Multiple co‐transfection and co‐expression of human β‐1,3‐ N ‐acetylglucosaminyltransferase with human calreticulin chaperone cDNA in a single step in insect cells

Human β‐1,3‐ N ‐acetylglucosaminyltransferase 2 (β3GnT2) 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, β3GnT2 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 μg/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 β3GnT2 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 β3GnT2. In this study we correlated the activity of β3GnT2 with the amount of β3GnT2 and human calreticulin cDNA in a stably expressing insect cell line simultaneously expressing calreticulin chaperone. When the amounts of chaperone and β3GnT2 cDNA were in a rough ratio of 1:1, the β3GnT2 activity was at a high level. In order to achieve better expression levels of β3GnT2 with less cost and time, efficient ways have to be devised.