Hitrap desalting column

Plasmid (pET28a-MSP1D1ΔH5) was purchased from Addgene (USA), used for transformation of E. coli BL21(DE3) strain. 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was obtained from Tokyo Chemical Industry, Japan. Bio-beads SM-2 was obtained from BIORAD, USA. HiTrap^TM Desalting Column was purchased from GE Healthcare Japan Ltd. Ferric ammonium citrate (FAC) was obtained from Wako Pure Chemical Industries, Ltd, Japan. 2-Nitroso-5- [N-n-propyl-N-(3-sulfopropyl) amino] phenol (Nitroso-PSAP) and DDM were obtained from Dojin Chemical Research Laboratory, Japan.

Ubiquitin was expressed and purified as described previously.⁴¹ (link) The unlabeled, ¹⁵N-labeled and ¹³C/¹⁵N doubly-labeled LC3B was expressed as described previously for UBA domain constructs of SQSTM1.^30,41 (link) Thrombin cleavage was used to remove the GST-tag from glutathione-Sepharose-immobilized LC3B fusions. Purification of the eluted proteins using a salt gradient (0 to 2 M NaCl) on a cationic column (GE Healthcare, 17115201) in 5 mM potassium phosphate, pH 7, allowed fractions of purified LC3B to be concentrated by lyophilization. Fractions were then subsequently desalted on a 5 × 5 ml HiTrap^TM desalting column (GE Healthcare, 17-1408-01) in 25 mM ammonium acetate before further lyophilization.

The reformatting, expression, and purification of full-length antibodies was performed as described previously [31 (link),35 (link)]. Isolated yeast vectors were sequenced, and the VL fragment was reformatted into pTT5-derived vectors by golden gate assembly. For the soluble expression of full-length chimeric antibodies, Expi293F^TM cells (Thermo Fisher Scientific) were transiently transfected using the ExpiFectamine^TM 293 Transfection Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. For purification, cell culture supernatants were collected five days post transfection, sterile-filtered, and applied to a MabSelect^TM PrismA HP column (GE Healthcare, Piscataway, NJ, USA) using an ÄKTA pure^TM chromatography system (GE Healthcare). One-armed molecules were captured by IMAC (HisTrap HP, GE Healthcare), followed by Strep-Tactin XT affinity chromatography according to the manufacturer’s protocol. Buffer exchange against PBS was performed using a HiTrap^TM Desalting column (GE Healthcare).

Preparation of all media and protocols for heterologous expression of aga-F75 in P. pastoris GS115 followed the Pichia expression manual (Invitrogen, Carlsbad, CA, USA). The gene fragment coding for mature Aga-F75 without the signal peptide-coding sequence was cloned into vector pPIC9 to construct the recombinant plasmid pPIC9-aga-F75, which was further linearized and transformed into P. pastoris GS115 competent cells by electroporation. Positive transformants were screened based on their α-galactosidase activities as described below. The transformant showing the highest activity was selected for large-scale fermentation in 1-l conical flasks. To purify recombinant Aga-F75, the crude enzyme was sequentially loaded onto the HiTrapTM Desalting column and HiTrap Q Sepharose XL FPLC column from GE Healthcare (Uppsala, Sweden). The purity of Aga-F75 was identified to be 90% based on SDS-PAGE. Purified Aga-F75 was deglycosylated with PNGase F following the instruction of manufacturer (New England Biolabs, Ipswich, MA, USA).

E. coli BL21 (DE3) harboring recombinant plasmids were cultured in Luria Bertani (LB) medium containing 50 μg/ml kanamycin at 37 °C and 200 rpm till the OD₆₀₀ reached to 0.6. Then isopropyl-β-d-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM to induce the expression of enzymes. Cultivation was continuously grown for another 12 h at 18 °C followed by cells harvest. The harvested cell pellets were washed with PBS buffer and disrupted by sonication. Then the soluble fractions of the cell lysate were collected by centrifugation at 4 °C and 12 000 rpm for 20 min. The supernatant was subjected to a 5 ml HisTrap^TM FF column (GE Healthcare) which was pre-equilibrated with 50 mM Tris-HCl (pH 9.0) containing 0.5 M NaCl and 30 mM imidazole. After being extensively washed with the same buffer, the binding proteins were eluted with 50 mM Tris-HCl (pH 9.0) containing 0.5 M NaCl and 300 mM imidazole. The desalting of the eluent was performed through a HiTrap^TM Desalting column (GE Healthcare) equilibrated with 50 mM Tris-HCl (pH 9.0). The molecular weight and homogeneity of the purified proteins were evaluated by SDS-PAGE, and the protein concentration was determined by Bradford assay using bovine serum albumin (BSA) as the standard17 (link).

The viva flow 200 ultrafiltration membrane system (Sartorius, Germany) with 5 kDa cut-off was used for the concentration and buffer exchange (to 0.1 M Tris-HCl, pH 8.0) of the crude enzymes. The recombinant proteins were desalted by HiTrapTM Desalting column and purified using the HiTrap Q Sepharose XL FPLC column (GE Healthcare) pre-equilibrated with 0.1 M Tris-HCl (pH 8.0). The gradient NaCl of 0–0.7 M at the flow rate of 3 mL/min was used to elute the proteins. Fractions showing cellulase activities were pooled and further desalted with a 5 kDa molecular cut-off concentration tube (Millipore) using 0.1 M McIlvaine buffer (pH 3.5 or 4.5). The purified proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for purity and molecular mass analysis. Protein concentration was determined by using the Bradford method.

For OPAA114644 expression, the BL21 (DE3) strain with recombinant plasmid was cultivated in 200 mL Luria-broth (LB) medium containing 50 µg/mL kanamycin and 0.2 mM MnCl₂ at 37 °C. OPAA114644 expression was induced with 1 mM IPTG, and the protein was purified by nickel affinity chromatography with a 5 mL HisTrapTM HP column (GE Healthcare, Uppsala, Sweden as described previously [24 (link)]. The binding buffer was 50 mM Bis-Tris propane buffer, 0.5 M NaCl and 20 mM imidazole (pH 8.0), and the elution buffer was 50 mM Bis-Tris propane buffer, 0.5 M NaCl and 0.5 M imidazole (pH 8.0). A 5 mL HiTrapTM desalting column (GE Healthcare, USA) was used to remove imidazole with 50 mM Bis-Tris propane buffer (pH 8.0). The purified enzyme was verified by 12.5% SDS–PAGE, and the protein concentration was determined by Modified Bradford Protein Assay Kit (Sangon Biotech, Shanghai, China), with bovine serum albumin as the standard. To determine the oligomeric state of OPAA114644, its molecular weight in active state was measured by sedimentation velocity experiment using a ProteomeLab XL-I analytical ultracentrifuge (Beckman, Brea, CA, USA).