Figure 6
Enzymatic synthesis of the artificial Sialoglycopolypeptide. α2,6-Sialoglycopolypeptide was enzymatically synthesized from asialoglycopolypeptide using recombinant ST6Gal1. A mixture containing 5.0 mg of asialoglycopolypeptide, 16.0 mM CMP-β-Neu5Ac, 80 mU/ml of the purified FLAG-tagged ST6Gal1, 2.5 mM MnCl2, 0.1 BSA and 10 U/ml of calf intestine alkaline phosphatase (Boehringer-Mannheim, Mannheim, Germany) in 50 mM MOPS buffer (pH 7.4) was incubated at 37°C for 48 h in a total volume of 1.0 ml. After heating at 100°C followed by centrifugation, the supernatant from the reaction mixture was directly loaded onto a Sephadex G-25M PD-10 column equilibrated with 100 mM PBS (pH 7.4).