Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Jan 15;90(2):427–430. doi: 10.1073/pnas.90.2.427

Rapid, high-level expression of biologically active alpha-trichosanthin in transfected plants by an RNA viral vector.

M H Kumagai 1, T H Turpen 1, N Weinzettl 1, G della-Cioppa 1, A M Turpen 1, J Donson 1, M E Hilf 1, G L Grantham 1, W O Dawson 1, T P Chow 1, et al.
PMCID: PMC45675  PMID: 8421670

Abstract

alpha-Trichosanthin, a eukaryotic ribosome-inactivating protein from Trichosanthes kirilowii, inhibits the replication of the human immunodeficiency virus (HIV) in vitro. The alpha-trichosanthin gene was placed under the transcriptional control of a tobamovirus subgenomic promoter in a plant RNA viral vector. Two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated alpha-trichosanthin to levels of at least 2% of total soluble protein. The recombinant alpha-trichosanthin was purified and its structural and biological properties were analyzed. The 23-amino acid signal peptide was recognized by N. benthamiana and the processed enzyme caused a concentration-dependent inhibition of protein synthesis in vitro. The high level of heterologous gene expression observed in these studies is due to the unique features of the RNA viral-based transfection system.

Full text

PDF
427

Images in this article

428Image
on p.428
429Image
on p.429
429Image
on p.429

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Chow T. P., Feldman R. A., Lovett M., Piatak M. Isolation and DNA sequence of a gene encoding alpha-trichosanthin, a type I ribosome-inactivating protein. J Biol Chem. 1990 May 25;265(15):8670–8674. [PubMed] [Google Scholar]
  3. Collins E. J., Robertus J. D., LoPresti M., Stone K. L., Williams K. R., Wu P., Hwang K., Piatak M. Primary amino acid sequence of alpha-trichosanthin and molecular models for abrin A-chain and alpha-trichosanthin. J Biol Chem. 1990 May 25;265(15):8665–8669. [PubMed] [Google Scholar]
  4. Dawson W. O., Beck D. L., Knorr D. A., Grantham G. L. cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1832–1836. doi: 10.1073/pnas.83.6.1832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dawson W. O., Lehto K. M. Regulation of tobamovirus gene expression. Adv Virus Res. 1990;38:307–342. doi: 10.1016/S0065-3527(08)60865-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dawson W. O., Lewandowski D. J., Hilf M. E., Bubrick P., Raffo A. J., Shaw J. J., Grantham G. L., Desjardins P. R. A tobacco mosaic virus-hybrid expresses and loses an added gene. Virology. 1989 Sep;172(1):285–292. doi: 10.1016/0042-6822(89)90130-x. [DOI] [PubMed] [Google Scholar]
  7. Donson J., Kearney C. M., Hilf M. E., Dawson W. O. Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7204–7208. doi: 10.1073/pnas.88.16.7204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Endo Y., Mitsui K., Motizuki M., Tsurugi K. The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J Biol Chem. 1987 Apr 25;262(12):5908–5912. [PubMed] [Google Scholar]
  9. Endo Y., Tsurugi K. RNA N-glycosidase activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. J Biol Chem. 1987 Jun 15;262(17):8128–8130. [PubMed] [Google Scholar]
  10. Hewick R. M., Hunkapiller M. W., Hood L. E., Dreyer W. J. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed] [Google Scholar]
  11. Hiatt A., Cafferkey R., Bowdish K. Production of antibodies in transgenic plants. Nature. 1989 Nov 2;342(6245):76–78. doi: 10.1038/342076a0. [DOI] [PubMed] [Google Scholar]
  12. Kahn J. O., Kaplan L. D., Gambertoglio J. G., Bredesen D., Arri C. J., Turin L., Kibort T., Williams R. L., Lifson J. D., Volberding P. A. The safety and pharmacokinetics of GLQ223 in subjects with AIDS and AIDS-related complex: a phase I study. AIDS. 1990 Dec;4(12):1197–1204. doi: 10.1097/00002030-199012000-00003. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. McGrath M. S., Hwang K. M., Caldwell S. E., Gaston I., Luk K. C., Wu P., Ng V. L., Crowe S., Daniels J., Marsh J. GLQ223: an inhibitor of human immunodeficiency virus replication in acutely and chronically infected cells of lymphocyte and mononuclear phagocyte lineage. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2844–2848. doi: 10.1073/pnas.86.8.2844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McGrath M. S., Santulli S., Gaston I. Effects of GLQ223 on HIV replication in human monocyte/macrophages chronically infected in vitro with HIV. AIDS Res Hum Retroviruses. 1990 Aug;6(8):1039–1043. doi: 10.1089/aid.1990.6.1039. [DOI] [PubMed] [Google Scholar]
  16. Miller W. A., Dreher T. W., Hall T. C. Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (-)-sense genomic RNA. Nature. 1985 Jan 3;313(5997):68–70. doi: 10.1038/313068a0. [DOI] [PubMed] [Google Scholar]
  17. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  18. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shaw P. C., Yung M. H., Zhu R. H., Ho W. K., Ng T. B., Yeung H. W. Cloning of trichosanthin cDNA and its expression in Escherichia coli. Gene. 1991 Jan 15;97(2):267–272. doi: 10.1016/0378-1119(91)90061-f. [DOI] [PubMed] [Google Scholar]
  20. Sijmons P. C., Dekker B. M., Schrammeijer B., Verwoerd T. C., van den Elzen P. J., Hoekema A. Production of correctly processed human serum albumin in transgenic plants. Biotechnology (N Y) 1990 Mar;8(3):217–221. doi: 10.1038/nbt0390-217. [DOI] [PubMed] [Google Scholar]
  21. Takamatsu N., Ishikawa M., Meshi T., Okada Y. Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA. EMBO J. 1987 Feb;6(2):307–311. doi: 10.1002/j.1460-2075.1987.tb04755.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Takamatsu N., Watanabe Y., Yanagi H., Meshi T., Shiba T., Okada Y. Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector. FEBS Lett. 1990 Aug 20;269(1):73–76. doi: 10.1016/0014-5793(90)81121-4. [DOI] [PubMed] [Google Scholar]
  23. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES