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 in CHO cells using a continuously operating fluidized bed system yielding 0.3 U/day. A one step 213 fold purification by affinity chromatography on IgG-Sepharose yielded a stable enzyme preparation with a specific 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 beta 1-->3GalNAc alpha 1-->R (core 1). Only little activity towards GlcNAc beta 1-->3Gal-NAc alpha 1-->R (core 3) (< 1%) and no incorporation on unsubstituted benzyl or peptide-bound GalNAc was detected. Zn2+ and to a lesser extent Mn2+ were found to be inhibitory whereas Mg2+ could activate the enzyme. The enzyme preparation proved suitable for in vitro application as a catalyst for the synthesis of core 2 structures.
Zeng
S
S
Institute of Physiology, University of Zürich, Switzerland.
Dinter
A
A
Eisenkrätzer
D
D
Biselli
M
M
Wandrey
C
C
Berger
E G
EG
D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D004187:Disaccharides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006023:Glycoproteins / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D051379:Mice; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D013205:Staphylococcal Protein A / Q000096:biosynthesis* / Q000302:isolation & purification; D013379:Substrate Specificity; D014162:Transfection / Q000379:methods; D015032:Zinc / Q000494:pharmacology
D004187:Disaccharides; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D008345:Manganese; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase; D008274:Magnesium; D015032:Zinc