Efficient production of human -1,3-N-acetylglucosaminyltransferase-2 fused with green fluorescence protein in insect cell

Human -1,3-N-acetylglucosaminyltransferase-2 ( 3GnT2) was produced in a baculovirus expression system as a secreted fusion protein with a green fluorescence protein variant, GFPuv, flanked by the (His)6 sequence and an enterokinase cleavage site. The expression of the 3GnT2–GFPuv fusion gene was rapidly detected using a fluorescence microscope without employing complicated assay methods. When Tn-5B1–4 cells were infected with a recombinant AcMNPV– 3GnT2–GFPuv virus at MOI 10, intracellular and extracellular 3GnT activities increased to 0.26 and 0.68 mU/ml, respectively, until 3 days post-infection (d.p.i.), and decreased markedly at 3 d.p.i. In contrast to Tn-5B1–4 cell culture medium, the extracellular 3GnT activity in Sf-9 cell culture medium increased to 0.86 mU/ml at 4 d.p.i. The fusion protein obtained from Tn-5B1–4 and Sf-9 cultures was confirmed based on the GFPuv of the fusion protein. The fusion protein was purified using a Ni2+ affinity column, and was concentrated by approximately 900-fold. The observed 3GnT activity and the specific 3GnT activity of the purified fusion protein were 77.6 mU/ml and 4.6 U/mg protein, respectively. When the purified fusion protein was treated with glycopeptidase F, its molecular weight decreased by 7–8 kDa, indicating that 3GnT2 is glycosylated. © 2003 Elsevier B.V. All rights reserved.