Figure 1.

Optimization of assay conditions for XXT2 and XXT5. A to C, Effects of sodium chloride concentration (A), pH (B), and temperature (C) on His-GB1-XXT2 or His-GB1-XXT5 activity. D, Effects of imidazole and HEPES on His-GB1-XXT2 activity. The imidazole sample was in Tris-HCl buffer. All assays were with XXT2 unless indicated. All buffers were adjusted to pH 7.4 unless indicated. All constructs were expressed in SoluBL21 cells and purified with Ni-NTA columns. Assays contained 25 μL with concentrated His-GB1-XXT2 (3 μm, 2.5 μL) or His-GB1-XXT5 (5.5 μm, 8.25 μL), 12.5 μL of 2× buffer, 2 mm UDP-Xyl, 0.5 mm cellohexaose, and 2 mm MnCl₂ and were incubated for 4 h; 2× buffer is a buffer that contains a concentration 2 times higher than that of the desired final concentration of all components in the assay reaction. The final concentration of buffers was 50 mm for His-GB1-XXT2 reactions; the buffer concentration of His-GB1-XXT5 was 250 mm to reduce the effect of the larger volume of His-GB1-XXT5 protein that was added to the reaction on the overall reaction conditions. Products of the enzyme assays were analyzed by high-performance anion-exchange chromatography (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.