Superdex 200 increase 10 300 gl gel

The Vip3Aa protein was expressed in E. coli BL21(DE3) at 25 °C for 48 h in autoinduction terrific broth medium. Bacterial cells were collected by centrifugation and resuspended in lysis buffer (20 mM Tris–HCl pH 8.0 and 300 mM NaCl). After the cells were lysed by a high-pressure cell crusher (Union-Biotech), the supernatant was collected, run through Ni-NTA agarose resin (Qiagen), and washed with 20 mM Tris–HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0. The small ubiquitin-like motif tag was removed with homemade His-tagged ULPI protease at room temperature (20–30 °C) for 2 h and proteins were then eluted with lysis buffer. The proteins were further purified by HiTrap Q HP ion-exchange chromatography and Superdex 200 Increase 10/300 Gl gel filtration chromatography using an AKTApure chromatography system (GE Healthcare Life Sciences). Proteins corresponding to the molecular weight of the tetramerized Vip3Aa were used for subsequent biochemical and bioassay analyses. The expression and purification steps of other Vip3Aa truncation variants and mutations were similar to those of Vip3Aa. The concentration of proteins was quantified by a NanoPhotometer N60 (Implen). The molar absorption coefficient of each protein was predicted by Protean (DNASTAR) based on the amino acid composition of the protein.

OASF proteins at 1 mg mL⁻¹ protein were injected into a Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare) using the ÄKTA Pure system (GE Healthcare) housed at 10 °C. The column was connected in-line to a Dawn HELEOS II MALS detector equipped with a 662 nm laser source and Optilab T-rEX differential refractometer equipped with 658 nm LED source (Wyatt Technology, Santa Barbara, CA, USA). Protein concentrations through the eluted peaks were determined using a refractive index increment of dn·dc⁻¹ = 0.185 mL·g⁻¹. Molecular weights were calculated by Zimm plot analysis using the ASTRA software (v6.1.5.22; Wyatt Technology).