Ktaprime plus chromatography system

The construction of the Xcl1-targeted and CCL3-targeted vaccibodies have been described previously^{5 (link),6 (link)}. Purification of NIP-, Xcl1- and CCL3- vaccibodies with mCherry or OVA as antigen were performed as described in Gudjonsson et al.²¹ . In brief, HEK293E cell were transiently transfected in 5-layer BD Multi-Flasks (Corning) using Lipofectamine 2000 (Invitrogen). Supernatants were harvested after 3–4 days and applied to a column containing CaptureSelect FcXL affinity Matrix (Life Technologies) connected to an ÄKTAprime plus chromatography system (GE healthcare). Bound vaccibodies were washed with PBS and eluted using 0.1 M glycin-HCl pH 2.7. Eluted proteins were dialyzed twice against PBS, and subsequently concentrated using Vivaspin20, 50.000 MWCO cutoff columns (Sartorius).

The fusion proteins (EGFP-LecA and LecA-EGFP) were produced in E. coli NiCo21 (DE3) (New England Biolabs GmbH, Frankfurt am Main, Germany) after the transformation of the plasmids pET-28a(+)-EGFP-LecA and pET-28a(+)-LecA-EGFP, respectively. The ÄKTA prime plus chromatography system (GE Healthcare, Leverkusen, Germany) was used for the first round of protein purification. A HisTrap FF Crude column (Cytiva, Freiburg im Breisgau, Germany), which uses an Ni-NTA agarose stationary phase, was utilized in this system. After purification, the fusion protein samples were transferred into a SnakeSkin dialysis tube (Life Technologies GmbH, Darmstadt, Germany) with a rated molecule weight cut-off of 10 kDa and dialyzed overnight with ice-cold DPBS^-/-. The next day, the dialysis buffer was renewed, and samples were continually dialyzed for another 5 h. Then, the fusion protein samples were collected and prepared for size exclusion chromatography (SEC) as a second round of purification. Gel filtration chromatography with DPBS^-/- as a buffer was used as a polishing step for fusion protein purification. Fractions eluted from the affinity column containing EGFP-LecA and LecA-EGFP were pooled together and additionally purified on a HiLoad 26/600 Superdex 200 pg column (Cytiva, Freiburg im Breisgau, Germany) using an ÄKTA avant system (Cytiva, Freiburg im Breisgau, Germany).

The TPC1 antibody was generated against the N-terminal Cys-peptide corresponding to amino acid region 774–790 of murine TPC1 (C-YQEEIQEWYEEHAREQE). Peptides were obtained from Intavis. The TPC2 antibody was generated by immunizing rabbits with the N-terminal Cys-peptide corresponding to amino acid region 705–726 of murine TPC2 (C-FRDILEEPKEEELMEKLHKHPH). Sera from immunized rabbits were peptide affinity purified employing the SulfoLink Immobilization Kit for Peptides (Thermo Scientific) according to the manufacturer´s protocol and an ÄKTAprime plus chromatography system (GE Healthcare Life Sciences). Antibody elution was performed by applying a linear pH gradient. The TPC1 antibody was first described in Arndt et al. (2014 (link)) and Castonguay et al. (2017 (link)); the TPC2 antibody first in Grimm et al. (2014 (link)).
Anti-alpha 1 sodium potassium ATPase antibody ab7671 was provided from abcam.

The purification of mAbs was performed by affinity chromatography. Briefly, novel mouse mAbs were purified by salt fractionation (solid ammonium sulphate ([NH₄]₂SO₄; 45% of saturation - 270 g/L; Fisher Scientific) followed by affinity chromatography using a 5 ml HiTrap Protein G HP column in an ÄKTAprime plus chromatography system (both from GE Healthcare, UK). The antibody sample was loaded into the HiTrap Protein G HP column pre-equilibrated with 25 ml binding buffer (0.1 M phosphate buffer, 0.15 M NaCl, pH 7.4) at a flow rate of 1 ml/min. Unbound proteins were removed by washing the column with 50 ml binding buffer and bound antibodies were eluted with 25 ml elution buffer (0.1 M glycine, pH 2.5). Selected fractions were pooled together and subjected to buffer exchange with PBS using the HiPrep 26/10 Desalting column (GE Healthcare Life Sciences). Following buffer exchange, the purified antibodies were filtered through a 0.2 μM syringe filter (Merck Millipore, UK), aliquoted and stored at -20°C for further studies.

The MW analysis was conducted by means of gel permeation chromatography using the method described by Hartmann et al. [36 (link)]. The setup comprises an ÄKTAprime plus chromatography system (GE Healthcare, Solingen, Germany) equipped with a Superdex 200 pg prepacked column (HiLoad 26/600; GE Healthcare, Solingen, Germany). A phosphate buffer (pH 7.0) obtained from Merck (Darmstadt, Germany).served as eluent at a flow rate of 2.2 mL min⁻¹. Calibration of the separation column regarding the molecular masses contained in the collected fractions was performed according to Kupetz and Gastl [37 ] using defined molecular size standards from Megazyme (Bray, Ireland). BSG hydrolysates were produced according to Section 2.2.3. Since the initial sample volume is highly diluted during chromatographic separation, the solids concentration of each sample subjected to MW analysis was adjusted individually. Based on the previously measured total β-glucan and AX content in the respective hydrolysate, the suspensions contained between 3 and 15% (w/w) solids. All samples were filtered (0.45 µm) and an initial volume of 3.0 mL was manually injected. The data were analyzed using PrimeView 5.0 software (GE Healthcare, Solingen, Germany). Measurements were performed in triplicate (n = 3).