Overexpression of active Syrian golden hamster prion protein PrPc as a glutathione S-transferase fusion in heterologous systems (original) (raw)

Abstract

This article describes a procedure which permits for the first time the isolation of the prion protein PrPc from the Syrian golden hamster in heterologous systems. Using a glutathione S-transferase (GST) fusion approach, milligram amounts of stable, soluble, and homogeneous GST::PrPc protein were obtained in Escherichia coli and with baculovirus-infected insect cells. Authentic PrPc was released from the immobilized fusion protein by direct cleavage with thrombin. GST::PrPc expressed in these two expression systems and also authentic PrPc released by thrombin cleavage were recognized by a polyclonal antibody directed against amino acid 95 to 110 of the golden hamster PrPc protein. GST::PrPc was not detected by a monoclonal antibody recognizing the region encompassing amino acids 138 to 152 of the human prion protein. The fusion protein was sensitive to proteinase K digestion, demonstrating that the cellular rather than the proteinase K-resistant scrapie isoform was produced.

Full Text

The Full Text of this article is available as a PDF (457.7 KB).

Selected References

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

  1. Baldwin M. A., Pan K. M., Nguyen J., Huang Z., Groth D., Serban A., Gasset M., Mehlhorn I., Fletterick R. J., Cohen F. E. Spectroscopic characterization of conformational differences between PrPC and PrPSc: an alpha-helix to beta-sheet transition. Philos Trans R Soc Lond B Biol Sci. 1994 Mar 29;343(1306):435–441. doi: 10.1098/rstb.1994.0041. [DOI] [PubMed] [Google Scholar]
  2. Basler K., Oesch B., Scott M., Westaway D., Wälchli M., Groth D. F., McKinley M. P., Prusiner S. B., Weissmann C. Scrapie and cellular PrP isoforms are encoded by the same chromosomal gene. Cell. 1986 Aug 1;46(3):417–428. doi: 10.1016/0092-8674(86)90662-8. [DOI] [PubMed] [Google Scholar]
  3. Büeler H., Aguzzi A., Sailer A., Greiner R. A., Autenried P., Aguet M., Weissmann C. Mice devoid of PrP are resistant to scrapie. Cell. 1993 Jul 2;73(7):1339–1347. doi: 10.1016/0092-8674(93)90360-3. [DOI] [PubMed] [Google Scholar]
  4. Büeler H., Fischer M., Lang Y., Bluethmann H., Lipp H. P., DeArmond S. J., Prusiner S. B., Aguet M., Weissmann C. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature. 1992 Apr 16;356(6370):577–582. doi: 10.1038/356577a0. [DOI] [PubMed] [Google Scholar]
  5. Caughey B., Race R. E., Vogel M., Buchmeier M. J., Chesebro B. In vitro expression in eukaryotic cells of a prion protein gene cloned from scrapie-infected mouse brain. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4657–4661. doi: 10.1073/pnas.85.13.4657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  7. Collinge J., Whittington M. A., Sidle K. C., Smith C. J., Palmer M. S., Clarke A. R., Jefferys J. G. Prion protein is necessary for normal synaptic function. Nature. 1994 Jul 28;370(6487):295–297. doi: 10.1038/370295a0. [DOI] [PubMed] [Google Scholar]
  8. Despreaux C. W., Manning R. F. The dacA gene of Bacillus stearothermophilus coding for D-alanine carboxypeptidase: cloning, structure and expression in Escherichia coli and Pichia pastoris. Gene. 1993 Sep 6;131(1):35–41. doi: 10.1016/0378-1119(93)90666-q. [DOI] [PubMed] [Google Scholar]
  9. Eisenberg D., Weiss R. M., Terwilliger T. C. The hydrophobic moment detects periodicity in protein hydrophobicity. Proc Natl Acad Sci U S A. 1984 Jan;81(1):140–144. doi: 10.1073/pnas.81.1.140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Groschup M. H., Langeveld J., Pfaff E. The major species specific epitope in prion proteins of ruminants. Arch Virol. 1994;136(3-4):423–431. doi: 10.1007/BF01321071. [DOI] [PubMed] [Google Scholar]
  11. Heukeshoven J., Dernick R. Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels. Electrophoresis. 1988 Jan;9(1):28–32. doi: 10.1002/elps.1150090106. [DOI] [PubMed] [Google Scholar]
