WO2010063460A1

WO2010063460A1 - Recombinant production of hydrophobic peptides and fusion proteins for use in producing same

Info

Publication number: WO2010063460A1
Application number: PCT/EP2009/008594
Authority: WO
Inventors: Verena Finder; Rudolf Glockshuber; Roger Nitsch; Ivana Vodopivec
Original assignee: Eidgenoessische Technische Hochschule Zurich ETHZ; Zurich Universitaet Institut fuer Medizinische Virologie
Current assignee: Eidgenoessische Technische Hochschule Zurich ETHZ; Zurich Universitaet Institut fuer Medizinische Virologie
Priority date: 2008-12-02
Filing date: 2009-12-02
Publication date: 2010-06-10
Anticipated expiration: 2011-06-02

Abstract

Described are a novel method and fusion proteins suitable for the production and purification of hydrophobic peptides, hi particular, authentic and highly purified preparation of hydrophobic peptides such as beta-amyloid peptide, the use of those peptides in imaging techniques as well as tests for screening amyloid toxicity-inhibiting drugs using said beta-amyloid peptide are described.

Description

Recombinant production of hydrophobic peptides and fusion proteins for use in producing same

Field of the invention The present invention generally relates to the technical field of medicine, in particular to the field of neurodegenerative, neurological and protein misfolding disorders such as amyloidosis. More specifically, the invention relates to a method for the production and purification of aggregating or hydrophobic peptides making use of the recombinant expression of a fusion protein comprising an enzymatic cleavage site located such so as to release the authentic peptide after cleavage and purification of the peptide on a hydrophobic matrix column. The recombinant hydrophobic peptides so produced are substantially devoid of any impurities and thus particularly suitable as therapeutic and diagnostic agents, for example as a vaccine or imaging agent. The method of the present invention is illustrated for the amyloid beta peptide (Aβl— 42), its recombinant production, aggregation in vitro and biological activity, i.e. neurotoxicity in vivo.

Background of the invention

A variety of diseases, the so-called protein misfolding diseases are associated with a specific structural form of a protein; e.g., a "misfolded protein" or a self-aggregated protein, while the protein in a different structural form, e.g., a "normal protein", is not harmful, hi many cases, the normal protein is soluble, while the misfolded protein forms insoluble aggregates. Examples of such insoluble proteins include prions in transmissible spongiform encephalopathy (TSE); amyloid beta (Aβ) peptide in amyloid plaques of Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), and cerebral vascular disease (CVD); α- synuclein deposits in Lewy bodies of Parkinson's disease; tau in neurofibrillary tangles in frontal temporal dementia and Pick's disease; superoxide dismutase in amyotrophic lateral sclerosis; Huntingtin in Huntington's disease; islet amyloid polypeptide in diabetes mellitus; and amyloid can be deposited in chronic rheumatic diseases; see, e.g., Glenner et al., J. Neurol. Sci. 94 (1989), 1-28; Haan et al., Clin. Neurol. Neurosurg. 92 (1990), 305-310.

Often, these insoluble proteins and peptides, respectively, form aggregates composed of non- branching fibrils with the common characteristic of a β-pleated sheet conformation, hi the CNS amyloid can be present in cerebral and meningeal blood vessels (cerebrovascular deposits) and in brain parenchyma (plaques). In tissues outside the CNS, such as the pancreas, for example, amyloid can be deposited in islet cells in diabetes mellitus and in the kidneys, amyloid can be deposited in chronic rheumatic diseases. Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized pathophysiological^ by the amyloid hypothesis¹ which proposes amyloid-β peptide (Aβ) aggregation as a central event associated with neurotoxicity and deposition of β-amyloid fibrils in both the neuropil and the cerebral vasculature. Aβ aggregation is an ordered assembly process during which monomers form soluble oligomers, protofibrils and fibrils².

While a large body of evidence in favor of the amyloid hypothesis is being accumulated, many questions about the aggregation process are however still unanswered for several reasons. Although Aβl-42 is the more amyloidogenic species and most likely responsible for the neuropathology, previous studies on Aβ amyloid formation were mainly focused on Aβl-

40 or fragments thereof, presumably because Aβl-42 is more difficult to handle in experimental settings due to its amphipathic character and distinct aggregation propensity³. Moreover, many findings obtained with synthetic Aβ are controversial and reproducibility of experiments has often been challenging. This may be related to the fact that almost all published studies had been done with preparations of synthetic Aβ; these are known to contain variable amounts of intrinsic impurities,³' ⁴. For example, Aβ aggregation properties were noticed to vary not only with synthetic batch-to-batch impurities due to the complex synthetic production process, but also with storage and solubilization conditions⁵.

In vivo, Aβ is generated via proteolytic cleavage from the amyloid precursor protein (APP). Considering the tendency to aggregate and the absence of an initial methionine, production of aggregation-competent Aβ without an N-terminal fusion partner is challenging. Therefore existing methods for recombinant production of Aβ⁴' ^6"π' ²⁰' ²¹ either led to production of modified Aβ (e.g. oxidation of methionine), yield low amounts, require enormous efforts, or overcome the strong aggregation tendency and toxicity by the persisting presence of either point mutations or fusion tags or an initial methionine at the N-terminus of the recombinant peptide with variable pathophysiological relevance. Moreover, some of these methods are exclusively developed for production of Aβl-40 or Aβl-40 fragments, but not for the pathophysiological^ relevant Aβl-42.

Thus, as illustrated by way of Aβl-42 the lack of success in manufacturing, purifying and handling authentic hydrophobic peptides substantially free of impurities such as chemical residuals have represented limitations of their potential applications including, but not limited to, their use in diagnostics, drug forms and in vaccine formulation. This is a particular problem in to-date vaccine formulation since many important antigens are membrane proteins and hence have important distinguishing hydrophobic epitopes, hi these applications it is also of great importance to achieve efficient presentation of protein epitopes to the host immune system, and therefore successful structure recovery and avoidance of premature and unspecific protein aggregation are desirable.

The above-mentioned problems are solved by the embodiments characterized in the claims and described further below.

Summary of the invention

The present invention generally relates to a method for expression, purification, and structure recovery and handling of self-aggregating or hydrophobic peptides. The method of the present invention includes the steps of designing and recombinant expression of a fusion protein which contains in its amino acid sequence an enzymatic cleavage site located between a hydrophilic polypeptide and the hydrophobic peptide of interest, and preferably an affinity tag such as a hexa-His tag at its N-terminus. The fusion protein is then isolated using hydrophobic chromatography, preceded by affinity chromatography, e.g. nickel-chelate if applicable, and then subjected to enzymatic cleavage under non-denaturing conditions so as to release the desired product. The recombinant peptides are then purified using hydrophobic chromatography. The resultant peptide preparation is comprised of monomelic forms of the peptide. Λ

The present invention is based inter alia on the approach involving the production of a fusion protein consisting of an affinity tag, a soluble fusion partner, and an enzymatic cleavage site for separation of the C-terminal hydrophobic amyloid beta peptide (Aβ). For enzymatic cleavage the tobacco etch virus (TEV) protease exhibiting high cleavage efficiencies was used. The produced Aβ is readily purified by using reversed-phase HPLC (RP-HPLC), and lyophilized for storage. Using E. coli for the recombinant expression of the fusion protein the method yields more than 20 mg per liter of E. coli culture.

Surprisingly, the physicochemical properties of recombinantly produced Aβl-42 compared to commercially available synthetic Aβl-42 are improved and supposed to more compare to native Aβl-42. Recombinant Aβl-42 aggregates faster than synthetic Aβl-42. Without intending to be bound by theory it is believed that this difference is due to the presence of a variety of impurities in the synthetic batch, which have similar masses and characteristics as authentic Aβl-42. The morphology of fibrils formed by recombinant Aβl-42 observed by using electron microscopy is more homogenous than in preparations of synthetic Aβl-42. Aβl-42 is more toxic in rat primary neurons and it accelerates abnormal phosphorylation of tau hyperphosphorylation and the formation of neurofibrillary tangles in brains of P301L tau transgenic mice. An initially monomeric Aβl-42 preparation is more toxic in the cell cultures than preformed fibrils and a mixture of different Aβ assembly forms.

Hence, the method of the present invention provides authentic hydrophobic peptides in unaggregated form with natural conformation and secondary structure and optimal aggregation kinetics as well as the capability of forming regular peptide fibrils. As demonstrated in the examples, these features of the recombinant hydrophobic peptide of the present invention are particular advantageous for the provision of recombinant Aβ which will help to further investigate Aβ as a target, drug, diagnostic agent and particularly as a vaccine in the treatment and diagnosis of the pathogenesis of Alzheimer's disease (AD).

While in the following the present invention will be explained in more detail with respect to Aβ and AD, it is to be understood that unless indicated otherwise, the embodiments disclosed herein are equally applicable to any other hydrophobic peptide associated with a disease such as neurodegenerative, neurological or neuropsychiatric disorder, which involve the presence and aggregation of hydrophobic peptides.

Brief descriptions of the drawings:

Fig. 1: Purification and analysis of recombinant Aβl-42 wild-type (wt) and arctic (indicated by a star) (a-d), and comparison to synthetic preparations (e, f). (a) Fusion construct for cytoplasmic expression in E. coli consisting of an N-terminal His₆-tag, a soluble fusion partner ((NANP) ^), and a TEV protease cleavage site (CS) containing the residues ENLYFQ for separation of Aβl-42, which is fused to the C-terminus. The sequence of Aβl-42 is shown; the presence of the arctic mutation (E22G) is indicated by a star, (b) RP-HPLC analysis of the fusion proteins after purification via Ni²⁺-NTA affinity chromatography. The elution profiles of fractions containing fusion protein with Aβl-42 wt and Aβl-42 arctic are shown, indicated by a black and a grey line, respectively. The peaks containing the proteins are indicated by a cartoon representing the fusion constructs and an arrow, (c) Further purification of the fusion proteins via RP-HPLC. The elution profiles of the fusion proteins containing Aβl-42 wt and arctic are indicated by a black and a grey line, respectively. Cartoons of the fusion constructs and an arrow indicate the peaks containing the corresponding proteins, (d) After cleavage of the fusion proteins with TEV protease, the produced Aβl-42 can be separated from the reaction mixture via RP-HPLC as it elutes later than the fusion protein and the other components. The proteins containing Aβl-42 wt and arctic are indicated by a black and a grey line, respectively. Cartoons of the fusion constructs and the corresponding Aβl-42 variants as well as an arrow indicate the peaks containing the respective proteins, (e) MALDI-MS analysis of recombinant Aβl-42 wt (black line) and arctic (shown in grey). Both preparations contain authentic Aβ wt and arctic with calculated masses of 4514.1 Da and 4442.0 Da, respectively. The peaks at the calculated masses + 206 represent an adduct with the MALDI-matrix sinapinic acid. Cartoons indicate the peaks of the Aβ variants, (f)

RP-HPLC analysis of recombinant and synthetic Aβl-42 wt. (g) Mass spectrum of synthetic Aβl-42 wt (shown in black), the spectrum of a batch analyzed in 2005 is shown in grey. The spectra contain peaks at a variety of m/z values, indicating the presence of impurities. A cartoon marks the position of authentic Aβl-42.

