Complexes comprising a prion protein and a peptidyl prolyl isomerase chaperone, and method for producing and using them Number:7,094,757 from the United States Patent and Trademark Office (PTO) owispatent The present invention relates to the diagnosis of transmissible spongiform encephalopathies (TSEs). It especially teaches the production of a soluble prion protein (PrP)-chaperone complex and the advantageous use of such chaperone-PrP complex, especially in the detection of PrP in an immunoassay, as well as its use as and immunogen.<H comprising, producing, Complexes, compr, 7094757.html, Patent, pto, 7094757

Title: Complexes comprising a prion protein and a peptidyl prolyl isomerase chaperone, and method for producing and using them

Abstract: The present invention relates to the diagnosis of transmissible spongiform encephalopathies (TSEs). It especially teaches the production of a soluble prion protein (PrP)-chaperone complex and the advantageous use of such chaperone-PrP complex, especially in the detection of PrP in an immunoassay, as well as its use as and immunogen.

Patent Number: 7,094,757 Issued on 08/22/2006 to Faatz, et al.

Inventors: Faatz; Elke (Huglfing, DE), Scholz; Christian (Penzberg, DE), Stock; Werner (Graefelfing, DE), Schaarschmidt; Peter (Uffing, DE)
Assignee: Roche Diagnostics Corporation (Indianapolis, IN)

Appl. No.: 10/745,393

Filed: December 23, 2003

Foreign Application Priority Data


Jun 22, 2001 [EP]			01115225
Aug 31, 2001 [EP]			01120939

Current U.S. Class: 514/12 ; 435/183; 435/320.1; 530/350

Current International Class: C07K 14/00 (20060101); C12N 9/00 (20060101)

Field of Search: 514/2 530/350 435/183,320.1 536/23.1

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WO 98/01349	Jan., 1998	WO
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Primary Examiner: Carlson; Karen Cochrane
Attorney, Agent or Firm: Brinks Hofer Gilson & Lione

Parent Case Text

RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No. 10/179,905, filed on Jun. 24, 2002, and entitled "Soluble Complexes of Target Proteins and Peptidyl Prolyl Isomerase Chaperones and Methods of Making and Using Them," which claims the benefit of application Ser. No. 10/167,774, filed on Jun. 10, 2002 and now abandoned. Priority is also claimed, under 35 U.S.C. .sctn. 119 to EPO applications: EPO 01115225.3, filed on Jun. 22, 2001 and EPO 01120939.2, filed on Aug. 31, 2001. The disclosures of the priority applications are incorporated by reference herein in their entireties

Claims

What is claimed is:

1. A protein which is soluble to at least 100 nM in a solution which has a pH of 7.4 and consists of 20 nM sodium phosphate and 150 nM sodium chloride, said protein comprising: a prion protein (PrP), and a peptidyl prolyl isomerase chaperone, wherein the PrP and the peptidyl prolyl isomerase chaperone are covalently linked.

2. The protein of claim 1, wherein the protein is soluble to at least 1 .mu.M.

3. The protein of claim 1, wherein the PrP and the peptidyl prolyl isomerase chaperone are linked recombinantly.

4. The protein of claim 1, wherein the ratio of PrP to peptidyl prolyl isomerase chaperone is 1:1.

5. The protein of claim 1, wherein the ratio of PrP to peptidyl prolyl isomerase chaperone is 1:2.

6. The protein of claim 1, wherein the PrP is selected from the group consisting of mPRP and hPrP.

7. The protein of claim 1, wherein the peptidyl prolyl isomerase chaperone is an FKBP chaperone.

8. The protein of claim 7, wherein the FKBP chaperone is selected from the group consisting of SlyD, FkpA, and trigger factor.

9. The protein of claim 1, said protein further comprising a label.

10. An immunoassay reagent for the detection of PrP comprising the protein of claim 1 as a standard material.

11. A recombinant polypeptide which is soluble to at least 100 nM in a solution which has a pH of 7.4 and consists of 20 nM sodium phosphate and 150 nM sodium chloride, said recombinant polypeptide comprising: a prion protein (PrP), a peptidic linker, and a peptidyl prolyl isomerase chaperone.

12. The recombinant polypeptide of claim 11, wherein the ratio of PrP to peptidyl prolyl isomerase chaperone is 1:1.

13. The recombinant polypeptide of claim 11, wherein ratio of PrP to peptidyl prolyl isomerase chaperone is 1:2.

14. The recombinant polypeptide of claim 11, wherein the PrP is selected from the group consisting of mPRP and hPrP.

15. The recombinant polypeptide of claim 11, wherein the peptidic linker is 10 to 50 amino acids in length.

16. The recombinant polypeptide of claim 11, wherein the peptidic linker is 15 to 35 amino acids in length.

17. The recombinant polypeptide of claim 11, wherein the peptidyl prolyl isomerase chaperone is an FKBP chaperone.

18. The recombinant polypeptide of claim 17, wherein the chaperone is selected from the group consisting of FkpA, SlyD, and trigger factor.

19. The recombinant polypeptide of claim 11, said recombinant polypeptide further comprising a label.

20. An immunoassay reagent for the detection of PrP comprising the recombinant polypeptide of claim 11 as a standard material.

