title: Multiple co-transfection and co-expression of human beta-1,3-N-acetylglucosaminyltransferase with human calreticulin chaperone cDNA in a single step in insect cells.
abstract: Human beta-1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is indispensable for the conversion of lacto-N-triose II into lacto-N-tetraose and lacto-N-neotetraose. In this paper, we report multiple co-transfection in a single step using two different human cDNAs in an insect cell, beta3GnT2 and calreticulin chaperone respectively. This minimized the time required to isolate stably expressing cell line from 12 weeks to 4 weeks and simplified the isolation technique to a one-step process. We tried to insert as much cDNA as possible and used various concentrations of two antibiotics, Blasticidin and Geneticin, at 25-1500 microg/ml respectively during co-transfection for the selection of an efficiently expressing stable cell line with no adverse effects. A stably expressing cell line was isolated which expressed beta3GnT2 and chaperone simultaneously, which gave an activity of 10.1 m-units/ml compared with 6.7 m-units/ml by a cell only carrying beta3GnT2. In this study we correlated the activity of beta3GnT2 with the amount of beta3GnT2 and human calreticulin cDNA in a stably expressing insect cell line simultaneously expressing calreticulin chaperone. When the amounts of chaperone and beta3GnT2 cDNA were in a rough ratio of 1:1, the beta3GnT2 activity was at a high level. In order to achieve better expression levels of beta3GnT2 with less cost and time, efficient ways have to be devised.
authors.affiliation: Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan.
authors.forename: Vipin Kumar
authors.initials: VK
authors.lastname: Deo
authors.forename: Enoch Y
authors.initials: EY
authors.lastname: Park
mesh_terms: D000818:Animals; D037282:Calreticulin / Q000096:biosynthesis* / Q000235:genetics; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007915:Lepidoptera / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000235:genetics; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection / Q000379:methods*
chemical_list: D037282:Calreticulin; D018076:DNA, Complementary; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D017351:N-Acetylglucosaminyltransferases; C428132:B3GNT2 protein, human