Expression and characterization of recombinant β-secretase from Trichoplusia ni BTI Tn5B1-4 cells transformed with cDNAs encoding human β1,4-galactosyltransferase and Gal β1,4-GlcNAc α 2,6-sialytransferase

β-Secretase (βSEC) was expressed in Trichoplusia ni BTI Tn5B1-4 (Tn5B1-4) cells transformed with cDNAs encoding β1,4-galactosyltransferase (GalT) and Gal β1,4-GlcNAc α 2,6-sialyltransferase (ST). The apparent molecular weight of recombinant β-secretase was increased from 57 to 59 kDa. A lectin blot analysis indicated that recombinant β-secretase from Tn5B1-4βSEC/GalT-ST cells (Tn5B1-4 cells co-transformed with cDNAs encoding β-secretase, glycosyltransferases, GalT, and ST) contained the glycan residues of β1,4-linked galactose and α2,6-linked sialic acid. Two-dimensional electrophoresis revealed that recombinant β-secretase from Tn5B1-4β SEC/GalT-ST cells had a lower isoelectric point than β-secretase from control Tn5B1-4βSEC cells (Tn5B1-4 cells transformed only with β-secretase cDNA). The enzyme activity of recombinant β-secretase from Tn5B1-4βSEC/GalT-ST cells was enhanced up to 77% compared to control Tn5B1-4βSEC cells. The concentrations at half-maximum inhibition (IC50) values estimated from inhibition analyses using purified β-secretases from Tn5B1-4/βSEC and Tn5B1-4/βSEC/GalT-ST cells were 32 and 290 nM, respectively.