21. A recombinantly-produced fusion polypeptide comprising: a prion protein (PrP), and an FKBP chaperone polypeptide, wherein said FKBP chaperone polypeptide is selected from the group consisting of FkpA, SlyD, and trigger factor.

22. The fusion polypeptide of claim 21, wherein the PrP is selected from the group consisting of mPRP and hPrP.

23. A recombinant polypeptide which is soluble to at least 100 nM in a solution which has a pH of 7.4 and consists of 20 nM sodium phosphate and 150 nM sodium chloride, said recombinant polypeptide comprising: at least one PrP domain of a prion protein (PrP), a peptidic linker, and at least one chaperone domain of a peptidyl prolyl isomerase chaperone.

24. The recombinant polypeptide of claim 23, wherein the polypeptide comprises at least one PrP domain and at least two chaperone domains.

25. The recombinant polypeptide of claim 23, wherein the PrP is selected from the group consisting of mPRP and hPrP.

26. The recombinant polypeptide of claim 23, wherein the peptidic linker is 10 to 50 amino acids in length.

27. The recombinant polypeptide of claim 23, wherein the peptidic linker is 15 to 35 amino acids in length.

28. The recombinant polypeptide of claim 23, wherein the peptidyl prolyl isomerase chaperone is an FKBP chaperone.

29. The recombinant polypeptide of claim 28, wherein the chaperone is selected from the group consisting of FkpA, SlyD, and trigger factor.

30. The recombinant polypeptide of claim 23, said recombinant polypeptide further comprising a label.

31. An immunoassay reagent for the detection of PrP comprising the recombinant polypeptide of claim 23 as a standard material.

32. An expression vector comprising: at least one nucleic acid sequence encoding a prion protein (PrP), at least one nucleic acid sequence encoding a peptidic linker, and at least one nucleic acid sequence encoding an FKBP chaperone selected from the group consisting of FkpA, SlyD, and trigger factor.

33. The expression vector of claim 32, wherein the nucleic acid sequence encoding FKBP chaperone is inserted in said expression vector upstream of the nucleic acid sequence encoding the peptidic linker and the nucleic acid sequence encoding a PrP.

34. An expression vector comprising: at least one nucleic acid sequence encoding a prion protein (PrP), at least one nucleic acid sequence encoding a peptidic linker, and at least one nucleic acid sequence encoding a peptidyl prolyl isomerase chaperone.

35. The expression vector of claim 34, wherein the nucleic acid sequence encoding peptidyl prolyl isomerase chaperone is inserted in said expression vector upstream of the nucleic acid sequence encoding the peptidic linker and the nucleic acid sequence encoding a PrP.

36. An expression vector comprising: at least one nucleic acid sequence encoding at least one PrP domain of a prion protein (PrP), at least one nucleic acid sequence encoding a peptidic linker, and at least one nucleic acid sequence encoding at least one chaperone domain of a peptidyl prolyl isomerase chaperone.

37. The expression vector of claim 36, wherein the nucleic acid sequence encoding at least one chaperone domain of the peptidyl prolyl isomerase chaperone is inserted in said expression vector upstream of the nucleic acid sequence encoding a peptidic linker and the nucleic acid sequence encoding a PrP domain of a PrP.

38. A method for producing a soluble prion protein (PrP)-chaperone protein comprising: incubating a polypeptide comprising PrP covalently linked to a peptidyl prolyl isomerase chaperone in a buffer wherein both the PrP and the chaperone are solubilized to at least 100 nM, said buffer comprising a pH of 7.4, 20 mM sodium phosphate, and 150 mM sodium chloride.

39. The method of claim 38, wherein the polypeptide is solubilized with a chaotropic reagent.

40. The method of claim 39, wherein the chaotropic agent is 7.0 M guanidinium chloride.

41. The method of claim 38, wherein the PrP is produced recombinantly.

42. The method of claim 38, wherein the peptidyl prolyl isomerase chaperone is produced recombinantly.

43. The method of claim 38, wherein the PrP and the peptidyl prolyl isomerase chaperone are linked recombinantly.

44. The method of claim 38, wherein the PrP is selected from the group consisting of mPrP and hPrP.

45. The method of claim 38 wherein the peptidyl propyl isomerase chaperone is an FKBP chaperone.

46. The method of claim 38, wherein the peptidyl prolyl isomerase chaperone is a binding-competent fragment of the peptidyl prolyl isomerase chaperone.

47. The method of claim 38, wherein the peptidyl prolyl isomerase chaperone is of human origin.

48. The method of claim 38, wherein the peptidyl prolyl isomerase chaperone is derived from an organism selected from the group consisting of Yersinia pestis, Vibrio cholerae, Pasteurelia multocida, and Treponema pallidum.

49. The method of claim 38, wherein the peptidyl prolyl isomerase chaperone is an FKBP chaperone.

50. The method of claim 49, wherein the FKBP chaperone is selected from the group consisting of FkpA, SlyD, and trigger factor.

51. A method for eliciting an immune response in a subject comprising administering a composition comprising the protein of claim 1 to said subject, thereby eliciting antibodies in said subject, said antibodies having the ability to bind the PrP.

