Enhanced activity of recombinant beta-secretase from Drosophila melanogaster S2 cells transformed with cDNAs encoding human beta1,4-galactosyltransferase and Galbeta1,4-GlcNAc alpha2,6-sialyltransferase.
beta-Secretase (betaSEC) was expressed in Drososphila melanogaster Schneider 2 (S2) cells transformed with cDNAs encoding beta1,4-galactosyltransferase (GalT) and Galbeta1,4-GlcNAc alpha2,6-sialyltransferase (ST). The apparent molecular weight of recombinant beta-secretase was increased from 56kDa to 61kDa. A lectin blot analysis indicated that recombinant beta-secretase from S2betaSEC/GalT-ST cells (S2 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 S2betaSEC/GalT-ST cells had a lower isoelectric point compared to beta-secretase from control S2betaSEC cells (S2 cells transformed only with beta-secretase cDNA). Recombinant beta-secretase from transformed S2 cells was also present as heterogeneous forms. The enzyme activity of recombinant beta-secretase from S2betaSEC/GalT-ST cells was enhanced up to 260% compared to control S2betaSEC cells. We have shown that an exogeneous human glycosyltransferases cDNA can be introduced into S2 cells to extend the N-glycan processing capabilities of the insect cell line, and that the extended glycosylation improves the activity of recombinant beta-secretase.
Chang
Kyung Hwa
KH
Department of Genetic Engineering and Plant Metabolism Research Center, Kyung Hee University, Suwon 449-701, Republic of Korea.
Yang
Jai Myung
JM
Chun
Hyung O K
HO
Chung
In Sik
IS
D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D002460:Cell Line; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics*; D010450:Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation; D024861:Genetic Enhancement / Q000379:methods*; D006801:Humans; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D029721:Drosophila Proteins; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human