Specific expression of GFPuv-beta1,3-N-acetylglucosaminyltransferase 2 fusion protein in fat body of Bombyx mori silkworm larvae using signal peptide.
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.
Kato
Tatsuya
T
Laboratory of Biotechnology, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan.
Park
Enoch Y
EY
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics; D002460:Cell Line; D005216:Fat Body / Q000201:enzymology* / Q000254:growth & development; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007814:Larva / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017351:N-Acetylglucosaminyltransferases