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

摘要

Human beta-1.3-N-acetylglucosaminyltransferase-2 (beta3GnT2) was produced in a haculovirus 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 assa) methods.When Tn-5B 1-4 cells were infected with a recombinant AcMNPV-beta3GnT2-GFP_uv virus at MOI 10,intracellular and extracellular beta3GnT activities increased to 0.26 and 0.68 mil/ml,respectively,until 3 days post-infection (d.p.i),and decreased markedly at 3 d.p.i.In contrast to Tn-5Bl-4 cell culture medium,the extracellular beta3GnT activity in Sf-9 cell culture medium increased to 0.86mU/ml at 4 d.p.i.The fusion protein obtained from Tn-5Bl-4 and Sf-9 cultures was confirmed based on the GFP11V of the fusion protein.The fusion protein was purified using a Ni~2+ affinity column,and W7as concentrated by approximately 900-fold.The observed beta3GnT activity and the specific beta3GnT activity of the purified fusion protein were 77.6mU/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.