Yeast-prepro-alpha-factor-leader-region-directed synthesis and secretion of truncated human macrophage colony-stimulating factor in the silkworm Bombyx mori.
Human macrophage colony-stimulating factor (hM-CSF) cDNA joined to the leader region of the precursor of the yeast mating pheromone alpha-factor (MF alpha L) was expressed at high levels in BmN cells and in silkworm (Bombyx mori) larvae, using recombinant Bombyx mori nuclear polyhedrosis virus, as a vector. The biological activity of rhM-CSF detected in the haemolymph was 1 x 10(6) colony-formation units/ml, approximately half of the expression level directed by the native signal peptide of hM-CSF in silkworm larvae. The secreted rhM-CSF was purified to homogeneity. N-terminal analysis showed that the signal peptide had been removed, indicating that insect cells possess the enzymic activity necessary to cleave the pro-alpha-factor leader region from the fusion protein at the carboxy side of Lys-Arg dibasic residues, which is the cleavage site recognized by KEX2 endopeptidase in yeast cells.
Qiu
P
P
Department of Biochemistry, Nanjing University, People's Republic of China.
Qin
J
J
Ding
Y
Y
Zhu
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D017403:In Situ Hybridization; D007814:Larva / Q000166:cytology / Q000378:metabolism; D016173:Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000072235:Mating Factor; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism
D010455:Peptides; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000072235:Mating Factor; D016173:Macrophage Colony-Stimulating Factor; D004247:DNA