Fig. 2: Aggregation of recombinant and synthetic Aβl-42 monitored by thioflavin T fluorescence, by analyzing the soluble Aβ concentration, and circular dichroism of concentrated Aβl-42 stock solutions and aggregation assays. Recombinant and synthetic Aβl-42 are indicated by a black and a grey line, respectively. The reactions were either stirred at 500 rpm (a, c, f) or incubated under quiescent conditions (b, d).

(a, b) The fluorescence of thioflavin T was followed, indicating the formation of β- sheet rich protofibrils and fibrils, (c, d) The peak area of the soluble amount of Aβl- 42 after ultracentrifugation at different time points was calculated from RP-HPLC elution profiles, (e) Circular dichroism of 74 μM Aβ stock solutions. Fresh stock solutions are indicated by solid lines, spectra of solutions incubated for more than 24 h are shown by a dashed line, (f) Circular dichroism of aggregation assays as performed in (c). Spectra measured at the beginning of the reaction and after 3 h are indicated by solid and dashed lines respectively. Fig. 3: Electron microscopy of aggregates formed in stirred reactions (a) or under quiescent conditions (b) from recombinant or synthetic Aβl-42 preparations. Samples were taken from aggregation assays at the beginning of the reaction (initial), at the fluorescence maximum (F_max) in corresponding thioflavin T assays, and after incubation for 17 h. A representative picture of each sample is shown. Scale bar, 100 nm.

Fig. 4: Aβ toxicity in rat primary cortical neuron cultures, (a) Cytotoxicity upon incubation with 7.4 μM monomelic synthetic and recombinant Aβl-42 was quantified by measuring the activity of lactate dehydrogenase (LDH) released from damaged cells into the culture medium. Recombinant Aβl-42 exhibited a significant neurotoxic effect in comparison to the synthetic peptide after 72 h (Fisher's LSD, **P=0.003). (b) Induction of apoptosis upon incubation with 7.4 μM monomelic synthetic and recombinant Aβl-42 was evaluated by the TUNEL assay. The apoptotic effect of recombinant Aβl-42 was significantly higher in comparison to the synthetic peptide after 24 h (Fisher's LSD, ***P<0.001). After 72 h, it reached plateau and did not differ from the synthetic peptide. Error bars indicate error propagation.

Fig. 5: Formation of hyperphosphorylated tau intraneuronal inclusions upon stereotactic intracerebral Aβl-42 injections. The mean age (months±SD) at the time of analysis and the number of mice per group are indicated. Horizontal bars indicate the median of each group, (a) Gallyas-positive neurofibrillary tangles (NFTs). Mann- Whitney U test: P=O.009 (two-tailed exact significance) comparing mice injected with recombinant and synthetic Aβl-42. (b) AT-100-positive perikarya. Mann- Whitney U test: P=0.03 (two-tailed exact significance) comparing mice injected with recombinant and synthetic Aβl-42.

Fig. 6: MALDI-MS of purified recombinant Aβl-42 and of synthetic preparations (a, b, compare Fig. 1) and comparison of the aggregation kinetics of different batches of recombinant Aβl-42 (c). (a) Mass spectra of recombinant Aβl-42 wt and arctic, indicated in black and grey, respectively. In addition to the peaks at 4514.1 m/z(wt) and 4442.5 m/z(arctic) caused by the monomelic proteins, the corresponding peaks of dimers and trimers are observed. Their position is indicated by cartoons and black arrows. The arctic mutation is shown by a star, (b) The synthetic preparations exhibit the peaks caused by monomers, dimers and trimers of the proteins, and minor peaks representing impurities. The position of the corresponding peaks are again indicated by cartoons representing Aβ and black arrows, (c) Stirred thioflavin T assays of three different batches of recombinant Aβl-42 show that there are no batch-to-batch variations in the aggregation behavior. The fluorescence intensity is normalized on the intensity at 30 min.

Fig. 7: Aggregation of recombinant and synthetic Aβl-42 preparations at identical concentrations of authentic Aβl-42. Recombinant and synthetic Aβl-42 are indicated in black and grey, respectively, (a) Adjustment of the Aβl-42 concentration in recombinant preparations to the authentic Aβl-42 concentration in synthetic preparations indicated by identical peak areas of the main peak containing authentic Aβl-42 in the RP-HPLC elution profiles. The concentration of recombinant Aβl-42 was reduced to 82.1 %. A blank run is shown by a dotted line, (b) RP-HPLC analysis of the soluble amount of Aβl-42 in stirred aggregation assays at 37 °C in the presence of equal amounts of Aβ. (c) Stirred thioflavin T assay of recombinant Aβl- 42 adjusted to the authentic concentration in the synthetic preparation. The aggregation reaction is not decelerated at the adjusted concentration of recombinant Aβl-42 (compare Fig. 2a).

Fig. 8: Further purification of synthetic Aβl-42 preparations increases the aggregation rate, but does not reconstitute the aggregation propensity of pure recombinant Aβl-42. (a) Synthetic Aβl-42 was further purified by RP-HPLC leading to elution as a single peak (grey line). A blank run without protein is indicated by a dotted line, (b) MALDI-MS of further purified synthetic Aβl-42 reveals that several impurities remain in the preparation. The position of Aβ 1-42 with a calculated molecular mass of 4514.1 g is indicated by a cartoon representing the protein and a black arrow. The peak at 4721.5 m/z represents an adduct with the matrix sinapinic acid, (c) Stirred thioflavin T assay of further purified Aβl-42. The aggregation rate could be increased by further purification, but did not recover that of the recombinant preparation (compare Fig. 2a).

Fig. 9: Aggregation kinetics in neurobasal medium with and without phenol red. Recombinant and synthetic Aβl-42 are indicated in black and grey, respectively, (a) Stirred thioflavin T assays at 37 °C in neurobasal medium, which was applied in cell cultures for toxicity studies in vitro. The dashed line indicates an assay, in which the same medium without phenol red was applied, showing that the aggregation rate is only slightly influenced by the presence of the indicator, (b) Quiescent thioflavin T assays in neurobasal medium performed as in (a).

Fig. 10: Primary neuronal cultures were exposed to vehicle only (a) or different concentrations of monomelic synthetic (b-d) and recombinant Aβl-42 (e-g), and were labeled for MAP-2 (green), cell nuclei (DAPI-blue) and Aβ (red). A clear difference in the cell morphology was observed at 0.74 μM Aβl-42, with the recombinant peptide showing a more detrimental effect on neurite lengths than the synthetic peptide. Additionally, recombinant Aβ aggregates had a plaque-resembling appearance. Scale bar, 50 μm.

Fig. 11: RP-HPLC analysis of the soluble amount of Aβl-42 after pre-aggregation for application in primary neuronal cell cultures (a) and of medium samples from cell cultures incubated with monomericAβl-42 (b). Samples were ultracentrifuged prior to analysis of the supernatant via RP-HPLC. The calculated peak areas correspond to the soluble amounts of Aβ 1-42. Recombinant and synthetic Aβ are indicated in black and grey in b, respectively; the time-points indicate the incubation period of cell cultures with Aβ before the medium samples had been analyzed, (a) Recombinant (R) and synthetic Aβ(S) were pre-aggregated until the time-point when half of the thioflavin T fluorescence amplitude had been observed (1/2 max) and until the time- point when the maximum fluorescence intensity had been obtained (max). + R and + S indicate a positive control run with the maximum Aβ content, (b) Medium samples from cell cultures incubated with aggregation assays that are at the beginning of the reaction.

Fig. 12: Twenty-one days after the stereotactic injections of aggregated Aβl-42 into mouse brains anti-amyloid β-peptide antibody revealed that Aβl-42 peptide was present at the injection sites (a), (b) Gallyas silver impregnation of neurofibrillary tangles (NFTs) (black arrows) and neuropil threads (white arrows) in the amygdale of Aβl- 42-injected mice, (c) Aβl-42 aggregates induced hyperphosphorylation of tauSer212/Thr214 epitope, as detected by ATlOO antibody. Recombinant Aβl-42, in comparison to the synthetic peptide, caused spatial progression of pathology to cortex (d), caudal subiculum (e) and caudal hippocampus (f). Scale bars, 50 μm (a); 100 μm (b); 200 μm (c-f).

Definitions

Unless otherwise stated, a term as used herein is given the definition as provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19 850673 2.

The term "peptide" is understood to include the terms "polypeptide" and "protein" (which, at times, may be used interchangeably herein) within its meaning. Similarly, fragments of proteins and polypeptides are also contemplated and may be referred to herein as "peptides". Nevertheless, the term "peptide" preferably denotes an amino acid polymer including at least 5 contiguous amino acids, preferably at least 10 contiguous amino acids, more preferably at least 15 contiguous amino acids, still more preferably at least 20 contiguous amino acids, and particularly preferred at least 25 contiguous amino acids. In addition, the peptide in accordance with present invention typically has no more than 100 contiguous amino acids, preferably less than 75 contiguous amino acids and more preferably less than 50 contiguous amino acids.

The term "hydrophobic peptide" as used in connection with the fusion proteins in accordance with the present invention relates to hydrophobic polypeptides, proteins or peptides which elute from reverse phase HPLC columns at concentrations between about 20% and about 60%, preferably around about 30% organic solvent in aqueous buffer, e.g. at a concentration of higher than about 30% ethanol in aqueous buffer. Most preferably, the hydrophobic peptide elutes from reverse phase HPLC columns at a concentration of about 28% (Aβl-40) and of about 30.5% (Aβl-42) acetonitrile at conditions as described in the appended Examples. The term "hydrophobic peptide" in accordance with present invention also includes peptides which per se may not be regarded as particularly hydrophobic but nevertheless tend to aggregate or self-aggregate. Preferred hydrophobic peptides are Aβ and variants and fragments thereof.

The term "hydrophilic polypeptide" as used in connection with fusion proteins in accordance with the invention relates to hydrophilic polypeptides which are characterized by their size and the content of hydrophilic amino acids giving rise to a well structured domain. The hydrophilic polypeptide of the fusion proteins in accordance with the invention serves a dual function: (a) to improve the solubility of hydrophobic fusion partners in an aqueous solution, and (b) to expose an enzymatic cleavage site to the enzyme in an aqueous solution. Advantageously, the hydrophilic polypeptide decelerates the spontaneous aggregation of the fusion protein in aqueous solution, in particular in the absence of polypeptide solubilizing denaturing agents. Due to this property, the hydrophilic polypeptide allows the specific enzymatic cleavage of a fusion protein in the desired products through the enzyme in an aqueous solution under non-denaturing conditions. Preferably, the hydrophilic polypeptide is amenable to high level recombinant expression and thus capable of conferring this property the fusion protein, thereby achieving a high yield of the desired hydrophobic peptide as well.

"Recombinant peptides or proteins" refer to peptides or proteins produced by recombinant DNA techniques, i.e., produced from cells, microbial or mammalian, transformed by an exogenous recombinant DNA expression construct encoding the fusion protein including the desired hydrophobic peptide. Proteins or peptides expressed in most bacterial cultures will typically be free of glycan. Proteins or polypeptides expressed in yeast may have a glycosylation pattern different from that expressed in mammalian cells.

"Native" or "naturally occurring" peptides refer to peptides, proteins or peptides thereof recovered from a source occurring in nature. A native peptide or protein would include post- translational modifications, including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, acylation, and cleavage. A wild type (wt) peptide commonly denotes a peptide which occurs in nature and/or which, for example because of being the prominent species among others, has been set as reference.

