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

Kyung Hwa Chang; Nam In Baek; Jai Myung Yang; Jong Min Lee; Jeon Hwang Bo; In Sik Chung

doi:10.1016/j.pep.2005.08.010

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

Protein Expr Purif. 2005 Dec;44(2):87-93. doi: 10.1016/j.pep.2005.08.010. Epub 2005 Sep 13.

Authors

Kyung Hwa Chang¹, Nam In Baek, Jai Myung Yang, Jong Min Lee, Jeon Hwang Bo, In Sik Chung

Affiliation

¹ Department of Genetic Engineering, Kyung Hee University, Suwon 449-701, Republic of Korea.

PMID: 16226891
DOI: 10.1016/j.pep.2005.08.010

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amyloid Precursor Protein Secretases
Animals
Aspartic Acid Endopeptidases
Cell Line
Electrophoresis, Gel, Two-Dimensional
Endopeptidases / biosynthesis*
Endopeptidases / chemistry
Endopeptidases / isolation & purification
Enzyme Inhibitors / chemistry
Fluorescent Dyes
Glycosylation
Humans
Molecular Weight
Moths / enzymology
Moths / genetics*
N-Acetyllactosamine Synthase / genetics*
N-Acetyllactosamine Synthase / metabolism
Plant Lectins / chemistry
Proteins / chemistry
Recombinant Proteins / biosynthesis*
Recombinant Proteins / chemistry
Recombinant Proteins / isolation & purification
Ribosome Inactivating Proteins
Sialyltransferases / genetics*
Sialyltransferases / metabolism
Transfection
beta-D-Galactoside alpha 2-6-Sialyltransferase

Substances

Enzyme Inhibitors
Fluorescent Dyes
Plant Lectins
Proteins
Recombinant Proteins
Ricinus communis agglutinin-1
Sambucus nigra lectins
amyloid beta-protein precursor inhibitor
N-Acetyllactosamine Synthase
Sialyltransferases
Ribosome Inactivating Proteins
Amyloid Precursor Protein Secretases
Endopeptidases
Aspartic Acid Endopeptidases
BACE1 protein, human
beta-D-Galactoside alpha 2-6-Sialyltransferase