(515bk) Specific Expression of GFPuv-beta1,3- N -acetylglucosaminyltransferase2 Fusion Protein in Fat Body of Bombyx Mori Larvae Using Signal Peptides

In the field of the production of recombinant proteins, signal peptide are important for the entrance to host's secretory pathway, and improvement and replacement of signal peptide can lead to the mass production of transmembrane and secretory protein. In this study, mutated bombyxin (bx) and polyphenoloxidase-activating enzyme (ppae) signal peptide from Bombyx mori, which bx and ppae signal peptide was used previously for protein production in silkworm larvae, and synthetic signal peptides (S. Barash et al. Biochem. Biophys. Res. Commun. 294, 2002, 835 - 842) were investigated using cysteine protease deficient Bombyx mori nucleopolyhedrolysis virus (BmNPV-CP^-) and its bacmid containing GFP_uv-beta1,3-N-acetylglucosaminyltransferase2 (GGT2) fusion gene in Bombyx mori Bm5 cells and larvae. The secretion efficiencies of all signal peptides including bombyxin (bx) and polyphenoloxidase-activating enzyme (ppae) signal peptides were 15 - 30 % in Bm5 cells and 24 -30 % in silkworm larvae except for +16 signal peptide, 0 % in Bm5 cells and only 1 % in silkworm larvae. In cell fractionation experiment, in fractions of all precipitations, 600, 8,000, 114,000 x g preciptations, 94 % of total intracellular beta3GnT activity was detected, especially in 114,000 x g precipitation 13% of activity was detected. +38 signal peptide, which showed a similar behavior as bx and ppae signal peptides, 60 % of total intracellular activity was detected in 114,000 x g supernatant and in contrast only 1 % was in 114,000 x g precipitation. It suggested that +16 signal peptide might be transmembrane region and not cleaved by signal peptidase, therefore the fusion protein connected with +16 signal peptide could not be secreted extracellularly.