Hitrap cm ff column

The venom compositions of RVV were isolated for phospholipase A₂(PLA₂) and metalloproteinase (MP) as the dominant protein
families, and L-amino acid oxidase (LAAO) and phosphodiesterase (PDE) as minor
protein families, as previously described [19 (link)]. Briefly, crude RVV was divided for isolation through
fractionation methods. One hundred milligrams of pool-crude RVV were used to
obtain phospholipase A₂ (PLA₂), resulting in a protein
yield of 7.6 mg. Another 100 mg of pool-crude RVV was isolated using
fractionation methods for metalloprotease (MP), L-amino acid oxidase (LAAO), and
phosphodiesterase (PDE), yielding 4.8 mg, 0.7 mg, and 0.32 mg of protein,
respectively, for comparative purposes. The enzymatic activities of crude RVV
venoms were measured as previously described [19 (link)].
To isolate PLA₂ from crude RVV, ion-exchange chromatography was
performed on a HiTrap CMFF column (GE Healthcare, Sweden). PLA₂activity was assessed according to the method of Holzer and Mackessy [20 (link)]. The isolation of MP, PDE, and LAAO was
achieved through gel filtration on SuperdexTM 75 10/300GL and column
ion-exchange chromatography. The proteolytic activity and inhibitor assay for MP
were determined using the method described by Anson [21 (link)]. LAAO activity was determined according to the
Worthington Enzyme Manual [22 ]. PDE
activity was measured according to Lo et al. [23 ].

A cDNA sequence encoding murine Lect2 was amplified by polymerase chain reaction. The amplified fragments were cloned in-frame into the EcoRI/XhoI site of the pCMV-Myc-C vector (Clontech, Mountain View, CA, USA), in which a c-Myc tag was introduced at the C-terminus.
Murine LECT2 was expressed in Expi293 cells (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s protocol. The cells were grown for 5–7 days after transfection, and the cell culture supernatant was diluted in 20 mmol/L phosphate buffer without sodium chloride and potassium chloride, pH 7.2 (buffer A) and loaded onto a HiTrap CM FF column (GE Healthcare Bioscience, Piscataway, NJ, USA) equilibrated with buffer A. After the column was washed with buffer A, the protein was eluted with a linear gradient of 0–1 mol/L sodium chloride. Next, the eluted protein was loaded onto a c-Myc-tagged protein purification cartridge (MBL International, Woburn, MA, USA) equilibrated with PBS (Wako Chemicals, Tokyo, Japan). The cartridge was washed with PBS, followed by elution with 0.5 mg/mL c-Myc tag peptide in PBS. The purified LECT2 was concentrated using VivaSpin (GE Healthcare). To determine the protein concentration, absorbance at 280 nm was measured (1 absorption unit at 280 nm = 1 mg/mL for this study). The mass spectra were measured using Ultraflex III (Bruker Daltonics, Billerica, MA, USA).

To isolate the DC maturation factors, protein samples were applied to an AKTA Explorer 100 FPLC system (GE Healthcare, Piscataway, NJ, USA). Briefly, 30 ml anti-CD3 Ab-activated supernatant from NKT cells was applied to a HiTrap diethylaminoethanol fast flow (DEAE FF) column (5 ml, GE Healthcare), and the DC maturation factors were eluted with 20 mM Tris-Cl (pH 8.0) containing 1 M NaCl. From each collection tube, 50 μl of supernatant was used to test the allogeneic immature DC maturation effect. Samples that showed a DC maturation effect were concentrated by dialysis with 20 mM Tris-Cl buffer at 4°C overnight after freeze-drying. The active region was again loaded to a HiTrap CM FF column (5 ml, GE Healthcare) and corrected by DEAE anion column chromatography. Active regions that were prepared for cation chromatography [27 (link)] were reloaded onto a HiTrap Blue HP (1 ml, GE Healthcare) and finally eluted with 1.5 M KCl (pH 7.0) containing 50 mM KH₂PO₄. The protein contents of the final active regions were analyzed by mass spectrometry (MS).

PEGylated rhG-CSF was prepared as control, referring to a previously reported method (Veronese et al., 2007 (link)). Briefly, rhG-CSF was dissolved in 0.05 M sodium phosphate buffer, pH 7.2, containing 4 M guanidinium chloride (Gu HCl). And, 10 kDa PEG-MAL was added at a mole ratio of PEG-MAL to rhG-CSF of 5:1. The solution was incubated at 30°C for 2 h. Before purification, the reaction mixture was dialyzed with buffer A (10 mM acetate buffers, pH 4.2), and then concentrated using Amicon Ultra-15 centrifugal filter devices with a 3 kDa MW cut-off (Millipore Corp. Bedford, MA, United States). Subsequently, the treated reaction mixture of 2 ml was loaded on a 5 ml HiTrap CM FF column (GE Healthcare, United States) pre-equilibrated with buffer A, using an AKTA purifier 10 system (GE Healthcare, United States) at room temperature. After the column was washed with three column volumes of the buffer A, PEGylated rhG-CSF fractions were eluted with a linear salt gradient from 0 to 50% buffer B (10 mM acetate buffer containing 1 M NaCl, pH 4.2) over 120 min, at a flow rate of 1 ml/min with UV detection at 280 nm. The collected fraction of purified PEG_10k-rhG-CSF was dialyzed with buffer A, and then concentrated using Amicon Ultra-15 centrifugal filter devices with a 3 kDa MW cut-off (Millipore Corp. Bedford, MA, United States), and stored at −20°C until use.