Glass econo column

WARS2 wild-type and WARS2(L53F) mutant proteins were prepared and purified by Genscript using the Baculovirus expression system. Recombinant WARS was prepared in house using an overnight Rosetta (Novagen) culture transformed with pEX-N-GST-WARS diluted in 1 L Luria-Bertani broth, shaking at 180 r.p.m. at 37 °C. Protein expression was induced with 1 mM IPTG and incubated at 16 °C for 16 h. The induced culture was harvested by centrifugation at 5,000g for 15 min, followed by resuspension in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% sodium deoxycholate, 1% triton, 25% glycerol, 1 mg ml⁻¹ lysozyme, 1 mM DTT and complete protease inhibitor cocktail (Roche)) followed with sonication. The lysate was cleared by centrifugation and the supernatant mixed with 2 ml of glutathione sepharose 4B resin (GE Healthcare) and resuspended in buffer (50 mM Tris pH 7.5, 150 mM NaCl, 25% glycerol, 1 mM DTT). The mixture was then applied to a glass econo-column (BioRad) and resin-bound WARS washed and then eluted in fractions of 2 ml elution buffer (50 mM Tris pH 8.0, 150 mM NaCl, 25% glycerol, 20 mM of L-glutathione reduced (Sigma) and 1 mM DTT). Fractions were run on SDS–PAGE gel and stained with Coomassie blue to check for purity and pure fractions pooled and buffer exchanged with 10 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol and 0.005% P20.

The oligosaccharides produced by L. lactis CCK940, L. lactis SBC001, and W. cibaria YRK005 strains were purified as previously described [11 (link),32 (link),33 (link)]. In this study, the oligosaccharides produced by L. lactis CCK940, L. lactis SBC001, and W. cibaria YRK005 strains were named CCK-, SBC-, and YRK-oligosaccharides, respectively. In brief, the culture supernatant of lactic acid bacteria was obtained by centrifuging at 9820× g for 15 min (Beckman Coulter, Brea, CA, USA), and the supernatant was concentrated under reduced pressure at 60 °C. The concentrated supernatant was then loaded onto Bio-Gel P2 in a Glass Econo-Column (1.5 × 120 cm), and the oligosaccharides were separated by gel-permeation chromatography (GPC) (Bio-Rad, Hercules, CA, USA). The flow rate was 0.5 mL/min, and the eluents were fractionated using a fraction collector (Gilson Inc., Middleton, WI, USA) with 5 mL per tube. The fractions were analyzed by TLC, and the active fractions containing oligosaccharides were collected and lyophilized (SunilEyela, Seongnam, Republic of Korea).

The medium of GAA-producing Arabidopsis alg3 culture was filtered through a Glass Econo-Column (Bio-Rad). NaCl was subsequently added to the medium until reaching a final concentration of 4 M. Then, the NaCl-containing medium was loaded into a hydrophobic interaction chromatography column (Toyopearl Phenyl-650 M, Tosoh Corporation) pre-equilibrated in 20 mM Tris-HCl pH 7.5 with 4 M NaCl. After washing the column using 4 M NaCl in 20 mM Tris-HCl pH 7.5 buffer, GAA was eluted by decreasing NaCl concentration. GAA-containing fractions were dialyzed to exchange the buffer into 20 mM sodium acetate pH 4.3. The dialyzed sample was applied to a cation exchange chromatography column (Toyopearl SP 550C, Tosoh Corporation) pre-equilibrated in 20 mM sodium acetate pH 4.3 buffer. After washing the column, GAA was eluted with 20 mM sodium acetate pH 4.3 containing 0.1–0.5 M NaCl. GAA-containing fractions were concentrated using a Vivaspin 20 with a 10 kDa cutoff (Sartorius Stedim Biotech GmbH).

The extracted nail proteins were transferred into a 2 mL boronate affinity column [Glass Econo-Column (Bio-Rad Laboratories Inc., Richmond, CA, USA)] to undergo a chromatographic separation into glycated nail proteins and nonglycated nail proteins. The wash buffer consisted of 0.25 mol/L ammonium acetate, containing 0.05 mol/L magnesium chloride, pH: 8.3. The elution buffer consisted of 0.1 mol/L Tris-HCl, containing 0.2 mol/L sorbitol (D-Glucitol, Sigma Aldrich, St. Louis, MO, USA) and 0.05 mol/L EDTA [34 (link)]. The concentration of proteins was assayed by the pyrogallol red-molybdate method on a Cobas 8000 analyzer [35 (link)]. The percentage of the glycated protein fraction was calculated from the protein content of both fractions. Following chromatography, binding and nonbinding fractions were analyzed using electrophoresis.