Stable production of an analog of human tissue plasminogen activator from cultured Drosophila cells.
We have studied the expression of an analog of human tissue plasminogen activator, FK2P, in Drosophila Schneider 2 cells. A number of promoters were tested, including the Drosophila metallothionein promoter (MTd), baculovirus immediate early promoter (IE), Drosophila copia promoter, mouse metallothionein promoter, cytomegalovirus immediate early promoter with or without intron, SV40 immediate early promoter, and human elongation factor 1 alpha promoter. Two of these promoters drove significant expression of FK2P. The MTd promoter is tightly regulated and upon induction with copper or cadmium expression of FK2P increases as much as 180-fold, accumulating in the culture medium to about 7 micrograms FK2P/10(6) cells/day as determined by ELISA. The IE promoter can direct the constitutive expression to yield about 0.4 microgram FK2P/10(6) cells/day. The production of FK2P in these cell lines remains at about the same level after repeated passages, even in the absence of selective pressure. The FK2P accumulated in the culture medium is fully active in an assay using a chromogenic substrate for serine proteases. Western immunoblot analysis shows that the product remains predominantly as single-chain molecules in serum-free medium, while in serum-containing medium two-chain material occurs as expected due to the presence of plasmin in serum. Judged from the size in Western immunoblots, the FK2P produced is glycosylated.
Molecular Biology Research, Upjohn Company, Kalamazoo, MI 49007.
M K
MK
Olsen
S K
SK
Rockenbach
H D
HD
Fischer
J G
JG
Hoogerheide
C S
CS
Tomich
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002934:Cinnamates; D016895:Culture Media, Serum-Free; D004331:Drosophila melanogaster / Q000166:cytology*; D004351:Drug Resistance; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D008668:Metallothionein / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000494:pharmacology
D002934:Cinnamates; D016895:Culture Media, Serum-Free; D011993:Recombinant Fusion Proteins; D006921:Hygromycin B; C026273:hygromycin A; D008668:Metallothionein; C080391:FK2P tissue plasminogen activator; D010959:Tissue Plasminogen Activator