52. The method of claim 31, wherein the PrP is selected from the group consisting of mPrP and hPrP.

53. A method for eliciting an immune response in a subject comprising administering a composition comprising the recombinant polypeptide of claim 11 to said subject, thereby eliciting antibodies in said subject, said antibodies having the ability to bind the PrP.

54. The method of claim 53, wherein the PrP is selected from the group consisting of mPrP and hPrP.

55. A method for eliciting an immune response in a subject comprising administering a composition comprising the recombinant polypeptide of claim 23 to said subject, thereby eliciting antibodies in said subject, said antibodies having the ability to bind the PrP.

56. The method of claim 55, wherein the PrP is selected from the group consisting of mPrP and hPrP.

57. A method for producing antibodies to a prion protein (PrP) comprising administering the protein of claim 1 to an animal, thus eliciting an immune response in the animal, and isolating antibodies having the ability to bind the PrP.

58. The method of claim 57, wherein the antibody to PrP is a monoclonal antibody.

59. The method of claim 57, wherein the antibody to PrP is a polyclonal antibody.

60. A method for producing antibodies to prion protein (PrP) comprising administering the recombinant polypeptide of claim 11 to an animal, thus eliciting an immune response in the animal, and isolating antibodies having the ability to bind the PrP.

61. The method of claim 60, wherein the antibody to PrP is a monoclonal antibody.

62. The method of claim 60, wherein the antibody to PrP is a polyclonal antibody.

63. A method for producing antibodies to prion protein (PrP) comprising administering the recombinant polypeptide of claim 23 to an animal, thus eliciting an immune response in the animal, and isolating antibodies having the ability to bind the PrP.

64. The method of claim 63, wherein the antibody to PrP is a monoclonal antibody.

65. The method of claim 63, wherein the antibody to PrP is a polyclonal antibody.

Description

BACKGROUND

1. Technical Field

The present invention relates to the diagnosis of transmissible spongiform encephalopathies (TSEs). It especially teaches the production of a soluble prion protein (PrP)-chaperone complex and the advantageous use of such chaperone-PrP complex, especially in the detection of PrP in an immunoassay, as well as its use as an immunogen.

2. Background Information

Transmissible spongiform encephalopathies (TSEs) include kuru-kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI) in humans, bovine spongiform encephalopathy (BSE), and scrapie in sheep. It is believed that the TSEs are caused by a novel class of infectious pathogens the so-called prion proteins or prions. According to the protein-only hypothesis the "infectious agent" is the prion protein in an abnormal oligomeric form which is called prion protein scrapie type (=PrP.sup.Sc). The mammalian prion protein is highly conserved during evolution and normally is a monomeric cell surface glycoprotein which is termed PrP.sup.C, wherein .sup.C stands for cellular. Both isoforms share the same amino acid sequence but differ in their secondary and tertiary structure. It is believed, that only the PrP.sup.Sc can transmit the disease, whereas PrP.sup.C appears not to be correlated with any disease mechanism leading to TSE.

Bovine spongiform encephalopathy (BSE) is a fatal, neuro degenerative, transmissible brain disease of cattle. The disease is fatal for cattle within weeks to months of its onset.

BSE first came to the attention of the scientific community in November 1986 with the appearance in cattle of a newly recognized form of neurological disease in the United Kingdom. Between November 1986 and November 2002, about 180 000 cases of BSE were confirmed in the United Kingdom.

Since 1989, when the first BSE case was reported outside the United Kingdom, relatively small numbers of cases (about 1500 in total) have also been reported in native cattle in many European countries and Japan. However, all but a couple of dozen have been reported in four countries--France, Ireland, Portugal and Switzerland.

Small numbers of cases have also been reported in Canada, the Falkland Islands, and Oman, but solely in animals imported from the United Kingdom.

Creutzfeldt-Jakob disease (CJD) is a rare and fatal neurodegenerative disease of unknown cause. Patients are usually aged between 50 and 75 and typical clinical features include a rapidly progressive dementia associated myoclonus and a characteristic electroencephalographic pattern. Neuropathological examination reveals cortical spongiform change, hence the term "spongiform encephalopathy".

H. G. Creutzfeldt is credited with the first description of the disorder in 1920, although by current diagnostic criteria his case would be highly atypical. A year later another German neurologist, A. Jakob described four cases, at least two of whom had clinical features suggestive of the entity today recognized as CJD.

Although CJD appears to occur as a predominantly sporadic disorder it can also occur as a dominantly inherited or infective condition. The latter mode of transmission was first elucidated during the study of kuru-kuru in the 1950's. This neurodegenerative condition occurs only in the people of the Fore region of Papua New Guinea and is thought to have resulted from the consumption of brains during endocannabalistic funeral rituals. The similarities between kuru-kuru and scrapie, the transmissible spongiform encephalopathy of sheep, prompted a veterinary neuropathologist, Hadlow, to suggest that transmission studies of kuru-kuru be performed. The success of those studies and the recognition that the neuropathological changes in kuru-kuru were similar to those of CJD, was followed by the transmission of CJD to the chimpanzee by intracerebral inoculation of brain tissue. In 1974 a case of iatrogenic CJD due to corneal transplantation occurred and subsequently contaminated neuro-surgical instruments, dural grafts, and brain depth electrodes have all been recognized as transmitting the disease. In 1985 the first case was reported in a recipient of contaminated human derived growth hormone and subsequently over 60 similar cases have arisen world-wide in addition to 4 cases associated with human derived gonadotrophin. The familial occurrence of CJD has been recognized for many years but was unexplained. The discovery of linkage to a region on the short arm of chromosome 20 has led recently to the elucidation of various dominantly inherited mutations.

Variant Creutzfeldt-Jakob disease (vCJD) is a rare and fatal human neurodegenerative condition. Like Creutzfeldt-Jakob disease (CJD), vCJD is classified as a transmissible spongiform encephalopathy (TSE) because of characteristic spongy degeneration of the brain and the transmissibility of the disease. First described in March 1996, vCJD appears to be strongly linked to exposure to the BSE agent.

From October 1996 to early November 2000, 85 cases of vCJD have been reported in the United Kingdom, 3 in France and 1 in Ireland. Insufficient information is available at present to make any well-founded prediction about the future number.

The nature of the transmissible agent in CJD is the matter of some controversy. Previously considered a "slow virus" no viral agent has ever been convincingly demonstrated and no evidence of an immunological response seen. Additionally the infectious pathogen shows a remarkable resistance to treatments that would normally be expected to inactivate viruses. The viral hypothesis has been elegantly challenged by the prion ("proteinaceous infectious particle") theory which states that the infectious agent is derived from a protease-sensitive protein (designated PrP.sup.C) which is a constituent of the normal cell membrane. It is postulated that the normal protein undergoes a post-translational conformational change forming the insoluble pathogenic form of the prion protein (PrPS.sup.Sc). This in turn induces more of the normal PrP.sup.C to form PrP.sup.Sc--hence a chain reaction is set in motion with the exponential production of the insoluble prion protein being formed. The initial abnormal prion protein needed to seed this process may occur spontaneously as a rare event (which would account for the low incidence of sporadic CJD), following inoculation (accounting for observed transmission phenomena) or when initiated by a genetic abnormality of the PrP gene. The mechanism by which PrP.sup.Sc induces the pathological changes in CJD-spongiform change, gliosis, neuronal loss and (infrequently) plaques remains unclear. Although the prion hypothesis neatly explains many of the observed phenomena of transmissible spongiform encephalopathies (TSEs) it has one particular weakness. Scrapie is known to exhibit various "strains" characterized by different incubation periods, clinical features and pathology when transmitted. This is much more in keeping with a virus-like agent and strain variation, independent of the host genome, is difficult to reconcile with the prion theory.

The majority of CJD-cases are sporadic (85%), between 10-15% are familial and the remainder are iatrogenic.

CJD occurs worldwide with a roughly even incidence of between 0.5-1.0 cases per million per year. Higher rates (up to 100-fold) have been reported in Slovakia and Libyan-born Israelis but this is explained by the high incidence of a certain mutation of the PrP gene in these groups. The geographical distribution of CJD in the United Kingdom over the past 25 years demonstrates no overall evidence of spatiotemporal aggregation of cases, despite the occurrence of local areas of relatively high incidence over short periods. There is no evidence of case to case transmission and spouses of sporadic cases do not have an increased incidence of the disease.

TSEs are known to affect various animal species including sheep, goats, mink, mule deer, cows and recently cats. Scrapie, a disorder of sheep and goats, has been known for over 300 years and is endemic in the British Isles. In 1938 experimental transfer of scrapie from one sheep to another by inoculation provided evidence of an infective etiology. However there is no evidence of transmission of scrapie from sheep to man and there is no increased incidence of CJD in countries with scrapie compared to those without (e.g. UK and Australia).

The close relationship in pathogenesis between BSE and CJD has in recent years triggered massive scientific and industrial research into further understanding disease transmission pathogenesis as well as into early diagnosis of the underlying disease.

Research, however, is hampered by the fact that both PrP.sup.C, as well as PrP.sup.Sc are difficult to isolate, to purify and especially to handle. PrP is a fairly sticky protein. Moreover, PrP is a metastable protein with a pronounced tendency to aggregate or precipitate in physiological buffers. Aggregation and precipitation of PrP is frequently observed under long term storage conditions and can be induced or accelerated by increasing storage temperature.

What render PrP even more critical is the biohazards associated with handling a PrP from a biological source, especially with regard to disease transmission.

Various attempts to obtain a prion protein by recombinant expression are known from the literature. Hornemann, S., et al., FEBS Letters 413 (1997) 277-281, for example describe the expression of full-length murine prion protein (mPrP (23-231)). These researchers expressed the complete amino acid sequence of the prion protein, mPrP (23-231), in the cytoplasm of Escherichia coli. The corresponding protein has been obtained in the form of inclusion bodies, which had to be solubilized by 8 M urea and oxidatively refolded. The human prion protein has been expressed in Escherichia coli by Zahn, R., et al., FEBS Letters 417 (1997) 400-404. In this paper the human prion protein (hPrP) from amino acid 23 to amino acid 231 has been expressed, as well as several fragments of hPrP. These investigators used polypeptides comprising a histidine tail fusion protein. This did facilitate the purification of the proteins which also had been obtained in the form of inclusion bodies. When purified, isolated and stored in a rather concentrated solution the hPrP thus obtained has been found to be stable.

However, if used as a standard material for immunoassays, hPrP, has to be diluted to comparatively low concentrations and nonetheless the diluted material has to be stable for several months.

Physiological, buffered salt solutions that mimic the extracellular fluids of human tissues have uniformly failed to solubilize PrP in solution if stored for weeks or months at 4.degree. C.

Immunoassays in general are performed at physiological pH. Hence polypeptides soluble at physiological buffer conditions are extensively used in various immunoassay methods, such as ELISA (enzyme-linked immunosorbent assay), for example in the diagnosis of and screening for a certain disease.

Due to its aggregation tendency under physiological buffer conditions, and due to its metastable nature the PrP is difficult to use in an immunoassay E.g., in a sandwich assay format PrP is difficult to handle, because it tends to aggregate or even precipitate. Due to its metastable nature PrP may also lead to false results caused by unspecific binding.

In addition the metastability of PrP under physiological buffer conditions renders this protein a difficult target for routine (bio-) chemical procedures. The vast majority of "labeling chemistries", i.e., the chemical procedures used for binding a label, e.g., a marker group to a polypeptide, are based on nucleophilic chemistry and thus rather restricted to a pH window from about pH 6 to about pH 8 and thus only work under more or less physiological buffer conditions. These routine procedures, e.g., as described in Aslam, M. and Dent, A., The preparation of protein-protein conjugates in "Bioconjugation", eds. M. Aslam and A. Dent, McMillan Reference, London (1998), pp. 216-363, are difficult to carry out with PrP.

Therefore a tremendous need exists to provide PrP in a thermodynamically stable rather than in its metastable form. In this thermodynamically stable form PrP is for example soluble at physiological pH, stable in solution and/or easier to produce and/or handle.

There is also an urgent need to provide such PrP without encountering the biohazards associated with PrP that has been isolated from a mammal.

SUMMARY

In one embodiment the invention is a complex which is soluble to at least 100 nM in a solution which has a pH of 7.4 and consists of 20 nM sodium phosphate and 150 nM sodium chloride. The complex comprises a prion protein (PrP), and a peptidyl prolyl isomerase chaperone, wherein the PrP and the peptidyl prolyl isomerase chaperone are covalently linked.

In another embodiment, the invention is a recombinant polypeptide which is soluble to at least 100 nM in a solution which has a pH of 7.4 and consists of 20 mM sodium phosphate and 150 mM sodium chloride. The recombinant polypeptide comprises a prion protein (PrP), a peptidic linker, and a peptidyl prolyl isomerase chaperone.

In yet another embodiment, the invention contemplates a recombinantly-produced fusion polypeptide. The fusion peptide comprises a prion protein (PrP), and an FKBP chaperone polypeptide selected from the group consisting of FkpA, Sly D, and trigger factor.

In yet another embodiment, the present invention is an expression vector. The expression vector comprises at least one nucleic acid sequence encoding a PrP, at least one nucleic acid sequence encoding FKBP chaperone selected from the group consisting of FkpA, SlyD and trigger factor, and a nucleic acid sequence encoding a peptidic linker.

In further embodiment, the present invention is a method for producing a soluble PrP-chaperone complex. The method includes incubating a polypeptide comprising a PrP covalently linked to a peptidyl prolyl isomerase chaperone in a buffer wherein both the PrP and the chaperone are solubilized. The method further includes adjusting the buffer to physiological conditions. The PrP-chaperone complex formed according to this method is soluble to at least 100 nM as measured in a solution which has a pH of 7.4 and consists of 20 mM sodium phosphate and 150 mM sodium chloride.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: UV spectrum of the fusion protein SS-hPrP (23-230) after matrix-assisted refolding and imidazole step elution. The spectrum was recorded on a Uvicon XS photometer using a pathlength of 1 cm. Buffer conditions were 50 mM sodium phosphate pH 8.0, 100 mM sodium chloride and .about.250 mM imidazole. The shape of the spectrum highlights the remarkable solubility of the chaperoned hPrP (23-230). Stray light effects which would indicate aggregation phenomena are not observed.

FIG. 2: Purification of SS-hPrP (23-230) as documented by SDS-PAGE. The Comassie-stained gel shows (from left to right) the protein standard M12 (Novagen), the E. coli crude extract, the IMAC flow-through, the imidazole eluate and the SS-hPrP (23-230) dimer fraction after gel filtration on a Superdex.RTM. 200 column.

FIG. 3: Differential CD-spectroscopy in the near UV region. Near UV CD spectra were recorded for the carrier module SS (thin line) and the fusion protein SSPrP (thick line). Subtraction of the SS spectrum from the SS-hPrP spectrum yields the broken thin line representing the CD signal contribution of the prion part of the fusion protein. When converted into mean residue weight ellipticity, a theoretical hPrP (23-230) spectrum results that is in good agreement with literature data. These results strongly suggest that SlyD (1-165) and human prion protein (23-230) behave as independent folding domains within the context of the fusion protein. Spectra were recorded on a Jasco-720 spectropolarimeter. The path length was 0.5 cm, the protein concentration of SS and SS-hPrp (23-230) was 33 .mu.M, respectively. Buffer conditions were 50 mM NaP pH 7.8, 100 mM NaCl, 1 mM EDTA, the response was 2 s. The spectra have been accumulated (9.times.) to improve the signal-to-noise ratio.

FIGS. 4A and 4B: Different residual solubility of S-hPrP and SS-hPrP after long-term incubation at elevated temperatures. The two fusion proteins were incubated at varying temperatures (from top to bottom: 8.degree. C., 35.degree. C., 45.degree. C., 50.degree. C. and 55.degree. C.) under identical buffer conditions and protein concentrations. Afterwards, they were assessed for aggregate formation by means of FPLC analysis on a Superdex.RTM.200 size exclusion column. FIG. 4A shows some aggregation tendency of S-hPrP at temperatures exceeding 45.degree. C. The resulting aggregate particles do not elute from the column, but obviously interact with the Superdex matrix. In contrast, the recovery of SS-hPrP is very high (4B). Moreover, the aggregation tendency is significantly reduced in the twin carrier fusion construct, SS-hPrP.

FIG. 5: The chaperone carrier module SS significantly increases the thermo tolerance of human prion protein. SS-hPrP and S-hPrP were subjected to thermal unfolding (max. temp. 80.degree. C.) and assessed for their solubility after cooling down to room temperature. Whereas S-hPrP elutes mainly as a high molecular aggregate (peak at 8.8 min), SS-hPrP elutes chiefly as a soluble dimer (peak at 13.15 min). The result clearly indicates that the SS carrier module increases the reversibility of thermally induced unfolding.

DETAILED DESCRIPTION OF THE DRAWINGS AND THE PRESENTLY PREFERRED EMBODIMENTS

It was the task of the present invention to investigate whether PrP can be provided in a form, which is both readily soluble under physiological buffer conditions and stable in solution even under long term storage conditions.

We found that folding helpers, e.g., many members of the peptidyl prolyl isomerase (PPI) class, especially from the FKBP family, not only exhibit catalytic activity, but also have substantial beneficial effects on the solubility of amyloidogenic proteins, or more generally speaking, of proteins which tend to aggregate, like the PrP. They do so by forming soluble complexes with such proteins that are otherwise (i.e. in an unchaperoned, isolated form) prone to aggregation. PrP which is both metastable and poorly soluble under physiological conditions turned out to be thermodynamically stable under mild physiological conditions (i.e. without the need for solubilizing additives such as detergents or chaotropic agents) once it is present as a complex with an appropriate PPI chaperone. Thus, for example we were able to produce soluble PrP-chaperone complexes comprising the prion protein (23-230)--which is otherwise prone to aggregation--and SlyD, FkpA or other FKBPs as solubility-conferring chaperones.

A soluble complex comprising a PrP and a PPI-class chaperone can for example be obtained from a single recombinant protein comprising both PrP and a PPI class chaperone. A recombinant protein comprising PrP and a chaperone selected from the peptidyl-prolyl-isomerase class of chaperones is described.

According to the procedure described here, it is now possible to provide a PrP in a readily soluble and stable form, e.g., for use as standard material in immunoassays.

The PrP-chaperone complexes described in the present invention provide a convenient means to produce a soluble and thermodynamically stable PrP for use in immunoassays irrespective of the detection format employed.

The novel complexes e.g., comprising PrP and SlyD, for example, are readily soluble, and thermodynamically stable under physiological conditions. They can be used to great advantage in the detection of PrP by immunological techniques or for immunization of laboratory animals with a PrP-PPI chaperone complex.

The present invention relates to a method for producing a soluble PrP-chaperone complex comprising a PrP covalently linked to a PPI chaperone, comprising: solubilizing said polypeptide, and adjusting the buffer to physiological conditions, wherein the PrP-chaperone complex formed is soluble to at least 100 nM as measured in a solution which has a pH of 7.4 and consists of 20 mM sodium phosphate and 150 mM sodium chloride.

A prion protein or "PrP" according to the present invention comprises amino acids 23-230 of the mouse or the human prion protein precursor. The sequence of the hPrP precursor is given in SEQ ID NO: 1.

In the following PrP is sometimes also referred to as "target protein".

It is obvious to the skilled artisan that PrP from non-human other mammalian species, as well as naturally occurring or synthetically produced variants of PrP may also be used with great advantage and shall also be encompassed by the present invention. The use of human PrP and naturally occurring variants thereof is most preferred.

A protein is considered "metastable" if it adopts different configurations in one and the same environment. For example PrP is metastable in a physiological buffer consisting of 20 mM sodium phosphate pH 7.4, 150 mM NaCl. Under long term storage conditions, even at 4.degree. C., PrP tends to aggregate in such buffers or even precipitates from such buffers.

The PrP-chaperone complex according to the present invention, however, is "thermodynamically stable" in a buffer consisting of 20 mM sodium phosphate pH 7.4, 150 mM NaCl. In such a buffer the target protein PrP, as comprised in the PrP-PPI-chaperone complex, is readily soluble and is stable under long term storage conditions.

The term "complex" is used to indicate that the peptide domain corresponding to PrP and the peptide domain corresponding to the chaperone interact with each other whereby the chaperone confers a solubilizing effect on the PrP.

Production of the soluble chaperone-target protein complex starts from a solubilizing buffer condition, i.e. from a buffer in which both the target protein and the chaperone are soluble. An appropriate buffer, which may be termed "non-physiological" or "solubilizing" buffer, has to meet the requirement that both the target protein and the PPI chaperone are not denatured or at least not irreversibly denatured. Starting from such buffer conditions, the chaperone binds to the target protein, and a change of the buffer conditions from non-physiological to physiological conditions is possible without precipitation of the target protein.

An appropriate (non-physiological) buffer, i.e., a buffer in which both the target protein and the PPI-chaperone are soluble either makes use of high or low pH, or of a high chaotropic salt concentration or of a combination thereof.

Although a chaperone and a PrP can be used as separate polypeptides, we have observed that it is advantageous to link both proteins covalently. Such covalent linkage is possible by conventional chemical cross-linking procedures; preferably, however, the covalent linkage is achieved by producing a recombinant polypeptide comprising a PrP and a chaperone.

In a further preferred embodiment, the present invention relates to a process for the production of a soluble PrP-chaperone complex comprising the steps of solubilizing, under appropriate buffer conditions, a protein comprising a recombinantly linked PrP and a chaperone protein selected from the peptidyl prolyl isomerase class and thereafter adjusting the buffer to physiological conditions. In this manner an intramolecular complex is obtained which is soluble to at least 100 nM in a buffer which has a pH of 7.4 and consists of 20 mM sodium phosphate and 150 mM sodium chloride. It is most preferred to perform this process starting from so-called inclusion bodies.

In the case of the production of an intramolecular complex comprising a PPI-chaperone and PrP the solubilizing buffer preferably is a buffer with a rather high concentration of a chaotropic salt, e.g., 6.0 M guanidinium chloride at a pH of about 7.8. Upon renaturation the target protein assumes its native-like structure and a soluble intramolecular complex forms.

The present invention teaches the use of chaperones derived from the class of folding helpers termed peptidyl prolyl cis/trans isomerases (PPIs or PPIases) (cf. Dartigalongue, C., and Raina, S., Embo J. 17 (1998) 3968-3980). Well-known examples of this family are members called CypA, PpiD (Dartigalongue, C. and Raina, S., supra; Schmid, F. X., Molecular chaperones in the life cyle of proteins, eds. A. L. Fink and Y. Goto, Marcel Decker Inc., New York (1998), pp. 361-389), FkpA (Danese, P. N., et al., Genes Dev. 9 (1995) 387-398) and trigger factor (Crooke, E., and Wickner, W., Proc. Natl. Acad. Sci. USA 84 (1987) 5216-5220; Stoller, G., et al., Embo J. 14 (1995) 4939-4948).

The peptidyl prolyl isomerases are subdivided into three families, the parvulines (Schmid, F. X., supra; Rahfeld, J. U., et al., FEBS Lett. 352 (1994) 180-184) the cyclophilines (Fischer, G., et al., Nature 337 (1989) 476-478, and the FKBP family (Lane, W. S., et al., J. Protein Chem. 10 (1991) 151-160). The FKBP family exhibits an interesting biochemical feature: its members were originally identified by their ability to bind to macrolides, e.g., FK 506 and rapamycin (Kay, J. E., Biochem J. 314 (1996) 361-385).

Some prolyl isomerases comprise different subunits or modules having different functions, e.g., a module exhibiting catalytic activity and a module exhibiting the chaperone or binding activity. Such modular members of the FKBP family are FkpA (Ramm, K., and Pluckthun, A., J. Biol. Chem. 275 (2000) 17106-17113), SlyD (Hottenrott, S., et al., J. Biol. Chem. 272 (1997) 15697-15701) and trigger factor (Scholz, C., et al., Embo J. 16 (1997) 54-58). Preferably members of the FKBP family of the PPI class of chaperones are used.

It is also well known and appreciated that it is not necessary to always use the complete sequence of a molecular chaperone. Functional fragments of chaperones (so-called modules) which still possess the required abilities and functions may also be used (cf WO 98/13496).

For instance, FkpA is a periplasmic PPI that is synthesized as an inactive precursor molecule in the bacterial cytosol and translocated across the cytoplasmic membrane. The active form of FkpA (mature FkpA or periplasmic FkpA) lacks the signal sequence (amino acids 1 to 25) and thus comprises amino acids 26 to 270 of the precursor molecule. Relevant sequence information relating to FkpA can easily be obtained from public databases, e.g., from "SWISS-PROT" under accession number P 45523.

A close relative of FkpA, namely SlyD (Swiss Prot accession number P30856), consists of a structured N-terminal domain responsible for catalytic and chaperone functions and of a largely unstructured C-terminus that is exceptionally rich in histidine and cysteine residues (Hottenrott, S., et al., J. Biol. Chem. 272 (1997) 15697-15701). We found that a C-terminally truncated variant of SlyD comprising amino acids 1-165 efficiently exerts its solubilizing functions on PrP. Unlike in the wild-type SlyD, the danger of compromising disulfide shuffling is successfully circumvented in the truncated SlyD-variant (1-165) used. We find that SlyD can be even further truncated (1-148) without compromising the chaperone activity and thus the solubility-conferring features.

