Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1992 Oct;1(10):1343–1352. doi: 10.1002/pro.5560011014

Purification and properties of the cellular prion protein from Syrian hamster brain.

K M Pan 1, N Stahl 1, S B Prusiner 1
PMCID: PMC2142104  PMID: 1363897

Abstract

The cellular prion protein (PrPC) is encoded by a chromosomal gene, and its scrapie isoform (PrPSc) features in all aspects of the prion diseases. Prior to the studies reported here, purification of PrPC has only been accomplished using immunoaffinity chromatography yielding small amounts of protein. Brain homogenates contain two PrPC forms designated PrPC-I and -II. These proteins were purified from a microsomal fraction by detergent extraction and separated by immobilized Cu2+ ion affinity chromatography. PrPC-II appears to be generated from PrPC-I by limited proteolysis of the N-terminus. Fractions enriched for PrPC-I were purified further by cation-exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Greater than 90% of the final product migrated as a broad band of M(r) 33-35 kDa as judged by silver staining after SDS-PAGE. Digestion of PrPC-I with peptide-N-glycosidase (PNGase) compressed the band and shifted its mobility giving an M(r) of 27 kDa. The protocol described should be amenable to large-scale preparation of PrPC, enabling physical comparisons of PrPC and PrPSc.

Full Text

The Full Text of this article is available as a PDF (5.4 MB).

