Vivaspin protein concentrator

IMAC (immobilized metal affinity chromatography) purified NT*-Bri2 BRICHOS was concentrated to 3.4 mg/mL with a Vivaspin protein concentrator (10 kDa cut-off, GE Healthcare) at 4000×g and 4 °C. Then, the different rh NT*-Bri2 BRICHOS oligomeric species were isolated by size exclusion chromatography (SEC) using a Superdex 200 26/600 column (GE Healthcare, Uppsala, Sweden), which was equilibrated with Buffer B (20 mM NaP_i buffer pH 8.0 containing 0.2 mM EDTA), and an ÄKTA Explorer liquid chromatographic system (GE Healthcare, Uppsala, Sweden). The individual peaks corresponding to the differently sized oligomeric species were narrowly collected. Then, the rh NT*-Bri2 BRICHOS species were cleaved separately with 1: 600 thrombin (w/w, Merck) in a cold room overnight. After cleavage, the NT* tag was removed by reapplying the sample onto an IMAC column with buffer B (reverse IMAC), where rh Bri2 BRICHOS was found in the flow-through, and the NT*-tag bound to the column was eluted with buffer B containing 200 mM imidazol. To polish the Bri2 BRICHOS species, they were further purified with SEC one more time, using a Superdex 200 26/600 (for oligomer, tetramer, and dimer), or a Superdex 75 26/600 (for monomer) column (GE Healthcare, Uppsala, Sweden). The purified Bri2-BRICHOS species were stored at −20 °C.

Selected AKTA fractions were concentrated using Vivaspin protein concentrator spin columns with 10 KDa MWCO (GE Healthcare) and protein was quantified using a Qubit protein assay (Thermo Fisher Scientific). Levels of Type 4 polysaccharide in vaccine preparations was quantified by ELISA using type 4 antiserum and a standard curve generated using purified type 4 polysaccharide (SSI, Denmark).

The aerial parts of the plants were harvested 5 days post agroinfiltration and homogenized in two buffer volumes of PBS (Lonza), supplemented with cOmplete^™ EDTA-free protease inhibitor (Roche). The crude homogenate was incubated for 1 h, at 4°C, with shaking and then filtered through four layers of Miracloth^™ (Merck). The crude plant sap was then clarified by sequential centrifugation steps; twice at 15,344 × g for 20 min and then again at 17,000 × g for 20 min. The supernatant was vacuum-filtered through a 0.45 µM Stericup-GP device (Merck Millipore) and applied to a Galanthuis nivalis lectin (GNL) column (Sigma) with a 0.5–1 ml/min flow rate. The column was sequentially washed with 100 ml of 0.5 M NaCl and then 100 ml of PBS (Lonza). The proteins were eluted in 1 M methyl α-D-manno-pyranoside (MMP) (Sigma), buffer exchanged into PBS and then concentrated using a Vivaspin Protein Concentrator with a 30 kDa cut-off (GE Healthcare). The purified proteins were quantified using the DC^™ Protein Assay (Bio-Rad).

A hHAO1 construct, encoding residues Met1-Ser368, with an engineered N-terminal His6-tag subcloned into the pNIC28-Bsa4 vector (Mackinnon et al., 2018 (link)), was transformed into E. coli BL21 (DE3) cells. hHAO1 was cultured in auto-induction Terrific Broth (Fox and Blommel, 2009 (link)) for 6 h before incubation at 18°C for 40 h. Cell pellets were harvested, homogenized in lysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP, 0.1 mM FMN), and centrifuged to remove insoluble material. The supernatant was purified by Nickel affinity (Thermo Fisher Scientific) followed by size exclusion (Superdex 200 Hi-Load 16/60, GE Healthcare) chromatography into crystallization buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP). The purified protein was concentrated to 13.7 mg/ml by cycles of centrifugation (15 min, 4,000 rpm, 4°C) and mixing in a Vivaspin protein concentrator with a molecular weight cut-off of 30 kDa (GE Healthcare).

All the material/chemicals used were of the highest quality and were purchased from Sigma or AppliChem. Enzymes for molecular biology were from New England Biolabs or Thermo Fisher Scientific. Anatrace was the primary supplier for all the detergents used, except for cholesteryl hemisuccinate Tris salt (CHS), which was purchased from Sigma. DNaseI was purchased from Roche. Empty disposable PD-10 columns, Superdex 200 10/300 GL, and Vivaspin protein concentrators were purchased from GE Healthcare. The tritiated agonist [³H] neurotensin and [³⁵S]GTPγS (1,250 Ci/mmol) was purchased from Perkin-Elmer. The unlabeled truncated neurotensin ligand (NT_8-13, RRPYIL) and its variant NT1 (GPGGRRPYIL) were purchased from Anaspec.