Expression and characterization of recombinant beta-secretase from Trichoplusia ni BTI Tn5B1-4 cells transformed with cDNAs encoding human beta1,4-galactosyltransferase and Gal beta1,4-GlcNAc alpha 2,6-sialytransferase.
Beta-Secretase (betaSEC) was expressed in Trichoplusia ni BTI Tn5B1-4 (Tn5B1-4) cells transformed with cDNAs encoding beta1,4-galactosyltransferase (GalT) and Gal beta1,4-GlcNAc alpha 2,6-sialyltransferase (ST). The apparent molecular weight of recombinant beta-secretase was increased from 57 to 59 k Da. A lectin blot analysis indicated that recombinant beta-secretase from Tn5B1-4 betaSEC/GalT-ST cells (Tn5B1-4 cells co-transformed with cDNAs encoding beta-secretase, glycosyltransferases, GalT, and ST) contained the glycan residues of beta1,4-linked galactose and alpha2,6-linked sialic acid. Two-dimensional electrophoresis revealed that recombinant beta-secretase from Tn5B1-4 beta SEC/GalT-ST cells had a lower isoelectric point than beta-secretase from control Tn5B1-4 betaSEC cells (Tn5B1-4 cells transformed only with beta-secretase cDNA). The enzyme activity of recombinant beta-secretase from Tn5B1-4 betaSEC/GalT-ST cells was enhanced up to 77% compared to control Tn5B1-4 betaSEC cells. The concentrations at half-maximum inhibition (IC(50)) values estimated from inhibition analyses using purified beta-secretases from Tn5B1-4/betaSEC and Tn5B1-4/betaSEC/GalT-ST cells were 32 and 290 nM, respectively.
Chang
Kyung Hwa
KH
Department of Genetic Engineering, Kyung Hee University, Suwon 449-701, Republic of Korea.
Baek
Nam In
NI
Yang
Jai Myung
JM
Lee
Jong Min
JM
Bo
Jeon Hwang
JH
Chung
In Sik
IS
D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D010450:Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000737:chemistry; D005456:Fluorescent Dyes; D006031:Glycosylation; D006801:Humans; D008970:Molecular Weight; D009036:Moths / Q000201:enzymology / Q000235:genetics*; D009237:N-Acetyllactosamine Synthase / Q000235:genetics* / Q000378:metabolism; D037121:Plant Lectins / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D054788:Ribosome Inactivating Proteins; D012799:Sialyltransferases / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D037121:Plant Lectins; D011506:Proteins; D011994:Recombinant Proteins; C017707:Ricinus communis agglutinin-1; C050600:Sambucus nigra lectins; C118914:amyloid beta-protein precursor inhibitor; D009237:N-Acetyllactosamine Synthase; D012799:Sialyltransferases; D054788:Ribosome Inactivating Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase