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

Human beta-1,3-N-acetylglucosaminyltransferase-2 (beta3GnT2) was produced in a baculovirus expression system as a secreted fusion protein with a green fluorescence protein variant, GFP(uv), flanked by the (His)(6) sequence and an enterokinase cleavage site. The expression of the beta3GnT2-GFP(uv) 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-p3GnT2-GFP(uv) virus at MOI 10, intracellular and extracellular beta3GnT 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-5B 1-4 cell culture medium, the extracellular beta3GnT activity in Sf-9 cell culture medium increased to 0.86 mU/ml at 4 d.p.i. The fusion protein obtained from Tn-5B 1-4 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 beta3GnT activity and the specific beta3GnT 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 beta3GnT2 is glycosylated. (C) 2003 Elsevier B.V. All rights reserved.