Selected References

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

  1. Ambler L., Peters G. E. An immunospecific enzyme assay for horseradish peroxidase. Anal Biochem. 1984 Feb;137(1):66–68. doi: 10.1016/0003-2697(84)90347-6. [DOI] [PubMed] [Google Scholar]
  2. Andersson L., Sulkowski E., Porath J. Facile resolution of alpha-fetoproteins and serum albumins by immobilized metal affinity chromatography. Cancer Res. 1987 Jul 15;47(14):3624–3626. [PubMed] [Google Scholar]
  3. Baldwin M. A., Stahl N., Reinders L. G., Gibson B. W., Prusiner S. B., Burlingame A. L. Permethylation and tandem mass spectrometry of oligosaccharides having free hexosamine: analysis of the glycoinositol phospholipid anchor glycan from the scrapie prion protein. Anal Biochem. 1990 Nov 15;191(1):174–182. doi: 10.1016/0003-2697(90)90405-x. [DOI] [PubMed] [Google Scholar]
  4. Barry R. A., Prusiner S. B. Monoclonal antibodies to the cellular and scrapie prion proteins. J Infect Dis. 1986 Sep;154(3):518–521. doi: 10.1093/infdis/154.3.518. [DOI] [PubMed] [Google Scholar]
  5. Barry R. A., Vincent M. T., Kent S. B., Hood L. E., Prusiner S. B. Characterization of prion proteins with monospecific antisera to synthetic peptides. J Immunol. 1988 Feb 15;140(4):1188–1193. [PubMed] [Google Scholar]
  6. 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]
  7. Bendheim P. E., Potempska A., Kascsak R. J., Bolton D. C. Purification and partial characterization of the normal cellular homologue of the scrapie agent protein. J Infect Dis. 1988 Dec;158(6):1198–1208. doi: 10.1093/infdis/158.6.1198. [DOI] [PubMed] [Google Scholar]
  8. Bolton D. C., Bendheim P. E., Marmorstein A. D., Potempska A. Isolation and structural studies of the intact scrapie agent protein. Arch Biochem Biophys. 1987 Nov 1;258(2):579–590. doi: 10.1016/0003-9861(87)90380-8. [DOI] [PubMed] [Google Scholar]
  9. Bolton D. C., Meyer R. K., Prusiner S. B. Scrapie PrP 27-30 is a sialoglycoprotein. J Virol. 1985 Feb;53(2):596–606. doi: 10.1128/jvi.53.2.596-606.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bolton D. C., Seligman S. J., Bablanian G., Windsor D., Scala L. J., Kim K. S., Chen C. M., Kascsak R. J., Bendheim P. E. Molecular location of a species-specific epitope on the hamster scrapie agent protein. J Virol. 1991 Jul;65(7):3667–3675. doi: 10.1128/jvi.65.7.3667-3675.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Borchelt D. R., Scott M., Taraboulos A., Stahl N., Prusiner S. B. Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. J Cell Biol. 1990 Mar;110(3):743–752. doi: 10.1083/jcb.110.3.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. CHANDLER R. L. Encephalopathy in mice produced by inoculation with scrapie brain material. Lancet. 1961 Jun 24;1(7191):1378–1379. doi: 10.1016/s0140-6736(61)92008-6. [DOI] [PubMed] [Google Scholar]
  14. Campbell D. G., Gagnon J., Reid K. B., Williams A. F. Rat brain Thy-1 glycoprotein. The amino acid sequence, disulphide bonds and an unusual hydrophobic region. Biochem J. 1981 Apr 1;195(1):15–30. doi: 10.1042/bj1950015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Caughey B. W., Dong A., Bhat K. S., Ernst D., Hayes S. F., Caughey W. S. Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy. Biochemistry. 1991 Aug 6;30(31):7672–7680. doi: 10.1021/bi00245a003. [DOI] [PubMed] [Google Scholar]
  16. Caughey B., Raymond G. J. The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive. J Biol Chem. 1991 Sep 25;266(27):18217–18223. [PubMed] [Google Scholar]
  17. Endo T., Groth D., Prusiner S. B., Kobata A. Diversity of oligosaccharide structures linked to asparagines of the scrapie prion protein. Biochemistry. 1989 Oct 17;28(21):8380–8388. doi: 10.1021/bi00447a017. [DOI] [PubMed] [Google Scholar]
  18. Fernando Bazan J., Fletterick R. J., Prusiner S. B. AIDS virus and scrapie protein genes. Nature. 1987 Feb 12;325(6105):581–581. doi: 10.1038/325581a0. [DOI] [PubMed] [Google Scholar]
  19. Gabizon R., McKinley M. P., Prusiner S. B. Purified prion proteins and scrapie infectivity copartition into liposomes. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4017–4021. doi: 10.1073/pnas.84.12.4017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goldmann W., Hunter N., Foster J. D., Salbaum J. M., Beyreuther K., Hope J. Two alleles of a neural protein gene linked to scrapie in sheep. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2476–2480. doi: 10.1073/pnas.87.7.2476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Goldmann W., Hunter N., Martin T., Dawson M., Hope J. Different forms of the bovine PrP gene have five or six copies of a short, G-C-rich element within the protein-coding exon. J Gen Virol. 1991 Jan;72(Pt 1):201–204. doi: 10.1099/0022-1317-72-1-201. [DOI] [PubMed] [Google Scholar]
  22. Haraguchi T., Fisher S., Olofsson S., Endo T., Groth D., Tarentino A., Borchelt D. R., Teplow D., Hood L., Burlingame A. Asparagine-linked glycosylation of the scrapie and cellular prion proteins. Arch Biochem Biophys. 1989 Oct;274(1):1–13. doi: 10.1016/0003-9861(89)90409-8. [DOI] [PubMed] [Google Scholar]
  23. Harris D. A., Falls D. L., Johnson F. A., Fischbach G. D. A prion-like protein from chicken brain copurifies with an acetylcholine receptor-inducing activity. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7664–7668. doi: 10.1073/pnas.88.17.7664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hemdan E. S., Zhao Y. J., Sulkowski E., Porath J. Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1811–1815. doi: 10.1073/pnas.86.6.1811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Hope J., Multhaup G., Reekie L. J., Kimberlin R. H., Beyreuther K. Molecular pathology of scrapie-associated fibril protein (PrP) in mouse brain affected by the ME7 strain of scrapie. Eur J Biochem. 1988 Mar 1;172(2):271–277. doi: 10.1111/j.1432-1033.1988.tb13883.x. [DOI] [PubMed] [Google Scholar]