Variants of the above-mentioned chaperones, bearing one or several amino acid substitutions or deletions, may also be used to perform a process according to the present invention, as long as the solubilizing effect is conserved.

Of course, the present invention is not restricted to the use of the specifically mentioned members of the peptidyl prolyl isomerase class, but can also be performed using homologues thereof, i.e., chaperones of the same class of chaperones but derived from a different species of bacteria.

Appropriate chaperones from alternative sources, and appropriate fragments or mutants of PPI chaperones can be easily selected by using the procedures as described in the Examples. Preferred alternative sources for PPI chaperones are Yersinia pestis, Vibrio cholerae, Pasteurella multocida, and Treponema pallidum. We surprisingly found that for example C-terminally truncated SlyD homologues from Y. pestis (1-165), V. cholerae (1-157), P. multocida (1-156) and T. pallidum (1-160) share the same beneficial chaperone features despite considerable differences in amino acid sequence. Moreover, all of the aforementioned PPI chaperones prove to be well suited as carrier modules according to the invention described here.

In a preferred embodiment according to the present invention, a binding-competent PPIase chaperone is recombinantly linked to a PrP in a manner designed to yield a high expression rate of the gene product in the bacterial cytosol. A binding-competent PPIase as referred to in the present invention encompasses at least the functional unit that mediates binding to the PrP substrate (i.e. the substrate binding or chaperone motif), irrespective of its catalytic PPIase activity.

It is known (e.g., Scholz et al., supra) that modular PPIs preferentially bind to denatured or partially denatured proteins. PPIases have now been found to have the striking property of not only catalyzing the folding of proteins, but also of forming stable complexes with such proteins, thereby conferring solubility. Surprisingly the PPIases studied (such as TF, SlyD and FkpA) bind to and thus for example solubilize native-like PrP.

There is a wealth of information on complex formation between model biomolecules, e.g., between an antibody and an antigen (for review see Braden, B. C., and Poijak, R. J., Faseb J. 9 (1995) 9-16). Usually, complex formation and dissociation occur in parallel; the complex and the binding partners coexist in free equilibrium. Likewise, the same seems true for complexes between PPI chaperones and amyloidogenic proteins, like PrP, as described in the present invention.

The formation of a complex, as described in the present invention comprising a thermodynamically stable PrP, is especially important. Whereas PrP alone is known to be a metastable protein with poor solubility it has now been found to be readily soluble and stable, e.g., under physiological buffer conditions, in the complex with a PPI chaperone.

Antigens that are soluble and stable under long term storage conditions, e.g., under simple and well-defined buffer conditions such as phosphate buffered saline, are of tremendous advantage in diagnostic applications. They can be used directly, e.g., as standard material in an immunoassay. Long term storage conditions are understood as storage at 4 to 8.degree. C. for 18 months. Usually liquid formulations of reagents used in immunoassays nowadays have to be stable under these conditions.

It is generally accepted that storage at elevated temperature for shorter periods of time is adequate to obtain a reasonable estimate of long term storage properties. As will be shown in the Examples section the PrP as comprised in a PrP-chaperone complex according to the present invention is clearly more stable than PrP alone.

Physiological buffer conditions are usually understood to correspond to salt and pH-conditions found in the plasma or serum of animals and are defined as a pH value of around 7.4 and a salt concentration of about 150 mM. The PrP-chaperone complex according to the present invention is readily soluble and thermodynamically stable under these buffer conditions. The PrP present therein is immunologically active, thus pointing to a native-like structure.

As the skilled artisan will appreciate the buffer conditions used for solubilization and renaturation may be modified as required and appropriate and must not be understood as an undue restriction of the invention, which can be carried out successfully over a broad range of buffer conditions.

The overall salt concentration of the physiological buffer is not critical as long as Prp is stabilized in its thermodynamically stable form. Preferably the physiological buffer comprises at least 10 mM of the buffer system and at most 200 mM. The other buffer constituents, if any, may be a salt without significant buffer capacity, e.g., sodium chloride. The physiological buffer preferably has a salt concentration between 20 and 500 mM, more preferably between 50 and 300 mM, and most preferably between 100 and 200 mM.

In a process according to the present invention, the physiological buffer may be varied to have a pH value in the range of 5.0 to 8.5; more preferably, the range of such buffer is between pH 5.5 and pH 8.3. Even more preferably, such physiological buffer conditions are defined as the salt concentrations stated above and a pH value between 6.0 and 8.0; most preferably, the pH of such a physiological buffer is between 6.5 and 7.8.

A preferred embodiment according to the present invention is a process for producing a soluble PrP-chaperone complex, comprising a PrP recombinantly linked to a peptidyl prolyl isomerase comprising: solubilizing said recombinant PrP-chaperone polypeptide and adjusting the buffer to physiological conditions. Whereas a PrP alone would spontaneously precipitate to a significant extent during this process, it surprisingly stays in solution when it is part of a recombinant protein according to the present invention. This important finding is most likely due to the formation of an intramolecular complex between PrP and the chaperone.

In the case of the recombinant production of PrP in E. Coli, the recombinantly pro