Superdex peptide 10 300 gl

Pectin oligosaccharide was prepared from RG-I, as previously reported^{30 (link)}. Briefly, acid hydrolysis treatment was performed on the side chains of RG-I. Cell lysates of YesX-expressing recombinant E. coli^{30 (link)} were added to 2% RG-I main chain solution containing 20 mM MnCl₂ and 50 mM tris-hydroxymethyl aminomethane-hydrochloric acid (Tris–HCl) buffer (pH 7.5) followed by enzymatic reaction at 30 °C for 24 h. The mixture was boiled for 10 min to inactivate the enzyme. After centrifugation, the supernatant was subjected to Centriprep Centrifugal Filter Unit (Merck Millipore) to collect the products with molecular weight of less than 3,000. The collected products were purified using Superdex Peptide 10/300 GL (GE Healthcare Life Sciences) and Bio-Gel P2 column (Bio-Rad). Elution was performed with distilled water. The collected fractions were subjected to TLC to confirm the purification of pectin oligosaccharides. The pectin oligosaccharide prepared in this study was found to include ΔtriGalUA (ΔGalUA-GalUA-GalUA) as a result of X-ray crystallography of the SPH1118-substrate complex.

To investigate GAG-PTS import activity, unsaturated hyaluronan disaccharide was prepared using recombinant hyaluronate lyase. A reaction mixture containing BL21(DE3)/pET21d-gbs1270 cell extract, 0.2% hyaluronan, and 20 mM Tris-HCl (pH 7.5) was incubated at 30°C for 2 days. The mixture was then boiled to stop the reaction and centrifuged at 20,000 g for 20 min to remove aggregated proteins. The resulting supernatant was concentrated by freeze-drying and subjected to gel filtration chromatography [Superdex Peptide 10/300 GL (GE Healthcare)]. The eluted fractions containing unsaturated hyaluronan disaccharide were identified by monitoring the absorbance (235 nm) from the C = C double bonds of the disaccharide. To confirm the presence of unsaturated hyaluronan disaccharide, pooled fractions were subjected to thin-layer chromatography (TLC) using a solvent system of 1-butanol:acetic acid:water (3:2:2, v:v:v), and hyaluronan breakdown products were visualized by heating the TLC plates [silica gel 60 F₂₅₄ (Merck)] at 130°C for 5 min after spraying with ethanol containing 10% sulfuric acid. The final disaccharide preparation was freeze-dried and dissolved in sterilized water to a final concentration of 200 mM calculated from the absorption coefficient.

The Sel-tagged Affibody molecules were ¹¹C-labeled specifically at the Sec residue, by the method used previously [19 (link), 20 (link), 29 (link), 46 ]. Briefly, the Sel-tagged Affibody molecule was reduced with DTT (1 mM) at 35–37 °C for ≥20 min. An aliquot (10–25 μl) of [¹¹C]CH₃ (prepared as in [47 (link)] from cyclotron-produced [¹¹C]CO₂ (PETtrace, GE Healthcare)) and trapped in DMSO (0.2 ml)) was added. After 20 min at 35–37 °C, the reaction was quenched with DTT (5 mM) and the [methyl-¹¹C]-Affibody-ST-CH₃ was desalted (NAP-5, PBS). Radiochemical purity was analyzed by radio-HPLC (Superdex Peptide 10/300 GL (GE Healthcare) and eluted with PBS, using ultraviolet- (210 nm) and radio-detectors in series).