Specific expression of GFPuv-beta1,3-N-acetylglucosaminyltransferase 2 fusion protein in fat body of Bombyx mori silkworm larvae using signal peptide |
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Authors: | Kato Tatsuya Park Enoch Y |
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Institution: | Laboratory of Biotechnology, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan. |
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Abstract: | 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(uv)-beta1,3-N-acetylglucosaminyltransferase 2 (GGT2) fusion protein in B. mori Bm5 cells and silkworm larvae using cysteine protease deficient B. mori multiple nucleopolyhedrovirus (BmMNPV-CP(-)) 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 beta1,3-N-acetylglucosaminyltransferase (beta3GnT) 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. |
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Keywords: | AcMNPV Autographa californica multiple nucleopolyhedrovirus BmMNPV Bombyx mori multiple nucleopolyhedrovirus BmMNPV-CP? cysteine protease deficient Bombyx mori multiple nucleopolyhedrovirus bx bombyxin ER endoplasmic reticulum GFPuv green fluorescent protein when excited with long-wave UV light β3GnT2 β1 3-N-acetylglucosaminyltransferase 2 GGT2 GFPuv-β3GnT2 HMM hidden Markov model PCR polymerase chain reaction ppae Bombyx mori prophenoloxidase-activating enzyme |
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