Use of the silkworm, Bombyx mori, and an insect baculovirus vector for high-level expression and secretion of biologically active mouse interleukin-3.
Using the virus vector derived from a baculovirus of Bombyx mori (Bm), we constructed an infectious recombinant virus carrying the mouse interleukin-3 (IL-3) cDNA placed downstream from the polyhedrin promoter. Silkworms infected in vivo with recombinant virus or the silkworm-derived BmN cell line infected in vitro secreted large amounts of IL-3 into hemolymph or culture medium, respectively. On a per volume basis, about 20-fold more activity was found in the culture supernatants of the infected BmN cells and 10000-fold more activity was detected in the hemolymph as compared to supernatants obtained from COS7 monkey cells transfected with plasmid pcD-IL3 using the SV40 early promoter [Yokota et al., Proc. Natl. Acad. Sci. USA 81 (1984) 1070-1074]. Three distinct species of Il-3 of molecular masses, 18, 20 and 22 kDa were produced and all were converted to a 15-kDa protein by N-glycanase digestion, indicating that silkworm cells glycosylated IL-3. The N-terminal amino acid sequences of the IL-3 purified from tissue culture medium and hemolymph were identical to that of mammalian-derived IL-3, showing that silkworm cells recognized the mammalian signal sequence and cleaved it at the correct position. The purified silkworm-produced IL-3 had biological activities indistinguishable from IL-3 produced by mammalian cells as assessed by mast-cell proliferation assays, colony-formation assays using mouse bone marrow cells, and by receptor-binding assays using [125I]IL-3.
Miyajima
A
A
Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto 94304.
Schreurs
J
J
Otsu
K
K
Kondo
A
A
Arai
K
K
Maeda
S
S
D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics* / Q000382:microbiology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004262:DNA Restriction Enzymes; D005796:Genes; D007303:Insect Vectors; D007304:Insect Viruses / Q000235:genetics*; D007377:Interleukin-3 / Q000235:genetics* / Q000494:pharmacology; D051379:Mice; D011994:Recombinant Proteins / Q000494:pharmacology; D014162:Transfection
D007377:Interleukin-3; D011994:Recombinant Proteins; D004247:DNA; D004262:DNA Restriction Enzymes