Specific expression of GFP{sub uv}-{beta}1,3-N-acetylglucosaminyltransferase 2 fusion protein in fat body of Bombyx mori silkworm larvae using signal peptide
- Laboratory of Biotechnology, Faculty of Agriculture, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529 (Japan)
- Laboratory of Biotechnology, Faculty of Agriculture, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529 (Japan) and Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529 (Japan)
Bombyxin (bx) and prophenoloxidase-activating enzyme (ppae) signal peptides from Bombyx mori, their modified signal peptides, and synthetic signal peptides were investigated for the secretion of GFP{sub uv}-{beta}1,3-N-acetylglucosaminyltransferase 2 (GGT2) fusion protein in B. mori Bm5 cells and silkworm larvae using cysteine protease deficient B. mori multiple nucleopolyhedrovirus (BmMNPV-CP{sup -} ) and its bacmid. The secretion efficiencies of all signal peptides were 15-30% in Bm5 cells and 24-30% in silkworm larvae, while that of the +16 signal peptide was 0% in Bm5 cells and 1% in silkworm larvae. The fusion protein that contained the +16 signal peptide was expressed specifically in the endoplasmic reticulum (ER) and in the fractions of cell precipitations. Ninety-four percent of total intracellular {beta}1,3-N-acetylglucosaminyltransferase ({beta}3GnT) activity was detected in cell precipitations following the 600, 8000, and 114,000g centrifugations. In the case of the +38 signal peptide, 60% of total intracellular activity was detected in the supernatant from the 114,000g spin, and only 1% was found in the precipitate. Our results suggest that the +16 signal peptide might be situated in the transmembrane region and not cleaved by signal peptidase in silkworm or B. mori cells. Therefore, the fusion protein connected to the +16 signal peptide stayed in the fat body of silkworm larvae with biological function, and was not secreted extracellularly.
- OSTI ID:
- 20991466
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 359, Issue 3; Other Information: DOI: 10.1016/j.bbrc.2007.05.137; PII: S0006-291X(07)01101-1; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
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