A "variant" includes peptides having an amino acid sequence sufficiently similar to the amino acid sequence of the natural peptide. The term "sufficiently similar" means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity. For example, amino acid sequences that comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, identical are defined herein as sufficiently similar. Preferably, variants will be sufficiently similar to the amino acid sequence of the preferred peptides of the present invention, in particular to Aβl-42. Such variants generally retain the functional activity of the peptides of the present invention. Variants include peptides that differ in amino acid sequence from the native and wt hydrophobic peptide, respectively, by way of one or more amino acid deletion(s), addition(s), and/or substitution(s). These may be naturally occurring variants as well as artificially designed ones. In particular, variants described in the literature for Aβ are included within the term Aβ such as fragments and Aβ variants involved in inherited forms of Alzheimer's disease, which may differ in their physical and biological characteristics from wt Aβl-42 to some extent, hi addition, hydrophobic peptides are included within the scope of the present invention, which may be a variant of a pathological self-aggregating peptide and which in a mixture are capable of interfering with the formation of fibrils by the wt peptide. Such peptides are most suitable for the development of peptide based therapeutics.

"Similarity" between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide. An amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution. Conservative substitutions include those described in Dayhoff, M.O., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D.C. (1978), and in Argos, EMBO J. 8 (1989), 779-785. For example, amino acids belonging to one of the following groups represent conservative changes or substitutions: -Ala, Pro, GIy, GIn, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr; -VaI, He, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe, Tyr, Trp, His; and -Asp, GIu.

The term "purity of 99%" refers to the degree of purity of the peptide as produced by the method of the present invention and preferably can be determined by analytical RP-HPLC and/or MALDI-MS as described in the Examples. An alternative or additional criterion can be the half-life period of spontaneous formation of aggregates, e.g. fibrillar structures as described for Aβ in Example 2 and shown in Fig. 2a. Generally, the recombinant peptide of the present invention will aggregate faster than its synthetic counterpart. Methods for conducting the quantitatively analytical determination of peptide aggregates are well known in the art, see, e.g., international application WO2003/054548. A peptide preparation may be considered 99% pure in accordance with the present invention if in an aggregation assay with the peptide at a concentration of 7.4 μM in 10 mM H₃PO₄-NaOH pH 7.4, 100 mM NaCl, and 50 μM thioflavin T in a stirred reaction at 500 rpm at 37°C fluorescence reaches a maximum after 25 minutes, preferably after 20 minutes or earlier.

Detailed description of the present invention

The present invention generally relates to a method for the production and purification of hydrophobic peptides making use of the recombinant expression of a fusion protein comprising an enzymatic cleavage site located such so as to release the authentic peptide after cleavage, and purification of the peptide on a hydrophobic matrix column. In one aspect, the present invention concerns a method for the production and purification of a hydrophobic peptide, which method comprises the steps of:

(a) expressing a recombinant nucleic acid molecule encoding a fusion protein of the formula: A-B-C or C-B-A, wherein (i) A is a soluble hydrophilic polypeptide;

(ii) B comprises a protease cleavage site; and (iii) C is a desired hydrophobic peptide;

(b) passing an aqueous solution containing the fusion protein recovered from step (a) through a hydrophobic matrix column; (c) subjecting the fusion protein obtained by step (b) to a solution containing said protease; and (d) purifying the resulting desired hydrophobic peptide through a hydrophobic matrix column. As described in the example, a method for recombinant production of human amyloid-β peptide Aβl-42 in Escherichia coli (E. colϊ) has developed providing highly pure material in milligram quantities, thereby overcoming a methodological bias related to a variety of impurities commonly present in typical preparations of synthetic Aβ). Characterization of the recombinant Aβl-42 revealed surprisingly improved biophysical properties and biological activities compared to synthetic Aβl-42. In particular, aggregation assays with recombinant Aβl-42 reached maximum thioflavin T fluorescence intensity at a threefold faster rate than synthetic Aβl-42. Electron microscopy analysis revealed a more uniform morphology of recombinant Aβ fibrils. The recombinant Aβl-42 was more toxic to cultured rat primary cortical neurons, and it was more toxic in vivo shown by increased induction of abnormal phosphorylation of tau and its related aggregation into neurofibrillary tangles in brains of P301L tau transgenic mice. These results establish significant biophysical and biological differences between synthetically and recombinantly produced Aβl-42, and they validate the use of recombinant Aβl-42 for both in vitro and in vivo studies designed to address mechanisms underlying Aβ aggregation and its related biological consequences for the pathophysiology, therapy and prevention of AD. Thus, while the general applicability of the present invention will be acknowledged, its further illustration has been exemplified for most embodiments with Aβ for the sake of conciseness only.

As described in the Examples, the fusion protein preferably comprises an affinity tag at its N- terminus if the fusion protein follows the formula A-B-C. This embodiment allows the fusion protein to be purified between steps (a) and (b) through affinity chromatography.

Alternatively, the affinity tag may be present at the C-terminus if the fusion protein follows the formula C-B-A. In view of the preferred embodiment of the present invention as illustrated in the Examples fusion proteins of the formula A-B-C are generally preferred in the method of the present invention.

Affinity tags are highly efficient tools for purifying proteins from crude extracts and have been used to facilitate the purification of proteins from, e.g. Escherichia coli, yeast, Drosophila, and HeLa extracts. Affinity tags that may be used in accordance with the present invention include, but are not limited to, His, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C) peptide tags, and the GST and MBP protein fusion tag systems; see for review, e.g., Lichty et al., Protein Expr. Purif. 41 (2005), 98-105. An overview on possible affinity tags affinity tags that may be used in accordance with the present invention is given in table 1 including information on the size and placement, hi this context, the person skilled in the art is well aware of the fact that some affinity tags due to their size and amino acid composition can also serve a hydrophilic polypeptide in the fusion protein according to the present invention; see table 1 below. Thus, in one embodiment fusion partner A already provides the feature of an affinity tag and thus allows the fusion protein to be purified via affinity chromatography.

Alternatively, or in addition, an affinity tag may be fused either directly or indirectly, for example through a peptide linker to the hydrophilic polypeptide, i.e. fusion partner A. For the sake of completeness, typical hydrophilic polypeptides are included in the table 1. In accordance with the Examples of the present invention a His tag is preferably used. The sequence of histidine residues binds selectively to nitrilotriacetic acid nickel chelate resins; see Hochuli and Dobeli, Biol. Chem. Hoppe-Seyler 368 (1987), 748 and European patent application EP 0 253 303, the disclosure contents being herein incorporated by reference.

Table 1 : Selection of affinity tags and hydrophilic peptides for the expression and purification of fusions proteins.

As described in the Examples, preferred hydrophilic polypeptides of the fusion proteins in accordance with the present invention are those with the peptide sequence of the formula (NANP)x, wherein x is 10-40, with 19 (SEQ ID NO: 3) being most preferred.

Suitable proteases and cleavage sites to be used as fusion partner B are known to the person skilled in the art; see for review, e.g., Barrett et al., Handbook of proteolytic enzymes, Academic Press (1998). Table 2 provides some proteases commonly used for tag removal including information about cleavage site, location, residual amino acids, pH range, chaotrope sensitivity, salt sensitivity and enzyme-to-target ratio.

Table 2: Proteases commonly used for tag removal.

Enzyme-

Cleavage Residual Chaotrope Salt

Protease Location pH range to-target amino acids sensitivity sensitivity

In order to identify a suitable protease and cleavage site, respectively, computer aided selection may be used such as the ExPASy PeptideCutter tool; see, e.g., Gasteiger et al., Protein Identification and Analysis Tools on the ExPASy Server; John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005). The enzyme, i.e. protease and its cognate cleavage site are preferably selected such that after cleavage the resultant peptide C is the authentic peptide and not a variant which for example extends at its N-terminus due to the remaining amino acids of the cleavage site of the enzyme. As shown in the Examples, it is also possible to adapt the cleavage site of a given enzyme such that after cleavage the remaining amino acids of the cleavage site at the C-terminal part of the fusion protein, i.e. the N-terminus of the peptide is identical with the amino acid sequence of the native peptide. Thus, in one embodiment of the method of the present invention with the fusion protein being of formula A-B-C fusion partner B comprises at least the N-terminal part of a unique protease cleavage site and the N-terminus of fusion partner C supplements the C-terminal part of said cleavage site where appropriate. An equivalent embodiment may be used for fusion protein being of formula C-B-A.

Preferably, the enzymatic cleavage site B does not interfere with the solubility conferred by the hydrophilic polypeptide A. Most preferably, fusion partner B is hydrophilic as well and thus adds to the solubility of the fusion protein. Hence, in some embodiments, fusion partner B may actually not be "fused" as a separate entity to fusion partners A and C but constituted after fusion of parts A and C.

In a preferred embodiment, said protease is a nuclear inclusion protein a (NIa) protease. Most preferably, the NIa protease of tobacco etch virus (TEV) protease is used, which is the 27 kDa catalytic domain of the NIa protein encoded by TEV. Because its sequence specificity is far more stringent than that of factor Xa, thrombin, or enterokinase, TEV protease is a very useful reagent for cleaving fusion proteins. It is also relatively easy to overproduce and purify large quantities of the enzyme. TEV protease recognizes a linear epitope of the general form E- Xaa-Xaa-Y-Xaa-Q-(G/S), with cleavage occurring between Q and G or Q and S. The most commonly used sequence is ENLYFQG (SEQ ID NO: 4). However, it has been described that high cleavage efficiencies after substitution of the glycine by other proteinogenic amino acids are retained in most cases. Upon substitution by aspartate the efficiency was found to be approximately 90 %. As explained in the Examples, because aspartate is the first amino acid of Aβ the cleavage site has been adapted accordingly. Thus, for the expression of Aβ and other peptides containing an aspartate at their N-terminus said cleavage site in the fusion protein in the method of the present invention preferably consists of the amino acid sequence ENLYFQD (SEQ ID NO: 6) and said NIa protease is derived from tobacco etch virus (TEV). In some embodiments of the method of the present invention the tobacco vein mottling virus (TVMV) protease and its recognition site may be a useful alternative to TEV protease when a recombinant protein happens to contain a sequence that is similar to a TEV protease recognition site; see Nallamsettya et al., in Prot. Expr. Puri. 38 (2004), 108-115.

In a particularly preferred embodiment the present invention makes use of a fusion protein, wherein A is (NANP) ₁₉, B is ENLYFQ(D), C is the hydrophobic peptide, in particular Aβ with having the N-terminal aspartate constituting the C-terminus of the cleavage site B, and having a 6xHis tag at the N-terminus of the fusion protein; see also the Examples. This fusion protein can be made on the basis of the expression vector and the nucleotide and amino acid sequence of the fusion protein shown in Figs. 4 and 5 of US patent no. 5,750,374, the disclosure content of which is incorporated herein by reference. In accordance with the present invention the methionine preceding the Aβ coding sequence identified with number 1 in Fig. 5 of US patent no. 5,750,374 is replaced by part of the recognition sequence of TEV protease, i.e. ENLYFQ (SEQ ID NO: 5), for example by site directed mutagenesis well known in the art. hi a particularly preferred embodiment of the present invention, the fusion protein comprises or consists of the amino acid sequence depicted in SEQ ID NO: 7.

As explained and demonstrated in the appended Examples, the method of the present invention can be performed at conditions which prevent or decrease the aggregation of the fusion protein and the hydrophobic peptide. Furthermore, modifications of reactive amino acids in the hydrophobic peptide, for example methionine are suppressed. This particularly concerns the conditions for the cleavage reaction, i.e. the temperature of the cleavage reaction being preferably below 30°C, more preferably below room temperature and most preferably between 0°C and 15°C, advantageously at about 4°C. Furthermore, the ratio of the substrate fusion protein to the enzyme is preferably at a micromolar concentration in the range of from about 100 : 1 to 100 : 10 and more preferably 100 : 5; see also appended Example 1. After the cleavage reaction aggregates can be sedimented by centrifugation and dissolved in 70% formic acid before being applied to hydrophobic interaction chromatograph; see also the appended Examples.

Hence, the method of the present invention is particularly suitable for producing hydrophobic peptides and peptides prone to self-aggregation in general since the method of the present invention prevents the formation of higher order protein and peptide aggregates. Examples of peptides which may be produced in accordance with the method of the present invention include but are not limited to amyloid islet polypeptide precursor protein, amyloid beta protein or Aβ peptide, serum amyloid A, insulin, amylin, non-amyloid beta component, prions, hemoglobin, immunoglobulins or fragments thereof, β2-microglobulin, α-synuclein, rhodopsin, αl-antichymotrypsin, cystallins, tau, p53, presenilins, low-density lipoprotein receptor, apolipoproteins, superoxide dismutase, neurofilament proteins, transthyretin, procalcitonin or calcitonin, atrial natriuretic factor, gelsolin, cystic fibrosis transmembrane regulator, Huntington's disease protein, fibrinogen alpha-chain, phenylalanine hydroxylase, collagen, beta-hexosaminidase, cystatin C protein, and any hydrophobic and/or self- aggregating fragment of any one thereof.

In a particular preferred embodiment of the present invention the desired hydrophobic peptide is Aβ selected from wild type Aβl-42 or a variant or fragment thereof. For example, variants comprise the arctic mutation E22G and said fragment may be Aβl-40; see also the appended Examples. However, any other variant of Aβ 1-42 and Aβl-40 are included within the scope of the present invention such as disclosed in the art; see Tjernberg et al., J. Biol. Chem. 274 (1999), 12619-12625; Wurth et al., J. MoI. Biol. 319 (2002), 1279-1290; Kim et al., J. Biol. Chem. 41 (2005), 35069-35076; and Lϋhrs et al., PNAS 102 (2005), 17342-17347, in particular in figure 2 and table 2 of the supporting information, the disclosure contents which are incorporated herein by reference in their entirety. Furthermore, Aβ fragments Aβ 16-22, Aβl6-35, and AβlO-35 are described in Ma and Nussinov, PNAS 99 (2002), 14126-14131. In addition, besides the Dutch Aβ variant E22Q the Flemish variant Aβ A21G is a prominent Aβ species. Hence, clinically relevant Aβ variants include particularly mutants at positions 21-23 of Aβl-42 and Aβl-40 (A21G, E22K, E22G, E22Q and D23N). A further clinically relevant Aβ variant has recently been described for an amyloid precursor protein mutation (E693D) in familial Alzheimer' s-type dementia. This mutation produces an Aβ variant lacking glutamate-22 (E22D) which shows enhanced oligomerization but no fibrillization; see Takuma et al., Neuroreport 19 (2008) 615-619 and Tomiyama et al., Ann. Neurol. 63 (2008), 377-387.

To express the fusion protein in a host cell, the nucleic acid molecule encoding the fusion protein may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al., Current Protocols in Molecular Biology (1989); see also the literature cited in the Examples section.

A variety of expression vector/host systems may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculo virus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

To express the fusion protein in a host cell, a procedure such as the following can be used. A restriction fragment containing a DNA sequence that encodes the fusion protein may be cloned into an appropriate recombinant plasmid containing an origin of replication that functions in the host cell and an appropriate selectable marker. The plasmid may include a promoter for inducible expression of the fusion protein (e.g., pTrc (Amann et al, Gene 69 (1988), 301 315) and pETl Id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), 60 89). The recombinant plasmid may be introduced into the host cell by, for example, electroporation and cells containing the recombinant plasmid may be identified by selection for the marker on the plasmid. Expression of the fusion protein may be induced and detected in the host cell using an assay specific for the fusion protein.

A suitable host cell for expression of the fusion protein may be any prokaryotic or eukaryotic cell; e.g., bacterial cells such as E. coli or B. subtilis, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cell (CHO). hi some embodiments, the DNA that encodes the peptide may be optimized for expression in the host cell. For example, the DNA may include codons for one or more amino acids that are predominant in the host cell relative to other codons for the same amino acid. Alternatively, the expression of the fusion protein may be performed by in vitro synthesis of the protein in cell-free extracts which are also particularly suited for the incorporation of modified or unnatural amino acids for functional studies; see also infra. The use of in vitro translation systems can have advantages over in vivo gene expression when the over- expressed product is toxic to the host cell, when the product is insoluble or forms inclusion bodies, or when the protein undergoes rapid proteolytic degradation by intracellular proteases. The most frequently used cell-free translation systems consist of extracts from rabbit reticulocytes, wheat germ and Escherichia coli. All are prepared as crude extracts containing all the macromolecular components (70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, etc.) required for translation of exogenous RNA. To ensure efficient translation, each extract must be supplemented with amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for the E. coli lysate), and other co-factors (Mg , K , etc.). Appropriate transcription/translation systems are commercially available, for example from Promega Corporation, Roche Diagnostics, and Ambion, i.e. Applied Biosystems.

In a preferred embodiment of the present invention, the nucleic acid molecule encoding the fusion protein is operatively linked to the expression control sequences of the T7 promoter and expression of the nucleic acid molecule is carried out in E. coli; see also the appended Examples.

As mentioned, the in vivo or in vitro expression of the recombinant nucleic acid molecule may be performed in the presence of at least one amino acid isotope, for example an isotope amino acid containing ²H, ¹³C and/or ¹⁵N.

The recombinant peptides produced according to this embodiment are particular useful for structural analysis through nuclear magnetic resonance (NMR) spectroscopy and other analytical methods, for example mass spectrometry (MS). The difference of molecular weight between the isotope labeled peptide of the present invention and non-labeled reagents allows researchers to detect and measure the relative amounts of the peptide in complex samples. Due to the potential of isotope reagents for various applications, commercial vendors have introduced the services of synthesis of non-radioactive isotope labeled peptides to research communities. Two major isotopes ¹⁵N and ¹³C are implanted in a specific amino acid of peptides. For example, a Leu amino acid can contain one N¹⁵ and six C¹³; and a peptide containing one such isotope labeled Leu amino acid will have seven units of molecular weight higher than the corresponding peptide without isotope labeled. This kind of peptide can be used in many biological research areas, especially in genomics, as an internal standard for quantification in combination with MS. Stable isotope labeled amino acids and methods for incorporating same into a target protein are also disclosed in US patent no. 7,022,310. Strategies for labeling proteins in vivo in cultured cell through metabolic incorporation of labeled amino acids into a protein are reviewed by Beynon and Pratt, MoI. Cell. Proteomics 4 (2005), 857-872. Uniform isotopic labeling as well as other patterns of isotope incorporation (¹³C and ¹⁵N) in an Aβ variant which may be used in structural studies of amyloid fibrils by solid-state NMR is described in Sharpe et al., Protein Expr. Purif. 42 (2005), 200-210. Dual amino acid-selective and site-directed stable isotope labeling of a protein by cell-free synthesis is described by Yabuki et al., J. Biomolecular NMR 11 (1998), 295-306. An improved cell-free protein synthesis for stable isotope labeling is described by Matsuda et al., J. Biomolecular NMR 37 (2007), 225-229.

Methods of hydrophobic interaction chromatography are well known to the person skilled in the art; see, e.g., Marie-Isabel Aguilar,HPLC of Peptides and Proteins: Methods and Protocols in Methods in Molecular Biology 251 (2003), ISBN 978-0-89603-977-3 (DOI: 10.1385/1- 59259-742-4:9); and Mant et al., Peptide Characterization and Application Protocols in Methods in Molecular Biology 386 (2007), 978-1-58829-550-7 (DOI: 10.1007/978-1-59745- 430-8). Hydrophobic matrix columns include, for example, cyanopropyl, cyclohexyl, phenyl, octyl or octadecyl group bonded silica matrix columns. In one embodiment of the present invention, RP-8 (C8 bound silica microparticle column) under reverse phase (RP) high performance liquid chromatography (HPLC) conditions may be used. Further suitable columns for this purpose include cyanopropyl, cyclohexyl, phenyl or octyl-columns (Bead size from about 5-30 mm). These columns are commercially available from Vydac or Macherey-Nagel under the trademarks VYDAC or NUCLEOSIL. Most preferably, a C8 RP- HPLC such as an A Zorbax 300 SB-C8 column (Agilent Technologies, Inc. USA ) as described in the Examples is used.

Prior to the loading with the fusion protein, the hydrophobic matrix column is conveniently equilibrated with an aqueous buffer. The equilibration buffer may contain a denaturing agent or a chaotropic agent, for example guanidine-HCl, urea or a detergent. The fusion protein in accordance with the present invention may be applied onto a hydrophobic matrix column in aqueous buffer which may also contain a denaturing agent or a detergent, for example guanidine-HCl, urea or TRITON. Preferably, the the fusion protein, for example as present in guanidinium chloride (GdmCl) -HCl when eluted from an afffinity column is applied to the RP-HPLC column in a buffer of aqueous acetonitrile (ACN) containing trifluoroacetic acid (TFA) as described in the Examples.

Elution of the fusion protein may be carried out using a gradient of an aqueous water miscible solvent. Suitable water miscible solvents for this purpose include alkanols, such as n- propanol, 2-propanol, ethanol, methanol, tert-butanol, or cyclic ethers, such as dioxane. Optimal elution conditions depend on desired hydrophobic peptide to be purified, hydrophobic matrix, column dimensions etc., and are conveniently determined on a case-by- case basis. Preferably, the elution buffer is substantially the same as the loading buffer; see also the appended Examples.

Applying the hydrophobic peptide after the cleavage reaction to hydrophobic interaction chromatography and elution of the same are performed substantially as described as descriebd for the fusion protein except that the buffer in which the peptide is dissolved may be diffferent, for example 70% formic acid.

After elution from the hydrophobic column the hydrophobic peptid can be lyophilized and stored. Lyophilized peptides can be solubilized in small volumes of for example 70 % formic acid and aliquoted. LoBind Eppendorf tubes (Vaudaux-Eppendorf) are preferably used for peptide solutions to minimize loss of peptide by adsorbance. The aliquots can be evaporated in a Speed Vac, frozen in liquid nitrogen and stored at -80 °C. For the production of peptide stock solutions, e.g. applicable for aggregation assays, the aliquots can be solubilized in 10 mM NaOH ad 100-200 μM peptide by vortexing, soni cation for 1 min (2 times) and a final vortexing step. The solubilized aliquots can then subjected to ultracentrifugation (135 500 g, 1 h, 4°C).

As demonstrated in the examples, hydrophobic peptides produced and purified in accordance with the method of the present invention are characterized by a high degree of purity, i.e. of at least 99% as determined by analytical RP-HPLC and/or MALDI-MS; see, e.g., appended Example 1 and Figs. Ie, f. Hence, the purity of the recombinant peptide of the present invention is higher than for any corresponding synthetic peptide described so far.

Furthermore, racemate analysis after complete hydrolysis of the purified, synthetic Aβl-42 revealed a content of 1.01% D-histidine, 0.14% D-arginine, and 0.1% D-methionine relative to racemate-free recombinant Aβl-42; see Example 2 and Table 1. Indeed, certain amino acids, in particular cysteine and histidine, show a tendency for conversion to D-amino acids during FMOC peptide synthesis; see, e.g., Veber, D.F. in Peptides: Chemistry, Structure and Biology, Walter and Meienhofer, eds.; Ann Arbor Science: Ann Arbor, Mi (1975); p. 307; and Jones, J. H. in Houben Weyl, Methods of Organic Chemistry, Vol. E22a "Synthesis of Peptides and Peptidomimetics", Goodman et al., eds., Thieme Stuttgart (2002), p. 334. Thus, most if not all synthetic peptide preparations suffer from the fact that they consist of racemic mixtures of peptides which have incorporated D-amino acids, which cannot be separated for the all-L-form by conventional, preparative reversed-phase HPLC. As demonstrated in the Examples, this is particularly true for synthetic Aβ. Thus, in a further aspect the present invention provides and relates to a preparation of hydrophobic peptides, which is pure and racemate-free, i.e. consisting only of the all-L-form of the peptide.

As shown in appended Examples 2 and 3 the lack of impurities of the Aβ peptide of the present invention and its provision as a racemic-free preparation compared to Aβ preparations hitherto available have also a significant influence on the biophysical and biological activity of the peptide. In particular, the peptide of the present invention is capable of forming regular fibril structures in vitro and has plaque-resembling appearance in vivo as illustrated for the Aβ peptide in Figs. 3, 9 and 10, and is more toxic in vivo as demonstrated in Examples 3 and 4, thus making it a more potent and reliable agent in the investigation of the pathogenesis of diseases such as Alzheimer's disease.

Accordingly, the present invention also relates to recombinant peptides produced and purified in accordance with a method of the present invention as described herein, hi particular, the present invention relates to a homogeneous monomelic hydrophobic peptide preparation obtainable by a method of the present invention with a purity of at least 99%. Preferably the petide is recombinant Aβ or a variant or fragment thereof. In a preferred embodiment, the present invention relates to a preparation of a recombinant hydrophobic peptide, prefererably Aβ or a variant or fragment thereof, which in an aggregation assay at a concentration of 7.4 μM in 10 mM H₃PO₄-NaOH pH 7.4, 100 mM NaCl, and 50 μM thioflavin T in a stirred reaction at 500 rpm at 37°C reaches a fluorescence maximum within 25 or preferably 20 minutes. For storage and sale the peptide preparation of the present invention may lyophilized; see also Example 1.

In a further embodiment the recombinant peptide of the present invention may comprise a label (e.g., fluorescent, radioactive, enzyme, nuclear magnetic, heavy metal) and may be used as a peptide probe to detect specific targets in vivo or in vitro including "immunochemistry" like assays in vitro.. The specific label chosen may vary widely, depending upon the analytical technique to be used for analysis including detection of the probe per se and detection of the structural state of the probe. The label may be complexed or covalently bonded at or near the amino or carboxy end of the peptide. One example of indirect coupling is by use of a spacer moiety. In using radioisotopically conjugated peptides of the invention for, e.g., immunotherapy, certain isotopes may be more preferable than others depending on such factors as leukocyte distribution as well as stability and emission. Depending on the autoimmune response, some emitters may be preferable to others. In general, α and β particle emitting radioisotopes are preferred in immunotherapy. Preferred are short range, high energy a emitters such as ²¹²Bi. Examples of radioisotopes which can be bound to the peptides of the invention for therapeutic purposes are ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹²Bi, ²¹²At, ²¹¹Pb, ⁴⁷Sc, ¹⁰⁹Pd and ¹⁸⁸Re. Most preferably, the radiolabel is ⁶⁴Cu. Other therapeutic agents which can be coupled to the peptides of the invention, as well as ex vivo and in vivo therapeutic protocols, are known, or can be easily ascertained, by those of ordinary skill in the art. For example, peptide labeling with a metal isotope or a radioactive halogen isotope is described in international application WO95/022341. hi addition, international application WO2004/013161 describes peptide aggregates that include assembling peptides optionally linked to metal binding moieties and/or target binding moieties as well as using such aggregates for magnetic resonance imaging. Methods of providing labeled Aβ and their use for detecting or monitoring Alzheimer's disease in a patient are also described in international application WO93/04194, which is incorporated herein by reference in its entirety.

In a still further embodiment the present invention relates to a peptide aggregate essentially consisting of one or more recombinant hydrophobic peptides provided herein. Preferably, peptide aggregates of the present invention possess regular fibrillar or pro-fibrillar morphology. Hence, as illustrated in the Examples peptide aggregates according to the present invention are advantageously characterized by their regular structure as illustrated for the Aβ peptide in Fig. 3 in contrast to corresponding Aβ fibrils produced with synthetic peptides which due to impurities contain bad spots in the otherwise regular structure of the individual Aβ peptides within the amyloid fibrils. Accordingly, the peptide aggregates of the present invention are also particularly useful as vaccines since because of their purity they are less immunogenic if at all and furthermore, more properly resemble the three dimensional structure of the natural antigen.

hi this context, the person skilled in the art is well aware of the fact that in the manufacture of vaccines, in particular against endogenous peptides or peptide epitopes the purity of the pathological structure used as the antigen is of particular relevance, since any impurity may result in undesired immune reactions, which can manifested themselves in the form of autoimmune-diseases up to septic shock with even lethal consequences. In particular, for preventive medical treatment but also for therapy the safety of a drug is a key criterion, for their development.

Thus, the present invention also relates to a composition for treating or diagnosing a disease, in particular neurodegenerative, neurological or neuropsychiatric disorder comprising the hydrophobic peptide as described above, and optionally a pharmaceutically acceptable carrier. In case of Aβ, for example, the preparation of vaccines may be reconsidered. Pharmaceutically acceptable carriers and administration routes can be taken from corresponding literature known to the person skilled in the art. The pharmaceutical compositions of the present invention can be formulated according to methods well known in the art; see for example Remington: The Science and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways. Examples include administering a composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods. Aerosol formulations such as nasal spray formulations include purified aqueous or other solutions of the active agent with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucous membranes. Formulations for rectal or vaginal administration may be presented as a suppository with a suitable carrier. Further guidance regarding formulations that are suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985) and corresponding updates. For a brief review of methods for drug delivery see Langer, Science 249 (1990), 1527-1533.

As mentioned, the recombinant Aβl-42 and variants thereof of the present invention hold great promise in the generation of vaccines against AD and other Aβ associated diseases as well as in the use as a means for the detection of the risk of disease, diagnosis of disease, and disease progression and etiology. The same applies to other hydrophobic and self-aggregating peptides. The following is a non-limiting list of diseases associated with specific structural protein/peptide states, followed parenthetically by the involved protein and peptide, respectively: Alzheimer's Disease (APP, Aβ peptide, αl-antichymotrypsin, tau, non-Aβ component, presenilin 1, presenilin 2, apoE); prion diseases, CJD, scrapie, and BSE (PrPSc); ALS (SOD and neurofilament); Pick's disease (Pick body); Parkinson's disease (α-synuclein in Lewy bodies); frontotemporal dementia (tau in fibrils); diabetes type II (amylin); multiple myeloma-plasma cell dyscrasias (IgGL-chain); familial amyloidotic polyneuropathy (transthyretin); medullary carcinoma of thyroid (procalcitonin); chronic renal failure (microglobulin); congestive heart failure (atrial natriuretic factor); senile cardiac and systemic amyloidosis (transthyretin); chronic inflammation (serum amyloid A); atherosclerosis (ApoAl); familial amyloidosis (gelsolin); serum amyloid A; and Huntington's disease (Huntingtin).

In a further aspect, the present invention relates to a method of identifying or obtaining compounds which bind to the hydrophobic peptide of the present invention and preferably interfere with their aggregation. Several strategies have been described in the prior art to detect and monitor, respectively, binding between molecules, and as a consequence detecting inhibition or modulation of said binding, respectively, which may be used in accordance with the present invention. Those strategies comprise for example tagging at least one partner with molecules the properties of which change upon binding such as illuminating molecules, wherein the detected signal might be light emittance such as fluorescence increase or decrease, or gaining additional or loosing former properties upon binding. Those strategies may of course also be used in accordance with the present invention, i.e. to detect and control, respectively, binding or non-binding of the hydrophobic peptide, e.g. Aβ to its interacting molecule. Concerning the screening applications of the present invention relating to the testing of pharmaceutical compounds in drug research, it is generally referred to the standard textbook "In vitro Methods in Pharmaceutical Research", Academic Press, 1997. In general, according to the present invention, the decrease of complex formation compared to performing the method without the test compound or collection of test compounds is indicative for a putative drug. Methods of using peptide probes for the diagnosis and treatment of a variety of diseases associated with conformationally-altered proteins and methods for identifying and deliver drugs useful for treating diseases associated with self- aggregating proteins and peptides, which methods may be adapted and applied in accordance with the recombinant hydrophobic peptide and peptide aggregates of the present invention are described in international application WO2008/013859, the disclosure content of which is incorporated herein by reference.

Thus, the present invention also contemplates screens for small molecules, analogs thereof, as well as screens for natural Aβ interacting molecules such as those that bind to and inhibit aggregation of Aβ in vivo. Suitable test agents for the screening methods may include antibodies, chelating agents, tridentate iron chelators, diketones, 2-pyridoxal isonicontinyl hydrazone analogues, tachypyridine, clioquinol, ribonucleotide reductase inhibitor chelators, 2,3-dihydroxybenzoic acid, Picolinaldehyde, Nicotinaldehyde, 2-Aminopyridine, 3- Aminopyridine, topical 2-furildioxime, n-Butyric acid, Phenylbutyrate, Tributyrin, suberoylanilide hydroxamic acid, 6-cyclohexyl-l-hydroxy-4-methyl-2(lH)-pyridinone, rilopirox, piroctone, benzoic acid-related chelators, salicylic acid, nicotinamide, Clioquniol, heparin sulfate, trimethylamine N-oxide, polyethylene glycol (PEG), copper cations, dimethylsulfoxide, Dexrazoxane, dopamine, tannic acid, triazine, levodopa, pergolide, bromocriptine, selegiline, glucosamine or analogs thereof, tetrapyrroles, nordihydroguaiaretic acid, polyphenols, tetracycline, polyvinylsulfonic acid, 1,3,-propanedisulfonic acid, β-sheet breaker peptide, e.g., 5-amino acid β-sheet breaker peptide (iAβ5p), nicotine, or salts or derivatives thereof to name a few. Furthermore, methods for screening, identifying, and/or quantifying modulators of amyloid and/or aggregates, fibrils or components thereof, in particular modulators of Aβ or Aβ fibrils, employing for example mass spectrometric methods for the screening of an Aβ target against compound libraries, in particular mixtures of compounds or combinatorial libraries are described in international application WO2007/134449, the disclosure content of which is incorporated herein by reference

As discussed above, a number of diseases are associated with self-aggregated proteins, such as insoluble protein aggregates or protein fibrils. For these conditions, peptide vaccines and peptide based imaging with the peptide being produced in accordance with the method of the present invention could be a means for detection of the disease. For example, Aβ binds to oligomer amyloid structures. Thus, detection of the peptide in self-aggregated peptide structures and/or Aβ fibrils could be a target structural state for detection of the disease, while remaining soluble and/or non-aggregated peptide could be a target structural state to confirm absence of the disease, or absence of an advanced stage of the disease. Many of the proteins identified in the preceding paragraph form self-aggregates and/or protein fibrils that are associated with disease states. Other such peptides and proteins include amyloid islet polypeptide precursor protein, serum amyloid A, insulin (e.g., which forms insulin-related amyloid), amylin, non-amyloid beta component, prions, hemoglobin (e.g. sickle cell anemia variant), immunoglobulins or fragments thereof (e.g., IgG L-chain), β2-microglobulin, α- synuclein, rhodopsin, αl- antichymotrypsin, cystallins, tau, p53, presenilins (e.g., presenilin 1 and presenilin 2), low-density lipoprotein receptor, apolipoproteins (e.g., apoA and apo E), superoxide dismutase, neurofilament proteins, transthyretin, procalcitonin or calcitonin, atrial natriuretic factor, gelsolin, cystic fibrosis transmembrane regulator, Huntington's disease protein (i.e., Huntingtin), fibrinogen alpha-chain, phenylalanine hydroxylase, collagen, beta- hexosaminidase, and cystatin C protein. Insoluble peptides and proteins generally exhibit β- sheet formation in the aggregate.

Hence, in further aspect the present invention relates to peptide probes useful for detecting a specific structural state of a target peptide or protein in a sample or in vivo, i.e., useful for detecting protein/peptide in a target structural state. Typically, the peptide probe includes an amino acid sequence substantially identical to the native peptide or corresponding to a region of the target protein which undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, and the peptide probe itself undergoes a conformational shift from an alpha-helical conformation to a beta-sheet conformation, for example because of its pronounced hydrophobicity. As discussed in more detail below, in some embodiments the peptide probes also are useful for identifying therapeutic agents and as therapeutic agents themselves. As noted above, one aspect of the invention provides peptide probes for detecting for detecting protein/peptide aggregates or fibrils in vitro and in vivo. For example, a peptide probe may be labeled such that it fluoresces when the peptide probe is an alpha-helix or random coil conformation (or soluble state), and does not fluoresce when the peptide probe is in a beta-sheet conformation (or insoluble aggregated state). Likewise, a peptide probe may be labeled such that it does not form excimers when the peptide probe is an alpha-helix or random coil conformation (or soluble state), but does form excimers when the peptide probe is in a beta-sheet conformation (or insoluble aggregated state). Exemplary labels include fluorophores such as pyrene, tryptophan, fluorescein, rhodamine, and numerous others known in the art. Alternatively, or in addition the peptide may be labeled by the introduction of isotope-coded affinity tags (ICAT) and the labeled peptide being determined with mass spectrometry (MS).

Traditionally, protein and peptide structures have been determined by a variety of experimental or computational methods described in the art; see, e.g., US patent application US 2006/0057671; US patent no. 6,448,087; Waldo et al., Nat. Biotech. 17 (1999), 691-695; Wurth et al., J. MoI. Biol. 319 (2002), 1279-1290; Kim et al., J. Biol. Chem. 280 (2005), 35069-35076, which are incorporated by reference herein in their entirety. For example, peptide structure may be assessed experimentally by any method capable of producing at least low resolution structures. Such methods currently include X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy.

In the context of the present invention, the native or altered (e.g., after contact with a target peptide or protein) conformation of a peptide probe may be determined by one or more methods such as "Circular dichroism" (CD), Fourier transform infra-red, ultra-violet, NMR, or fluorescence, light scattering, hydrophobicity detection using extrinsic fluors, such as 1- anilino-8-naphthalene sulfonate (ANS) or Congo Red stain, fluorescence resonance energy transfer (FRET), quenching of intrinsic tryptophan fluorescence through either conformational change or monomer or binding at an interface in an α-β heterodimer, equilibrium ultracentrifugation, and size-exclusion chromatography; see, e.g., Physical biochemistry-application to biochemistry and molecular biology, by David Freifelder. W. H. Freeman and Co., San Francisco, 2nd Edition, 1982, for descriptions of these techniques. As noted above, in some embodiments, the probe is modified to comprise labels that are detectable by optical means. Such labels may include tryptophan (an amino acid), pyrene or similar fluorophores, or a fluorescent protein, attached at or near the terminal positions of the peptide probe. Attachment of labels such as fluorophores is achieved according to conventional methods which are well known in the art. Another class of fluorescent probes called quantum dots (QD) may be used as well, which are a class of polymer-encapsulated and bioconjugated probes that can fluoresce at multiple wavelengths spanning the visible spectrum; see, e.g., for review Mansour and Kazmierczak, Clinical Biochemistry 40 (2007), 917-927.

In vivo near-infrared fluorescence imaging is also well known in the art; see, e.g., Frangioni, Current Opinion in Chemical Biology 7 (2003), 626-634. A review of imaging techniques such as ultrasound, CT (Computed Tomography), MRI (Magnetic Resonance Imaging), PET (Positron Emission Tomography), and molecular probes such as quantum dots and nanoshells and of their utility in system biology is given by Kherlopian et al., in BMC Systems Biology 2 (2008) 74 (DOI: 10.1186/1752-0509-2-74).

In in vivo embodiments, a labeled peptide probe is administered to a patient, such as by local injection, allowed to localize at any sites of target protein/peptide or higher order target protein/peptide structures such as a soluble oligomer of the protein/peptide or insoluble self- aggregates of the protein, e.g., insoluble amorphous self-aggregates, protofibrils, and fibrils, present within the patient, and then the patient can be scanned to detect the sites of labeled probe localized at sites of target protein or higher order target protein structures. Other routes of administration also are contemplated, including intranasal and oral. As discussed above, the probe can be labeled with any label suitable for in vivo imaging. The patient can be subject to a full body scan to identify any site of target protein. Alternatively, specific areas of the patient can be scanned to determine whether target protein is localized in the specific areas. Specific areas of interest may include vascular tissue, lymph tissue or brain (including the hippocampus or frontal lobes), or other organs such as the heart, kidney, liver or lungs.

Accordingly, the present invention relates to in vivo imaging techniques employing any one of the peptides of the present invention. For example, the medical imaging technique positron emission tomography (PET) which produces a three-dimensional image of body parts is based on the detection of radiation from the emission of positrons. Typically, a biomolecule is radioactively labeled, e.g. it incorporates a radioactive tracer isotope. Upon administration of the labeled biomolecule to the subject, typically by injection into the blood circulation, the radioactively labeled biomolecule becomes concentrated in tissues of interest. The subject is then placed in the imaging scanner, which detects the emission of positrons, hi one embodiment, a labeled, preferably ⁶⁴Cu labeled peptide, preferably Aβ is administered to a subject and detection of the peptide is performed by placing the subject in an imaging scanner and detecting the emission of positrons, thereby indicating a neurological disorder if for example emission within plaque-resembling structures is detected. The present invention thus encompasses a method for PET imaging, comprising the step of administering a ⁶⁴Cu-labelled or equivalent labeled peptide of the present invention to a subject.

Hence, the present invention generally relates to the use of the the peptide and peptide aggregate of the present invention for the preparation of a composition or kit for the prevention, amelioreation, treatment or diagnosis of a disease, monitoring of the progression or therpapy of a disease, in vitro or in vivo studies aiming at elucidation of the mechanisms underlying a disease, screening of peptide binding compounds, preferably antibodies, or for the screening drugs, preferably drugs interfering with self-aggregation of peptides. Preferably, said peptide is Aβ or avariant or fragment thereof and said disease Alzheimer's disease, hi one embodiment of the present invention, the use involves the detection of said peptide or a peptide aggregate comprising said peptide, for example by MRI, NIR or PET.

For example, in the context of AD, peptide probes can be used to identify oligomer amyloid structures, insoluble aggregates of Aβ, protofibrils and fibrils present in a sample. The ability to identify specific structural forms of Aβ protein offers significant clinical advantages. For example, the presence and load of Aβl-42 peptide and higher order Aβ structures (e.g., ADDLs, protofibrils, and fibrils) can be used to identify a patient at risk for AD or a patient suffering from AD, and/or the extent to which the disease has progressed. The same information also could be used to determine the need for a therapeutic regimen or for a more or less aggressive regimen than currently being used, and to monitor the efficacy of a given therapeutic regimen.

hi one embodiment, peptide probes are used to determine the location of Aβ 1-42 or higher order Aβ structures within the patient. For example, biological samples from specific segments of the brain can be obtained and analyzed for the presence of Aβl-42 or higher order Aβ structures. Alternatively, labeled probes can be administered to the patient, such as by local injection, allowed to localize at any sites of Aβ 1-42 or higher order Aβ structures present within the patient, and then the patient can be scanned to detect the sites of labeled probe localized at sites of Aβl-42 or higher order Aβ structures. Specific sites of interest might include the hippocampus or frontal lobes of the brain. Other sites of interest might include vascular tissue, lymph tissue, and other organs such as the heart, kidney, liver or lungs.

In some embodiments the recombinant hydrophobic peptide of the present invention or aggregates thereof as described above are immobilized on a solid support. This can be achieved by methods known in the art, such as methods comprising exposing a peptide to a solid support for a sufficient amount of time to permit immobilization of the probe to the solid support. The methods may further comprise removing unbound peptide, cross-linking the peptide to the solid support (e.g., chemically and/or by exposure to UV-irradiation), and drying the solid support and peptide. Methods of immobilizing peptides on solid supports are known in the art. In one embodiment, the probes are immobilized in a specific structural state, such as a specific conformation, i.e. as a peptide aggregate, predominantly β-sheet as described in US patent application US2006/0057671, which is incorporated herein by reference in its entirety. In an alternative embodiment, a peptide-based array may be used, which is for example loaded with hydrophobic peptides of the present invention in order to detect autoantibodies which may be present in patients suffering from a disease such as a neurological disorder, in particular Alzheimer's disease. For example, antigen microarray profiling of autoantibodies in rheumatoid arthritis has been reported by Hueber et al., Arthritis Rheum. 52 (2005), 2645-2655. Design of microarray immunoassays is summarized in Kusnezow et al., MoI. Cell Proteomics 5 (2006), 1681-1696.

Accordingly, the present invention also relates to microarrays loaded with hydrophobic peptides of the present invention, in particular Aβ. In one embodiment, the microarray of the present invention may contain different variants of Aβ known to be associated with a neurological disorder, in particular Alzheimer's disease and amyloidosis, respectively.

General methods in molecular and cellular biochemistry useful for diagnostic purposes can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996). Reagents, detection means and kits for diagnostic purposes are available from commercial vendors such as Pharmacia Diagnostics, Amersham, BioRad, Stratagene, Invitrogen, and Sigma- Aldrich as well as from the sources given any one of the references cited herein, in particular patent literature.

The present invention also relates to a kit for use in any one of the methods as described above, i.e. for identifying, isolating, determining and/or using the peptide and peptide aggregate of the present invention, said kits containing specific reagents such as those described hereinbefore, for example a fusion protein of the present invention, a recombinant nucleic acid molecule encoding said fusion protein, an expression vector comprising said nucleic acid molecule, which is operatively linked to an expression control sequence, a host cell comprising said nucleic acid molecule or expression vector, preferably the host cell is E. coli; and optionally a protease, preferably TEV or a corresponding expression host; see also the Examples. The kit may further comprise for example selectable markers, reference samples, microarrays, culture vessels, and maybe some monitoring means. The kit preferably comprises at least one recombinant hydrophobic peptide of the present invention, preferably Aβ, as well as reference molecules for indicating the potential drug efficacy of an added agent, wherein the reagents are preferably kept in single containers. The kit of the present invention is preferably suitable for commercial manufacture and scale and can still further include appropriate standards, positive and negative controls. It preferably further comprises at least one reagent which is selected from the group consisting of reagents that selectively detect the presence or absence of Aβ, for example an anti-Aβ antibody.

Preferably, the kit further comprises means for detecting a level, i.e. a decrease or increase of complex formation between the peptide, e.g., Aβ itself and/or and its at least one interacting molecule or an increased or decreased binding capacity compared to a control by, for example, labels comprising fluorescent label, phosphorescent label, radioactive label, which are known to those skilled in the art. Furthermore, the kit may comprise one or more reagents appropriate to perform a Thioflavin T assay as described in the Examples.

Such kit would further typically comprise a compartmentalized carrier suitable to hold in close confinement at least one container and the compounds of the kit may be sterile, where appropriate. The kit may further include a transfer means, such as pipes for transferring the reagents or cells. In other embodiments, there may be components for application of agents, compounds or compositions to an individual, preferably an animal, such as a syringe, a needle, and so forth. The kit may further comprise components for extracting for example cells from a tissue of interest. Furthermore, instructions can be provided to detail the use of the components of the kit, such as written instructions, video presentations, or instructions in a format that can be opened on a computer, e.g. a diskette or CD-ROM disk. These instructions indicate, for example, how to use the cell, agent, compound, composition and the like to screen test agents of interest. Most preferably, the instructions refer to the use of the kits in the methods concerning the identification and/or isolation of interacting molecules of Aβ or validation or assessment of potential drugs, agents, compositions or compounds influencing, either inhibiting or enhancing said interaction.

Naturally, in view of the fact that the recombinant peptide preparations in accordance with the present invention are substantially devoid of contaminants and more properly reflect the antigenic properties of the peptides in vivo they are also particularly suitable for raising antibodies and detection of antibodies, including auto-antibodies to the endogenous peptide or peptide epitope of a corresponding protein. Accordingly, the present invention also relates to the use of the recombinant hydrophobic peptide of the present invention or an aggregate thereof for the generation and detection of antibodies as well as other peptide binding molecules. Naturally, the present invention also extends to the antibodies and binding molecules so obtained.

As mentioned before, the recombinant peptides and peptide aggregates of the present invention are particularly useful as vaccines since because of their purity they are less immunogenic if at all and the latter properly resemble the three dimensional structure of the natural antigen, i.e. the pathological structure such as of a misfolded protein or of native peptide aggregates. Therefore, in a particular important aspect the present invention relates to vaccines comprising the recombinant peptide or peptide aggregate of the present invention, and optionally a suitable pharmaceutical carrier, preferably for the treatment or prevention of diseases caused by or involving protein misfolding or toxic and aggregated forms of peptides and proteins, respectively. Hence, protein misfolding and aggregation are pathological aspects of numerous neurodegenerative disorders such as those mentioned above with Alzheimer's and Parkinson's disease among others being the most common ones. It was shown that polyclonal antibodies that bind to a toxic oligomeric conformation of β-amyloid that has been implicated in Alzheimer's Disease, also bind to oligomeric structures of α-synuclein and other oligomeric proteins that are involved in Huntington's Disease, Type II diabetes and prion- related diseases. This suggests that those oligomeric forms of proteins share a common structural motif; see, e.g., Kayed et al., Science 300 (2003), 486-489. Thus, it is prudent to assume that a peptide comprising that structural motif and produced in accordance with the method of the present invention or the corresponding peptide aggregates thereof should be useful to evoke and emphasize the production of such auto-antibodies which advantageously would protect the body against several forms of misfolded or aggregated protein/peptide induced disorders. Peptide vaccination approaches have already provided some promising results; see, e.g., the discussion of Aβ immunization in the prevention and treatment of Alzheimer's disease by Holtzman et al. in Adv. Drug Deliv. Rev. 54 (2002), 1603-1613, the report on the vaccination with recombinant mouse prion protein delaying the onset of prion disease in mice by Sigurdsson et al., Am. J. Pathol. 161 (2002), 13-17, and alpha-synuclein immunization in a mouse model of Parkinson's disease described by Masliah et al., Neuron 46 (2005), 857-868.

Vaccine formulation is know to the peson skilled in the art; see, e.g., Vaccine Protocols.2nd Edition by Robinson et al., Humana Press, Totowa, New Jersey, USA, 2003; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems. 2nd Edition by Taylor and Francis. (2006), ISBN: 0-8493-1630-8. Preferably, the pharmaceutically acceptable carrier is KLH, tetanus toxoid, albumin binding protein, bovine serum albumin, or an adjuvant substance described in Singh et al. , Nat. Biotech. 17 (1999), 1075- 1081 (specifically those cited in table 1 of this document) and O'Hagan et al., Nature Reviews, Drug Discovery 2 (9) (2003) , 727- 735 (specifically the innate immune-potentiating compounds and the delivery systems described therein), or mixtures thereof. In addition, the vaccine composition may contain aluminium hydroxyde. The vaccine of the present invention may be administered by any suitable application mode, e.g. i.v., i.p., i.m., intranasal, oral, subcutaneous, etc. and in any suitable delivery device; see, e.g., O'Hagan et al., Nature Reviews, Drug Discovery 2(9) (2003), 727- 735. Typically, the vaccine contains the peptide or petide aggregate according to the present invention in an amount of 0.1 ng to 10 mg, preferably 10 ng to 1 mg, especially 100 ng to 100 μg or, alternatively e.g. 100 fmole to 10 μmole, preferably 10 pmole to 1 μmole, especially 100 pmole to 100 nmole. The vaccine may also comprise typical auxiliary substances, e.g. buffers, stabilizers, etc; see also supra. As discussed therefore, the vaccine of the present invention is especially expected to prove useful in the treatment or prevention of disordes realted to endogenous self-antigens with pathological conformations, in particular in aged subjects with compromised immune response. Such disorders are primarily being associated with neurodegenerative diseases, such as Alzheimer Disease, Down's syndrome, cerebral amyloid angiopathy, mixed dementia, or inclusion body myositis, glaucoma, or arteriosclerosis associated amyloidoses, or other forms of amyloidoses comprising fibrillaric proteins derived from at least one of the following precursor proteins SAA (Serum-Amyloid-Protein A), AL (k or 1-light chains of Immunoglobulins), AH (gl Ig-heavy chains), ATTR (Transthyretin, Serum-Prealbumin), AApo-A-1 (Apolipoprotein Al), AApoA2 (Apolipoprotein A2), AGeI (Gelsolin), ACys (Cystatin C), ALys (Lysozyme), AFib (Fibrinogen), Beta-amyloid (Amyloid precursor protein), Beta-amyloid2M (beta2 -microglobulin), APrP (Prion protein), ACaI (Procalcitonin), AIAPP (islet amyloid polypeptide); APro (Prolactin), AIns (Insulin); AMed (Lactadherin); Aker (Kerato-epithelin); ALac (Lactoferrin), Abri (AbriPP), ADan (ADanPP); or AANP (Atrial natriuretical peptide), or neurodegenerative diseases characterized by the deposition of abnormally aggregated forms of endogenous proteins including but not limited to beta- amyloid in Alzheimer's disease, Down's syndrome, cerebral amyloid angiopathy, hereditary cerebral hemorrhage with amyloidosis, Dutch type and Icelandic type alpha-synuclein in Parkinson's disease, Alzheimer's disease, dementia with Lewy body, multiple system atrophy; Prion protein in Creutzfeldt- Jakob disease and related prion diseases, Huntingtin in Huntington's disease, tau or other neurofibrillary tangle-related proteins in tauopathies including progressive supranuclear palsy (PSP), cortico-basal degeneration (CBD), agyrophilic grain disease (AGD), fronto-temporal dementia (FTD, frontotemporal dementia with Parkinsonism (FTDP 17), Pick bodies in Pick's disease, ataxin in Spinocerebellar ataxia and copper/zinc super oxide dismutase in amyotrophic lateral sclerosis. Thus, the vaccine in accordance with the present invention may be applied to any kind of disease which amelioration has been demonstrated to be associated with the presence of auto-antibodies or other protein binding molecule such as chaperones and the like as well as peptide inducable celluar repsonses against misfolded proteins, protein/peptide aggregates and/or toxic forms of peptides.

These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature concerning any one of the materials, methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries and databases, using for example electronic devices. For example the public database "Medline" may be utilized, which is hosted by the National Center for Biotechnology Information and/or the National Library of Medicine at the National Institutes of Health. Further databases and web addresses, such as those of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL) are known to the person skilled in the art and can also be obtained using internet search engines. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness are given in Berks, TIBTECH 12 (1994), 352-364.

The above disclosure generally describes the present invention. Several documents are cited throughout the text of this specification. Full bibliographic citations may be found at the end of the specification immediately preceding the claims. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application and manufacturer's specifications, instructions, etc) are hereby expressly incorporated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.

A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention.

EXAMPLES

The Examples which follow further illustrate the invention, but should not be construed to limit the scope of the invention in any way. Detailed descriptions of conventional methods, such as those employed herein can be found in the cited literature; see also "The Merck Manual of Diagnosis and Therapy" Seventeenth Ed. ed. by Beers and Berkow (Merck & Co., Inc., 2003).

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.

Methods in molecular genetics and genetic engineering are described generally in the current editions of Molecular Cloning: A Laboratory Manual, (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press); DNA Cloning, Volumes I and II (Glover ed., 1985); Oligonucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1984); Transcription And Translation (Hames and Higgins eds. 1984); Culture Of Animal Cells (Freshney and Alan, Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, eds.); Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Edition (Ausubel et al., eds.); and Recombinant DNA Methodology (Wu, ed., Academic Press). Gene Transfer Vectors For Mammalian Cells (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, VoIs. 154 and 155 (Wu et al., eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods hi Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-FV (Weir and Blackwell, eds., 1986). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, and Clontech. General techniques in cell culture and media collection are outlined in Large Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8 (1997), 148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); and Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251. Material and Methods

Materials

Synthetic Aβl-42 (Lot 1009102, purity 97.1 %, ordered in 2007 and Lot 0568732, purity 98.4 %, ordered in 2005), and N-acetyl tyrosine amide were delivered by Bachem. All other chemicals were commercially available and of highest purity.

Plasmids

Expression of the fusion construct containing Aβl-42 with an N-terminal methionine as cleavage site for BrCN has been described elsewhere⁶. The cleavage site was exchanged by the recognition sequence of TEV protease (ENLYFQ) applying the QuikChange© Kit (Stratagene). The arctic mutation (E22G in the Aβ sequence) was introduced in the same way. The resultant DNA construct for the expression of the fusion protein sequence MRGS(H)OGS(NANP)₁₉RSENLYFQDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGWIA (SEQ ID NO: 7) for wt Aβ and the corresponding fusion protein for the actic mutant were cloned into pRSETA expression vector (Invitrogen, San Diego, CA, USA). The plasmid applied for expression of TEV Protease was pRK793_TEV¹⁹.