  27. Hsiao K., Baker H. F., Crow T. J., Poulter M., Owen F., Terwilliger J. D., Westaway D., Ott J., Prusiner S. B. Linkage of a prion protein missense variant to Gerstmann-Sträussler syndrome. Nature. 1989 Mar 23;338(6213):342–345. doi: 10.1038/338342a0. [DOI] [PubMed] [Google Scholar]
  28. Kascsak R. J., Rubenstein R., Merz P. A., Tonna-DeMasi M., Fersko R., Carp R. I., Wisniewski H. M., Diringer H. Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins. J Virol. 1987 Dec;61(12):3688–3693. doi: 10.1128/jvi.61.12.3688-3693.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kretzschmar H. A., Prusiner S. B., Stowring L. E., DeArmond S. J. Scrapie prion proteins are synthesized in neurons. Am J Pathol. 1986 Jan;122(1):1–5. [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Lowenstein D. H., Butler D. A., Westaway D., McKinley M. P., DeArmond S. J., Prusiner S. B. Three hamster species with different scrapie incubation times and neuropathological features encode distinct prion proteins. Mol Cell Biol. 1990 Mar;10(3):1153–1163. doi: 10.1128/mcb.10.3.1153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McKinley M. P., Taraboulos A., Kenaga L., Serban D., Stieber A., DeArmond S. J., Prusiner S. B., Gonatas N. Ultrastructural localization of scrapie prion proteins in cytoplasmic vesicles of infected cultured cells. Lab Invest. 1991 Dec;65(6):622–630. [PubMed] [Google Scholar]
  33. Merril C. R., Dunau M. L., Goldman D. A rapid sensitive silver stain for polypeptides in polyacrylamide gels. Anal Biochem. 1981 Jan 1;110(1):201–207. doi: 10.1016/0003-2697(81)90136-6. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Mobley W. C., Neve R. L., Prusiner S. B., McKinley M. P. Nerve growth factor increases mRNA levels for the prion protein and the beta-amyloid protein precursor in developing hamster brain. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9811–9815. doi: 10.1073/pnas.85.24.9811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Oesch B., Westaway D., Wälchli M., McKinley M. P., Kent S. B., Aebersold R., Barry R. A., Tempst P., Teplow D. B., Hood L. E. A cellular gene encodes scrapie PrP 27-30 protein. Cell. 1985 Apr;40(4):735–746. doi: 10.1016/0092-8674(85)90333-2. [DOI] [PubMed] [Google Scholar]
  37. Porath J., Carlsson J., Olsson I., Belfrage G. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature. 1975 Dec 18;258(5536):598–599. doi: 10.1038/258598a0. [DOI] [PubMed] [Google Scholar]
  38. Prusiner S. B., Bolton D. C., Groth D. F., Bowman K. A., Cochran S. P., McKinley M. P. Further purification and characterization of scrapie prions. Biochemistry. 1982 Dec 21;21(26):6942–6950. doi: 10.1021/bi00269a050. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Prusiner S. B., Groth D. F., Cochran S. P., Masiarz F. R., McKinley M. P., Martinez H. M. Molecular properties, partial purification, and assay by incubation period measurements of the hamster scrapie agent. Biochemistry. 1980 Oct 14;19(21):4883–4891. doi: 10.1021/bi00562a028. [DOI] [PubMed] [Google Scholar]
  41. Prusiner S. B., Hadlow W. J., Eklund C. M., Race R. E., Cochran S. P. Sedimentation characteristics of the scrapie agent from murine spleen and brain. Biochemistry. 1978 Nov 14;17(23):4987–4992. doi: 10.1021/bi00616a020. [DOI] [PubMed] [Google Scholar]
  42. Prusiner S. B., Hadlow W. J., Garfin D. E., Cochran S. P., Baringer J. R., Race R. E., Eklund C. M. Partial purification and evidence for multiple molecular forms of the scrapie agent. Biochemistry. 1978 Nov 14;17(23):4993–4999. doi: 10.1021/bi00616a021. [DOI] [PubMed] [Google Scholar]
  43. Prusiner S. B., McKinley M. P., Bowman K. A., Bolton D. C., Bendheim P. E., Groth D. F., Glenner G. G. Scrapie prions aggregate to form amyloid-like birefringent rods. Cell. 1983 Dec;35(2 Pt 1):349–358. doi: 10.1016/0092-8674(83)90168-x. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Prusiner S. B., Scott M., Foster D., Pan K. M., Groth D., Mirenda C., Torchia M., Yang S. L., Serban D., Carlson G. A. Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication. Cell. 1990 Nov 16;63(4):673–686. doi: 10.1016/0092-8674(90)90134-z. [DOI] [PubMed] [Google Scholar]
  46. Puckett C., Concannon P., Casey C., Hood L. Genomic structure of the human prion protein gene. Am J Hum Genet. 1991 Aug;49(2):320–329. [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Safar J., Wang W., Padgett M. P., Ceroni M., Piccardo P., Zopf D., Gajdusek D. C., Gibbs C. J., Jr Molecular mass, biochemical composition, and physicochemical behavior of the infectious form of the scrapie precursor protein monomer. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6373–6377. doi: 10.1073/pnas.87.16.6373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stahl N., Baldwin M. A., Hecker R., Pan K. M., Burlingame A. L., Prusiner S. B. Glycosylinositol phospholipid anchors of the scrapie and cellular prion proteins contain sialic acid. Biochemistry. 1992 Jun 2;31(21):5043–5053. doi: 10.1021/bi00136a600. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Sulkowski E. The saga of IMAC and MIT. Bioessays. 1989 May;10(5):170–175. doi: 10.1002/bies.950100508. [DOI] [PubMed] [Google Scholar]
  52. Taraboulos A., Serban D., Prusiner S. B. Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells. J Cell Biol. 1990 Jun;110(6):2117–2132. doi: 10.1083/jcb.110.6.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. Turk E., Teplow D. B., Hood L. E., Prusiner S. B. Purification and properties of the cellular and scrapie hamster prion proteins. Eur J Biochem. 1988 Sep 1;176(1):21–30. doi: 10.1111/j.1432-1033.1988.tb14246.x. [DOI] [PubMed] [Google Scholar]
  55. Westaway D., Goodman P. A., Mirenda C. A., McKinley M. P., Carlson G. A., Prusiner S. B. Distinct prion proteins in short and long scrapie incubation period mice. Cell. 1987 Nov 20;51(4):651–662. doi: 10.1016/0092-8674(87)90134-6. [DOI] [PubMed] [Google Scholar]
  56. Williams A. F., Gagnon J. Neuronal cell Thy-1 glycoprotein: homology with immunoglobulin. Science. 1982 May 14;216(4547):696–703. doi: 10.1126/science.6177036. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES