Histrap ff crude column

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).

VP1 pentamer were produced using plasmid pET15b expression vector encoding BKPyV VP1 mutant amino acid sequences from positions 30–300 with an N-terminal hexahistidine tag (His-tag) and a thrombin cleavage site (BioCat Gmbh). The protein was expressed in E.coli BL21 DE3 by IPTG induction. Proteins were purified first by nickel affinity chromatography using a 5 mL HisTrap FF crude column (Cytiva). After protein sample loading, the column was washed with 20 mM Tris pH 7.5, 250 mM NaCl, 10 mM imidazole and 10% glycerol, and proteins were eluted by applying a gradient of elution buffer composed of 20 mM Tris pH 7.5, 250 mM NaCl, 500 mM imidazole and 10% glycerol. For glycan array analysis, the His-tag was retained while for crystallisation this tag was cleaved with 10 U/mg of thrombin protease (Cytiva) for 24 h at 20°C with agitation. Cleaved and uncleaved pentamers were finally purified by size-exclusion chromatography on a Superdex 200 16/600 column (Cytiva) by eluting protein with 20 mM HEPES pH 7.5 and 150 mM NaCl.

Protein purification was performed depending on the production scale in either 24-well filter plate with 0.5 mL resin (10 mL scale) or 1 mL column on Äkta go (Cytiva), Äkta Pure (Cytiva), or Profina System (BIO-RAD). MabSelect SuRe or HiTrap Fibro PrismA (Cytiva) was used as resins for Protein A purification. For His-tag purification of Expi293F, supernatant HisTrap FF Crude column (Cytiva) and for His-tag purification of insect cell supernatant HisTrap excel column (Cytiva) was used. All purifications were performed according to the manufacturer’s manual. Indicated antigens were further purified by SEC by a 16/600 Superdex 200 kDa pg (Cytiva). All antigens, antibodies, and scFv-Fc were run on Superdex 200 Increase 10/300GL (Cytiva) on Äkta or HPLC (Techlab) on an AdvanceBio SEC 300 Å 2.7 µm, 7.8 × 300 mm (Agilent) for quality control.

For crystallization trials,
protein purification tags were removed by overnight cleavage with
TEV protease at 30 °C as previously described.^{40 (link)} Cleaved proteins were purified by affinity-tag purification
using a HisTrap FF crude column (Cytiva) on an ÄktäPure
FPLC instrument, collecting the flow-through. Proteins were further
separated by size exclusion chromatography (HiLoad 26/600 Superdex
75, Cytiva) and concentrated using Ultra-4 or 15 mL centrifugal filter
devices (Amicon, Merck). The correct size and high purity were verified
via SDS–PAGE and LC-MS analysis. Protein labeling was performed
in activity buffer, overnight at room temperature using fluorophore
substrates at 10 μM (CA-TMR/CA-CPY and BG-TMR for HT7/HOB and
SNAP, respectively) in the presence of 5 μM (3 mg) protein.
After concentration to ∼200 μL, an excess of fluorophore
substrate was removed by buffer exchange using Illustra microspin
G-25 columns (Cytiva) according to the manufacturer’s instructions.
Protein labeling was verified by SDS–PAGE fluorescence scanning
and LC-MS analysis. Protein concentrations were adjusted between 10
and 20 mg/mL and submitted to crystallization trials using different
commercial screens via mixing in a 200 nL final volume protein solution/crystallization
solution (1:1) using a Mosquito robot (TTP Labtech).

To generate purified MPP8-10xHIS protein, pCMV6-MPP8-10xHIS was overexpressed in HEK293 by transfection with Lipofectamine 2000 according to the manufacturer’s protocol. After 24 h, cells were lysed with three cycles of snap freezing in liquid nitrogen and 2% triton X-100 with protease inhibitors. Recombinant MPP8-10xHis was purified using HisTrap FF crude columns (Cytiva Europe, Freiburg, Germany, #11000458) with a linear gradient of imidazole (from 20 to 500 mM, Merck, Burlington, United States, #104716) in an Äkta Prime Plus FPLC system (GE Healthcare/Cytiva Europe).