Hitrap column

Recombinant His-tagged endomannanases were over-expressed in E.coli BL21 (DE3) cells. Transformants were grown at 37°C with 200 rpm aeration in 500 ml of LB medium containing 50 μg/ml kanamycin until optical density measured at 600 nm (OD₆₀₀) reached 0.6–1.0. Protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG, 1 mM final concentration), followed by overnight agitation at 20°C. Cells were harvested and disrupted by sonication and the lysate was centrifuged at 2,360 x g for 20 min at 4°C and supernatant was loaded on a 5 ml Hi Trap column (GE Healthcare) for immobilized metal ion affinity chromatography (IMAC) purification. Protein elution was performed with a 0–500 mM imidazole gradient in 300 mM NaCl, 20 mM sodium phosphate buffer, pH 7.2 and protein concentration of pooled fractions was determined by Bradford method using BSA as protein standard [41 (link)].
The molecular mass of the enzymes was estimated by SDS-PAGE analysis. Endomannanase zymography was done according to Posta et al. [33 (link)], with minor modification: instead of carboxymethyl-cellulose 0,1% LBG mannan was added to the gel.

Anti-β7 (FIB504) and anti-α4β7 (DATK32) antibodies were ordered from ATCC and their sequences were individually cloned from hybridomas. Antigen-binding domain sequences from the parent hybridomas were fused to mouse IgG1 Fc in a pRK expression vector and transfected into HEK293 cells. Transfected supernatants were purified on a HiTrap column (GE healthcare) with Mabselect Sure resin (GE healthcare) with a phosphate-buffered saline (PBS) loading buffer. Antibodies were eluted with 0.1 M citrate (pH 3.0) and neutralized with 3 M Tris, pH 8.0, to a final pH of ∼7.0 prior to dialysis against PBS, pH 7.2. Each antibody was run on a Superdex S200 10/300 GL size exclusion column (SEC) (GE Healthcare) using PBS, pH 7.2, load buffer at a flow rate of 1 mL/min (30 cm/h) to remove any aggregates. Pooled fractions were filtered using a 0.2 mm filter and the final antibody preparation was assessed by analytical SEC carried out with a TSK-GEL, Super SW3000, 4.6 mm × 30 cm, 4 mm (Tosoh Bioscience) column using a Dionex Ultimate 3000 system (Thermo Fisher Scientific) to confirm > 95% homogeneity of monomeric antibody. Anti-αE blocking antibody (clone number M290) was from BioXcell and anti-E-cadherin antibody (clone number ECCD-2) was from Invitrogen.

The recombinant plasmids pWB980-20b-Xyn and pWB980-20b-XynB were transformed into the competent cells of B. subtilis WB800. The recombinant bacteria were grown in LB media containing 50 μg/mL Kanamycin at 37°C. The cells were harvested by centrifugation at 10,000 g for 10 min and then sonicated to release the intracellular proteins. The intracellular proteins and extracellular proteins were then heat treated at 60°C for 30 min and centrifuged (10,000 g for 20 min). The resulting supernatants were purified by affinity chromatography on Ä KTATPLC™ with a HiTrap column (GE Healthcare Life Sciences). To confirm the target proteins’ purity, SDS-PAGE was used. Bradford method was used to measure the concentration of the purified enzymes.

Single cysteine mutants of ColE9 or the isolated ColE9 DNase domain were desalted on a 5 ml HiTrap column (GE Healthcare) equilibrated with binding buffer (20 mM potassium phosphate pH 7, 500 mM NaCl) to remove DTT. The protein was diluted to 50 µM and labelled with a 5:1 molar excess of fluorophore:protein with either Alexa Fluor 488 C₅‐Maleimide (Invitrogen) (AF488), Alexa Fluor 568 C₅‐Maleimide (Invitrogen) (AF568), or Alexa Fluor 647 C₂‐Maleimide (Invitrogen) (AF647). Maleimide dyes were re‐suspended in DMSO and stored as a 10 mM stock. Labelling was allowed to proceed for 1–2 h at room temperature in the dark, and then quenched using 2 mM DTT for 15 min, before overnight dialysis at 4°C (20 mM potassium phosphate pH 7, 500 mM NaCl) to remove excess dye (Spectra Por 10K MWCO). Labelled protein was applied to a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with 20 mM potassium phosphate pH 7, 500 mM NaCl, to remove residual dye. Protein‐containing fractions were identified by SDS–PAGE. The protein concentration and labelling efficiency (typically > 90%) was estimated from absorbance readings using sequence‐based extinction coefficients, molecular weights, and dye correction factors (Invitrogen).

Anti-IMPDH1 antibody was generated against the bovine canonical recombinant protein (bIMPDH1-514aa). Bovine IMPDH1 cDNA corresponding to the canonical IMPDH1 (514aa) protein was amplified from total RNA from fresh bovine retinas and cloned into pET15b bacterial expression plasmid (Novagen, Madison, WI, USA) for expression in E. coli BL21 (DE3) cells. The expression of recombinant protein was induced in bacterial cultures at OD₆₀₀ = 0.6 for 5 hr at 37°C. Cells were then collected and bacterial inclusion bodies were obtained and solubilized in 6M guanidinium hydrochloride buffer as previously described (López-Begines et al., 2018 (link)). bIMPDH1-514aa.His was purified by metal chelation using a HiTrap column (GE Healthcare, Chicago, IL, USA) and dialyzed against dialysis buffer (1M urea, 0.4M L-arginine, 20 mM Hepes and 200 mM NaCl).
Purified bIMPDH1(514aa).His was used to immunize two New Zealand White rabbits following a standard 84 day protocol: initial injection of protein (0.5 mg) emulsified in Freund’s complete adjuvant, and three boosts of protein (0.25 mg) in incomplete adjuvant at three-week intervals. At exsanguination, blood serum was affinity purified with bIMPDH1-crosslinked Aminolink coupling resin (Thermo Fisher Scientific, Waltham, MA, USA); dialyzed against 0.1M sodium phosphate, 0.15M NaCl, pH 7.2, and concentrated with 10K Amicon devices.