Figure 2.

Protein expression and enzyme activity of XXT1, XXT2, and XXT5. All His-GB1-XXT proteins were expressed in SoluBL21 cells and purified on Ni-NTA resin. A, SDS-PAGE of XXT1, XXT2, and XXT5 with 10 μL (5.6 μg), 20 μL (12 μg), and 25 μL (12.5 μg) of the elution loaded in each well, respectively. The positions of XXT proteins are shown with asterisks. B, HPAEC of XXT enzyme assay products. XXT1 and XXT2 concentration in assay reactions was 3 μm; XXT5 was concentrated 6-fold, and the concentrated protein was assayed at 11.5 μm. PAD, Pulsed-amperometric detection. C, MALDI-TOF chromatograms of enzyme assay products. Mass in MALDI-TOF chromatograms represents the mass of the oligosaccharide plus a sodium ion; the negative control consists of the activity assay reaction with no enzyme. D, Enzyme assay of XXT1, XXT2, and XXT5 in combination. All XXT concentrations were 8.5 μm, with reaction time of 20 h. Black bars indicate monoxylosylated cellohexaose, white bars indicate dixylosylated cellohexaose, and gray bars indicate trixylosylated cellohexaose. E, XXT5 activity with various acceptors. Acceptor substrates were synthesized as described previously (Fauré et al., 2007). All reactions contained 2 mm UDP-Xyl, 2 mm MnCl₂, 0.5 mm acceptor substrate, and 14 μm XXT5 with reaction time of 4 h. Products of the enzyme assay were analyzed by HPAEC, quantified by peak integration, and are presented as pmol UDP-Xyl min⁻¹ μg⁻¹ XXT protein. All enzyme assays were performed in duplicate.