PILOT-SCALE EXPRESSION AND PURIFICATION OF SOLUBLE-PROTEIN-A TAGGED BETA-1,6N-ACETYLGLUCOSAMINYLTRANSFERASE IN CHO CELLS

Expression of recombinant soluble protein A tagged mouse core 2 beta 6 -N-acetylglucosaminyltransferase (E.C. 2.4.1.102) has been scaled-up i n CHO cells using a continuously operating fluidized bed system yieldi ng 0.3 U/day. A one step 213 fold purification by affinity chromatogra phy on IgG-Sepharose yielded a stable enzyme preparation with a specif ic activity of 44 mU/mg. The enzyme was shown to belong to the L-type with a highly restricted specificity for the acceptor substrate Gal be ta 1-->3GalNAc alpha 1-->R (core 1). Only little activity towards GlcN Ac beta 1-->3Gal-NAc alpha 1-->R (core 3) (<1%) and no incorporation o n unsubstituted benzyl or peptidebound GalNAc was detected. Zn2+ and t o a lesser extent Mn2+ were found to be inhibitory whereas Mg2+ could activate the enzyme. The enzyme preparation proved suitable for in vit ro application as a catalyst for the synthesis of core 2 structures.(2 ) (C) 1997 Academic Press.