-6_N, amino acids 1–168) or the N-terminal domain plus a short stretch of the coiled-coil interface (CeSAS-6_N-CC, amino acids 1–215) were cloned in a modified pET15b vector containing an N-terminal His₆-tag, transformed into Escherichia coli BL21 (DE3) cells grown in Luria-Bertani (LB) media, and protein expression was induced for 16 h with 0.25 mM final concentration of isopropylb-D-1-thiogalactopyranoside at 18 °C. Cell pellets were resuspended in lysis buffer containing 20 mM Tris HCl buffer pH 7.5, 500 mM NaCl, 0.5% v/v Triton X-100 and Complete protease inhibitor tablets (Roche), and sonicated for cell lysis. Metal affinity purification of clarified lysates was performed using His-Trap HP columns (GE LifeSciences), followed by His₆-tag cleavage using thrombin protease (Sigma-Aldrich) and size exclusion chromatography on Sephadex G75 columns (GE LifeSciences) equilibrated in PBS (20 mM sodium phosphate buffer pH 7.0, 150 mM NaCl and 2 mM DTT). For the production of isotopically labelled protein samples E. coli cells were grown in M9 minimal media supplemented with ¹⁵NH₄Cl and ¹³C₆-glucose (Isotech) as necessary.

The isolation and purification procedures were performed according to the method of Kuang et al. (1 (link)), with minor changes (Figure 1A). Sevag reagents precipitated ethanol (4°C, 24 h) and deproteinized to acquire crude EPS. Then, the crude lyophilized EPS was dissolved in pure water at 20 mg/ml concentration and filtered by a 0.22 μm filter. The EPS solution was eluted with gradient concentrations of sodium chloride solutions (0, 0.1, 0.3, and 0.5 M) at a flow rate of 1.5 ml/min by dicthylaminoethyl (DEAE)-Cellulose-52 anion exchange column (2.6 cm × 40 cm, GE Healthcare, Beijing, China). The eluate (10 ml/tube) was gathered and analyzed by the phenol-sulfate method. The crude EPS-LP2 was purified by Sephadex G-75 column (1.6 cm × 50 cm, GE Healthcare, Beijing, China) as described by Huang et al. (12 (link)) and eluted with pure water (0.5 ml/min). The eluent from each tube was gathered and analyzed. The purified EPS-LP2 with the highest absorption peak was collected and freeze-dried for further analysis.
The molecular weight of the purified EPS-LP2 was determined by adopted the method of Huang et al. (12 (link)) by high-performance gel permeation chromatography (HPGPC) with a TSK-gel-G3000_PWXL column (0.78 cm × 30 cm, Tokyo, Japan). The diverse dextran standards from 5.0 to 670 kDa were used to calculate the molecular weight.