Blue sepharose 6 fast flow

When the fermentation process was completed, the culture medium was harvested and centrifuged at 4°C and 8000 rpm for 20 min. The supernatant was concentrated by ultrafiltration using Millipore Cogent M1 Tangential Flow Filtration System (molecular weight cutoff, 30kDa) and then loaded onto a SephadeG-25 column(2.6cm×60cm) to remove pigment. A weak cation exchanger (Capto™ MMC, GE Health, 2cm×25cm) pre-equilibrated with sodium acetate -acetic acid buffer (25mM, pH 4.6) was then applied. The bound protein fractions were eluted using Na₂HPO₄-NaH₂PO₄ buffer (50mM, pH7.2) containing 1.0 M NH₄Cl. Fractions containing the target protein were pooled and further loaded on a Blue-Sepharose™ 6 Fast Flow (GE Health, 2cm×25cm) column pre-equilibrated with 0.05M citric acid-0.1M Na₂HPO₄ (pH 7.0). The column was washed with the same buffer to baseline and the bound protein was eluted with 0.05M KH₂PO₄ containing 1.5M KCl (pH 7.0). The collection of target protein was stored at 4°C for further analysis. SDS-PAGE was carried out to determine the homogeneity of purification and the molecular mass of the recombinant fusion protein as previously described [25 ]. Coomassie brilliant blue R-250 was used for staining.

Blue Sepharose 6 Fast Flow was from GE Healthcare (Tokyo, Japan). Potassium warfarin (Eisai Co., Tokyo, Japan), L-tryptophan (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and diazepam (Nippon Roche K.K., Tokyo, Japan) were obtained. All other chemicals were of the highest grade commercially available, and all solutions were prepared using deionized and distilled water.

Sea-ICR mice (5 weeks, male) were obtained from Kyudo Co., Ltd. (Saga, Japan). Blue Sepharose 6 Fast Flow, HiTrap Phenyl HP column and PD-10 desalting column were obtained from GE Healthcare (Tokyo, Japan). Chloral hydrate was obtained from Sigma (Tokyo, Japan). Diff-Quick reagents were purchased from Kokusai Shiyaku (Kobe, Japan). Coomassie Brilliant Blue solution and HistoVT One were obtained from Nacalai Tesque (Kyoto, Japan). Interferon-γ (IFN-γ) ELISA kit was purchased from Biolegend (San Diego, CA, USA). Mayer’s hematoxylin, a 1% eosin alcohol solution, mounting medium for histological examinations (malinol) were from Muto Pure Chemicals (Tokyo, Japan). 4′,6-Diamidino-2-phenylindole (DAPI) was obtained from Dojindo (Kumamoto, Japan). All other chemicals were of the highest analytical grades available.

Expression of rHA-Infestin-4 was performed as described previously [26 (link)], with the modification that the coding sequence had been recloned into expression vector pIRESneo3 and a stable Chinese hamster ovary (CHO) clone had been selected for high yield fermentation. Purification was started by concentrating the cell culture supernatant using the Centramate 500S Tangential Flow Filtration system with 30 kD membranes (Pall, Crailsheim, Germany). The concentrate was thereafter purified using Blue Sepharose 6 Fast Flow (GE Healthcare, Freiburg, Germany) and CaptureSelect Human Albumin (Life Technologies, Leiden, the Netherlands) chromatography, both according to protocols provided by the manufacturers. The eluate was finally dialysed and concentrated to around 50 mg/mL of protein.

Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) studies were performed in accordance with CLSI Guideline EP17‐A2. Five pooled serums (each pooled serum is prepared using different serums) were treated with resin (Blue Sepharose 6 Fast Flow; GE Healthcare) to remove albumin (ALB) analyte to prepare blank samples for the LoB study. A scalar dilution with saline was used to prepare samples for LoD and LoQ studies.