Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Stable production of an analog of human tissue plasminogen activator from culturedDrosophila cells

Abstract

We have studied the expression of an analog of human tissue plasminogen activator, FK2P, inDrosophila Schneider 2 cells. A number of promoters were tested, including theDrosophila 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 1α 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 μg FK2P/106 cells/day as determined by ELISA. The IE promoter can direct the constitutive expression to yield about 0.4 μg FK2P/106 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 predominately 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.

This is a preview of subscription content, log in to check access.

References

  1. Allday MJ, Sinclair JH, MacGillivray AJ and Sang JH (1985) Efficient expression of an Epstein-Barr nuclear antigen inDrosophila cells transfected with Epstein-Barr virus DNA. EMBO J 4: 2955–2959.

  2. Angelichio ML, Beck JA, Johansen H and Ivey-Hoyle M (1991) Comparison of several promoters and polyadenylation signals for use in heterologous gene expression in culturedDrosophila cells. Nucleic Acids Res 19: 5037–5043.

  3. Bourouis M and Jarry B (1983) Vectors containing a prokaryotic dihydrofolate reductase gene transformDrosophila cells to methotrexate-resistance. EMBO J 2: 1099–1104.

  4. Brinster RL, Chen HY and Trumbauer M (1981) Somatic Expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 27: 223–231.

  5. Bunch TA, Grinblat Y and Goldstein LSB (1988) Characterization and use of theDrosophila metallothionein promoter in culturedDrosophila melanogaster cells. Nucleic Acids Res 16: 1043–1061.

  6. Culp JS, Johansen H, Hellmig B, Beck J, Matthews TJ, Delers A and Rosenberg M (1991) Regulated expression allows high level production and secretion of HIV-1 gp120 envelope glycoprotein inDrosophila Schneider cells. Bio/Technology 9: 173–177.

  7. Di Nocera PP and Dawid IB (1983) Transient expression of genes introduced into cultured cells ofDrosophila. Proc Natl Acad Sci USA 80: 7095–7098.

  8. Flavell AJ, Levis R, Simon MA and Rubin GM (1981) The 5′ termini of RNAs encoded by the transposable element copia. Nucleic Acids Res 9: 6279–6291.

  9. Giordano TJ and McAllister W (1990) Optimization of the hygromycin B resistance-conferring gene as a dominant selectable marker in mammalian cells. Gene 88: 285–288.

  10. Jarvis DL, Fleming JAGW, Kovacs GR, Summers MD and Guarino LA (1990) Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells. Bio/Technology 8: 950–955.

  11. Johansen H, van der Straten A, Sweet R, Otto E, Maroni G and Rosenberg M (1989) Regulated expression at high copy number allow 20 production of a growth inhibitory oncogene product inDrosophila Schneider cells. Genes and Development 3: 882–889.

  12. Kim DW, Uetsuki T, Kaziro Y, Yamaguchi N and Sugano S (1990) Use of the human elongation factor 1α promoter as a versatile and efficient expression system. Gene 91: 217–223.

  13. Lastowsky-Perry D, Otto E and Maroni G (1985) Nucleotide sequence and expression of aDrosophila metallothionein. J Biol Chem 260: 1527–1530.

  14. Maisonhaute C and Echalier G (1986) Stable transformation ofDrosophila Kc cells to antibiotic resistance with the bacterial neomycin resistance gene. FEBS Letters 197: 45–49.

  15. Maniatis T, Fritsch EF and Sambrook J (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Habor, NY.

  16. Maroni G, Otto E and Lastowsky-Perry D (1985) Molecular and cytogenetic characterization of a metallothionein gene ofDrosophila. Genetic 112: 493–504.

  17. Marotti KR and Tomich CSC (1989) Simple and efficient oligonucleotide-directed mutagenesis using one primer and circular plasmid DNA template. Gene Anal Techn 6: 67–70.

  18. Morse LS, Pereira L, Roizman B and Schaffer PA (1978) Anatomy of herpes simplex virus DNA. X. Mapping of viral genes by analysis of polypeptides and functions specified by HSV-1×HSV-2 recombinants. J Virol 26: 389–410.

  19. Otto E, Allen JM, Young JE, Palmiter RD and Maroni M (1987) A DNA segment controlling metal-regulated expression of theDrosophila melanogaster metallothionein gene. Mtn Mol Cell Biol 7: 1710–1785.

  20. Rehberg EF, Theriault NY, Carter JB, Palermo DP, Hubert EV, Bergum PW, Wunderlich CJ, Erickson LA and Marotti KR (1989) Construction of a tissue plasminogen activator gene having unique restriction sites to facilitate domain manipulation: A model for the analysis of multi-domain proteins. Protein Engineering 2: 371–377.

  21. Rio DC and Rubin GM (1985) Transformation of culturedDrosophila melanogaster cells with a dominant selectable marker. Mol Cellular Biol 5: 1833–1838.

  22. Sinclair JH (1987) The human cytomegalovirus immediate early gene promoter is a strong promoter in culturedDrosophila melanogaster cells. Nucleic Acids Res 15: 2392.

  23. Sugden B, Marsh K and Yates J (1985) A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-Barr virus. Mol Cell Biol 5: 410–413.

  24. van der Straten A, Johansen H, Rosenberg M and Sweet RW (1989) In: Methods in Molecular and Cellular Biology, John Wiley & Sons Inc., pp. 1–8.

  25. Wallace RB, Johnson MJ, Suggs SV, Miyoshi DI, Bhatt R and Itakura K (1981) A set of synthetic oligodeoxyribonucleotide primers for DNA sequencing in the plasmid vector pBR322. Gene 16: 21–26.

Download references

Author information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Olsen, M.K., Rockenbach, S.K., Fischer, H.D. et al. Stable production of an analog of human tissue plasminogen activator from culturedDrosophila cells. Cytotechnology 10, 157–167 (1992). https://doi.org/10.1007/BF00570892

Download citation

Key words

  • Drosophila cell culture
  • gene expression