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

Content Provider	Semantic Scholar
Author	Kato, Tatsuya Murata, Takeomi Usui, Taichi Park, Enoch Y.
Copyright Year	2004
Abstract	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, GFP uv, 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 GFP uv of the fusion protein. The fusion protein was purified using a Ni 2+ 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
Starting Page	15
Ending Page	23
Page Count	9
File Format	PDF HTM / HTML
DOI	10.1016/j.bej.2003.09.009
Volume Number	19
Alternate Webpage(s)	http://www.agr.shizuoka.ac.jp/c/biotech/park/assets/files/BEJv19p15-23.pdf
Alternate Webpage(s)	https://doi.org/10.1016/j.bej.2003.09.009
Language	English
Access Restriction	Open
Content Type	Text
Resource Type	Article