Enhanced production of secretory β1,3-N-acetylglucosaminyltransferase 2 fusion protein into hemolymph of Bombyx mori larvae using recombinant BmNPV bacmid integrated signal sequence

Abstract

The enhanced secretion of beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) fusion protein into the hemolymph of Bombyx mori larvae was studied using a recombinant B. mori nucleopolyhedrovirus (BmNPV) bacmid integrating seven signal sequences. When the BmNPV bacmid encoding the signal sequences from the silkworm B. mori bombyxin (bx) and B. mori prophenoloxidase-activating enzyme (ppae) was injected into silkworm larvae, 56.1 and 51.5mU/ml beta3GnT, respectively, were secreted into the hemolymph of silkworm larvae. For bx, 97.3% of the total beta3GnT activity was secreted into hemolymph, and only 1.1% remained in the intestines of silkworm larvae. For ppae, 90.8% of the total beta3GnT activity was secreted to the hemolymph, but 7.8% remained in the intestines of silkworm larvae. Using the BmNPV bacmid encoding bx, the amount of secreted beta3GnT was 91mug per larva, which was 2.5% of the total amount of protein in the hemolymph.

Molecular Chaperone-Assisted Production of Human α-1,4-N-Acetylglucosaminyltransferase in Silkworm Larvae Using Recombinant BmNPV Bacmids

“…In a previous study, we confirmed that the bx signal peptide allowed recombinant proteins to be secreted into culture supernatants of Bm5 cells, and into the hemolymph of silkworm larvae, but the gp signal peptide did not [10]. Therefore, the bx signal peptide was adopted for silkworm larvae expression.…”

“…1b), indicating that a4GnT productivity of Tn-pXgp/GFP uv -a4GnT in cell line 5 was higher than that in cell line 3. A previous report found that a GFP uvb3GnT2 fusion protein was not secreted by the gp signal peptide in Bm5 cells isolated from B. mori ovaries [10]. This indicates that the bx signal peptide may not be functional in Tn-5B1-4 cells, but Bm5 cells, because bx signal peptide is from bombyxin of silkworm.…”

“…To resolve this problem, human a4GnT was produced in a baculovirus expression system (BES) using insect cells and larvae. Insect larvae, especially silkworm larvae, are used as living factories for the production of recombinant proteins [9][10][11]. Protein production using insect larvae is easy to manipulate and to scale-up.…”

Improved secretion of molecular chaperone‐assisted human IgG in silkworm, and no alterations in their N‐linked glycan structures

“…This was also shown when using BmNPV bacmids containing a viral protease: more than 90% of total protein was detected in the hemolymph of silkworm larvae. 28 However, the secreted proteins appear to be especially susceptible to intracellular aggregation because of the complexity of the secretion pathway. It has been reported that secreted levels of expressed protein can be 10 to 100 times lower than intracellular levels.…”

Versatility of chitosan/BmNPV bacmid DNA nanocomplex as transfection reagent of recombinant protein expression in silkworm larvae

“…130 kbp, which is much larger than that of plasmid vectors; the transfection of large DNA such as the BmNPV bacmid has not previously been conducted. To confirm the transfection efficiency, GFP uv -b1,3-N-acetylglucosaminyltransferase 2 (b3GnT2) fusion protein (GGT2) was expressed in Bm5 cells and silkworm larvae (Park et al 2007) using the chitosan/BmNPV bacmid DNA nanocomplexes. To demonstrate the versatility of these chitosan-based nanocomplexes, rat a2,6-sialyltransferase (ST6), hemagglutinin (HA) from an influenza A virus, and the Neospora caninum surface protein (NcSRS2) were expressed using the chitosan/BmNPV bacmid DNA nanocomplexes.…”