  12. Hope J., Morton L. J., Farquhar C. F., Multhaup G., Beyreuther K., Kimberlin R. H. The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP). EMBO J. 1986 Oct;5(10):2591–2597. doi: 10.1002/j.1460-2075.1986.tb04539.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jameson B. A., Wolf H. The antigenic index: a novel algorithm for predicting antigenic determinants. Comput Appl Biosci. 1988 Mar;4(1):181–186. doi: 10.1093/bioinformatics/4.1.181. [DOI] [PubMed] [Google Scholar]
  14. Kocisko D. A., Come J. H., Priola S. A., Chesebro B., Raymond G. J., Lansbury P. T., Caughey B. Cell-free formation of protease-resistant prion protein. Nature. 1994 Aug 11;370(6489):471–474. doi: 10.1038/370471a0. [DOI] [PubMed] [Google Scholar]
  15. Meyer R. K., McKinley M. P., Bowman K. A., Braunfeld M. B., Barry R. A., Prusiner S. B. Separation and properties of cellular and scrapie prion proteins. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2310–2314. doi: 10.1073/pnas.83.8.2310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pan K. M., Baldwin M., Nguyen J., Gasset M., Serban A., Groth D., Mehlhorn I., Huang Z., Fletterick R. J., Cohen F. E. Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10962–10966. doi: 10.1073/pnas.90.23.10962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Prusiner S. B., Groth D. F., Bolton D. C., Kent S. B., Hood L. E. Purification and structural studies of a major scrapie prion protein. Cell. 1984 Aug;38(1):127–134. doi: 10.1016/0092-8674(84)90533-6. [DOI] [PubMed] [Google Scholar]
  18. Prusiner S. B. Molecular biology of prion diseases. Science. 1991 Jun 14;252(5012):1515–1522. doi: 10.1126/science.1675487. [DOI] [PubMed] [Google Scholar]
  19. Prusiner S. B. Novel proteinaceous infectious particles cause scrapie. Science. 1982 Apr 9;216(4542):136–144. doi: 10.1126/science.6801762. [DOI] [PubMed] [Google Scholar]
  20. Rogers M., Serban D., Gyuris T., Scott M., Torchia T., Prusiner S. B. Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system. J Immunol. 1991 Nov 15;147(10):3568–3574. [PubMed] [Google Scholar]
  21. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Scott M. R., Butler D. A., Bredesen D. E., Wälchli M., Hsiao K. K., Prusiner S. B. Prion protein gene expression in cultured cells. Protein Eng. 1988 Apr;2(1):69–76. doi: 10.1093/protein/2.1.69. [DOI] [PubMed] [Google Scholar]
  24. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  25. Sreekrishna K., Potenz R. H., Cruze J. A., McCombie W. R., Parker K. A., Nelles L., Mazzaferro P. K., Holden K. A., Harrison R. G., Wood P. J. High level expression of heterologous proteins in methylotrophic yeast Pichia pastoris. J Basic Microbiol. 1988;28(4):265–278. doi: 10.1002/jobm.3620280410. [DOI] [PubMed] [Google Scholar]
  26. Stahl N., Baldwin M. A., Teplow D. B., Hood L., Gibson B. W., Burlingame A. L., Prusiner S. B. Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry. 1993 Mar 2;32(8):1991–2002. doi: 10.1021/bi00059a016. [DOI] [PubMed] [Google Scholar]
  27. Stahl N., Borchelt D. R., Hsiao K., Prusiner S. B. Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell. 1987 Oct 23;51(2):229–240. doi: 10.1016/0092-8674(87)90150-4. [DOI] [PubMed] [Google Scholar]
  28. Taraboulos A., Rogers M., Borchelt D. R., McKinley M. P., Scott M., Serban D., Prusiner S. B. Acquisition of protease resistance by prion proteins in scrapie-infected cells does not require asparagine-linked glycosylation. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8262–8266. doi: 10.1073/pnas.87.21.8262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Wang Y. H., Davies A. H., Jones I. M. Expression and purification of glutathione S-transferase-tagged HIV-1 gp120: no evidence of an interaction with CD26. Virology. 1995 Apr 1;208(1):142–146. doi: 10.1006/viro.1995.1137. [DOI] [PubMed] [Google Scholar]
  31. Weissmann C. Molecular biology of prion diseases. Trends Cell Biol. 1994 Jan;4(1):10–14. doi: 10.1016/0962-8924(94)90032-9. [DOI] [PubMed] [Google Scholar]
  32. Whitford M., Stewart S., Kuzio J., Faulkner P. Identification and sequence analysis of a gene encoding gp67, an abundant envelope glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol. 1989 Mar;63(3):1393–1399. doi: 10.1128/jvi.63.3.1393-1399.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]