Expression and purification of Aβ as a fusion protein

E. coli BL21(DE3) cells bearing the plasmid for expression of the fusion protein were grown at 37 °C overnight in TB medium (12 g/1 tryptone, 24 g/1 yeast extract, 4 g/1 glycerol, 17 mM KH₂PO₄, 72 mM K₂HPO₄, pH 7.2) containing 0.1 mg/ml ampicillin until an OD₆₀₀ of -3.5 was obtained. Protein expression was induced by addition of 1 mM IPTG, growth was continued for 4 h. The cells were harvested by centrifugation (6000 g, 10 min, 4 ⁰C; yield: wt: 5.2 g/1, arctic: 9.2 g/1), and frozen in liquid nitrogen prior to storage at -20 °C.

After resuspension in 6 M GdmCl-NaOH pH 8.0 (10 ml per g of fresh cells), the homogenized cell lysate was stirred at 4 °C for 90 min; the cell debris was removed by ultracentrifugation (149 000 g, 1 h, 4 °C). The soluble fraction of the cell extract was loaded on a Ni²⁺-NTA agarose column (1 ml resin per 0.6 g of processed cells) equilibrated with 6 M GdmCl-NaOH pH 8.0. After washing with 4 column volumes (CV) of 6 M GdmCl-NaOH pH 6.0, the fusion protein was eluted in 3 CV of 6 M GdmCl-HCl pH 2.0. The obtained fractions were stored at -20 °C. The yield could be determined by RP-HPLC analysis (see description below): wt: 137 mg/1 of cell culture, arctic: 114 mg/1. For further purification the fractions were loaded onto a semi-preparative Zorbax SB300 C8 column (Agilent) in 10 % aqueous ACN containing 0.1 % TFA at 80 °C and a flow rate of 1 ml/min. Elution of the fusion protein was performed at an increased flow rate of 4 ml/min and simultaneous increase of the ACN concentration to 30.5 %. The eluting fusion protein was collected and lyophilized, the yields could be calculated via the peak area: wt: 89.1 mg/1 of cell culture, arctic: 98.2 mg/1.

Cleavage of the fusion protein

Cleavage of 100 μM fusion protein with 5 μM TEV protease was performed in 10 mM Tris- HCl pH 8.0, 0.5 mM EDTA, 1 mM DTT at 4 °C overnight. The lyophilized fusion proteins were solubilized in the reaction buffer, and the reaction was initiated by addition of the protease.

Purification of Aβ The cleavage reaction was applied to RP-HPLC purification as described for purification of the fusion proteins. Aggregates in the cleavage assays had been sedimented by centrifugation (4500 g, 10 min, 4 °C) and dissolved in 70 % formic acid or 8 M guanidinium chloride. The eluting Aβ was collected and lyophilized. The yields (calculated via the peak areas of the eluate at 280 nm) were: wt: 21.5 mg/1 of cell culture; arctic: 20.7 mg/1. The corresponding cleavage yields are 77 % and 68 %, respectively.

MALDI-MS

MALDI-MS spectra were measured with sinapinic acid as matrix. The matrix for the analysis of synthetic Aβl-42 from 2005 was HCCA.

Solubilization of Aβ

For the production of an Aβl-42 stock solution applicable for aggregation assays, aliquots of the lyophilized peptide were solubilized in 10 mM NaOH ad 100-200 μM Aβ by vortexing and sonication for 1 min (2 times) and a final vortexing step. The solubilized aliquots were then subjected to ultracentrifugation (135 500 g, 1 h, 4 ⁰C).

The Aβl-42 concentration of the supernatants (pH 12) was determined via the specific absorbance at 280 nm (E₂₈₀= 1730 M^-1Cm^"1), which is determined by the single tyrosine residue in the peptide. This extinction coefficient differs from that at physiological pH (1280 M^-1Cm^"1) due to the deprotonation of the tyrosine side chain (pK_a= 10.46) at pH 12). The solutions were diluted with 10 mM NaOH to 74 μM Aβl-42 and served as stock solutions for aggregation assays. Stock solutions were kept on ice and used within 24 h after solubilization.

Thioflavin T assays Aggregation assays in the presence of thioflavin T were performed in stirred quartz cuvettes (500 rpm or quiescent) at 37 °C. The assays contained 7.4 μM Aβl-42, 10 mM H₃PO₄-NaOH pH 7.4, 100 mM NaCl, and 50 μM thioflavin T. The fluorescence emission at 482 nm was monitored on a PTI fluorescence spectrometer with excitation at 440 nm (slit width: 2 nm). The fluorescence intensity was recorded every 3 min for 30 seconds and averaged. The cuvettes were inverted 5 times prior to recording the thioflavin T fluorescence of 30 seconds.

RP-HPLC analysis

Aggregation assays were performed in the same way as the thioflavin T assays, but without the dye. Samples of 120 μl were removed after different times and subjected to ultracentrifugation (135 500 g, 30 min, 4 °C). 108 μl of the supernatants were mixed with 12 μl of 1 M HCl, 100 μl were injected on an analytical Zorbax 300 SB C8 column (Agilent) in 30.5 % aqueous acetonitrile containing 0.1 % TFA and chromatographed at 80 °C and a flow rate of 1 ml/min.

Electron microscopy

Aggregation assays without thioflavin T were performed as described above. Samples for electron microscopy were removed from the cuvettes, adsorbed to carbon-coated copper grids for 1 min and stained with 2 % uranyl acetate for 30 seconds after washing with water. Electron microscopy was performed at 100 MeV.

Circular dichroism

Far-UV circular dichroism spectra were recorded on a Jasco J-715 polarimeter at 25 ⁰C. 12 spectra were recorded and averaged. Spectra of 74 μM stock solutions of Aβ were recorded in a cuvette with 0.2 mm path length; for 7.4 μM aggregation assays a cuvette with 1 mm path length was applied.

Toxicity assays in cultures of rat primary cortical neurons

Aggregation assays with 74 μM Aβl-42 were performed as described for the 7.4 μM assays without thioflavin T (or with the dye for monitoring the fluorescence) at 10 mM NaCl concentration. The same assay containing no Aβ was applied as control solution. The assays were either directly diluted 1 :10 into cell culture or pre-diluted with control solution for further 1 :10 dilution into cell culture. Dilution of aggregation assays, which are at the beginning of the reaction, into the cell culture was performed by mixing the assay components on ice followed by immediate addition to cell culture. Pre-aggregation at 37 ⁰C in stirred quartz cuvettes at 500 rpm was performed either until the time point where half of the maximum thioflavin T fluorescence amplitude had been achieved (1/2 max; recombinant: 16 min, synthetic: 40 min) or until the time point, where the maximum fluorescence intensity was observed in assays with thioflavin T (max; recombinant: 45 min, synthetic: 3 h). The reactions were stopped by cooling the cuvettes on ice.

Ultracentrifugation of 120 μl of culture medium was performed (107 400 g, 30 min, 4 °C). 108 μl of the supernatant were diluted with 12 μl of 1 M HCl, 100 μl were analyzed via RP- HPLC as described above.

Primary cortical neuron cultures

Primary cortical neuron cultures were prepared from cortices of embryonic day 18 Wister rats. Cortices were dissected out and exposed to 2.4 U/ml Dispase II (Roche Applied Science) for 10 min and titurated to isolate cells. Neurons were plated in Neurobasal media with B-27 and N-2 supplements (GIBCO, Invitrogen) on 24-well plates containing glass coverslips pre- coated with poly-L-ornithine (Sigma- Aldrich) at a density of 100,000 cells per 13 mm coverslip. Cultures were maintained in a humidified 7 % CO₂ incubator.

Aβ treatment, cytotoxicity assays and immunocvtochemistry

Primary cortical neuron cultures were treated with Aβ or vehicle on DIV 5. The influence of two Aβ-related factors on cytotoxicity was analyzed at two time points: after 24 and 72 h of incubation with Aβ or vehicle. First factor was the Aβ aggregation state, as determined by the thioflavin T assay. Three different aggregation states were therefore prepared: (i) monomelic, (ii) ¹A max (see above) (iii) max (see above). Aβ concentrations, the second factor analyzed, were the following: 7.4 μM, 0.74 μM and 74 nM. There were two culture wells on a 24-well plate receiving the same treatment for each time point analyzed.

Cytotoxicity was assessed by three different methods. First, by measuring the activity of the cytosolic enzyme, lactate dehydrogenase (LDH), released into culture medium by damaged cells, using In vitro Toxicology Assay Kit, Lactic Dehydrogenase based (Sigma-Aldrich) according to the manufacturer's instructions. Briefly, half volume of the culture medium was collected from culture wells 24 and 72 h after the Aβ treatment and was incubated with an equal volume of LDH substrate solution for 30 min. The absorbance was measured at 490 run. Second, either the TUNEL assay or immunofluorescence cell morphology study were performed with the same cells fixed with 4 % paraformaldehyde after the removal of the medium for the LDH assay after 24 and 72 h. The TUNEL assay was performed using In Situ Cell Death Detection Kit, Fluorescein (Roche Applied Science) following the manufacturer's instructions. Nuclei were counterstained with 4,6'-diamidino-2-phenylindole-2 HCl (DAPI). Fluorescent microscopy was performed to sequentially visualize DAPI staining and the TUNEL positive apoptotic cells. At least 6 visual fields per culture in duplicate cultures were analyzed per individual experiment. The percentage of apoptotic cells of the total cells (stained with DAPI) was calculated after automated particle counting with the ImageJ software (http://rsb.info.nih.gov/ij/) and used for assessing Aβ effects on cell apoptosis. Alternatively, the morphology study was based on staining neuronal cells with mouse anti- MAP-2 antibody (1 :1000; Sigma), Aβ with anti-amyloid β-peptide antibody (1:500; Zymed, Invitrogen) and nuclear staining with DAPI.

Stereotactic injections of fibrillar Aβ into P301L mouse brains

Aggregation assays without thioflavin T containing 74 μM Aβ were performed as described until the time point when the maximum thioflavin T fluorescence had been observed. Fresh assays were performed for each mouse. Assays containing 10 mM NaOH instead of Aβ stock solution were applied as control.

Ultracentrifugation of a small volume of the applied aggregation assays was performed (107 400 g, 30 min, 4 ⁰C). 6 μl of the supernatant were diluted with 114 μl of 100 mM HCl, 100 μl were analyzed via RP-HPLC as described above.

Animals and surgical procedure

Animal experiments were approved by the veterinary office of the Swiss cantonal Health Department. Thyl.2-tauP301L mice, line pR5-183, carrying human pathogenic P301L tau mutation, bred in our animal care facility, were used in this study. Fifteen 3.7- 5.3 -months old mice were anesthetized with a mixture of fentanyl, midazolam and medetomidine (100 μl/10 g body weight) and stereotactically injected with 1.5 μl of either aggregated Aβ or vehicle into the parietal association cortex of the right hemisphere (coordinates: AP: -1.9 mm from the bregma, LAT -1.0 mm, DV -1.5 mm) with a 10-μl Hamilton syringe driven by a mini pump with an injection speed of 0.15 μl/min. The needle was kept in the injection site for another 10 min and then slowly withdrawn. Operated animals were given the antidote, were treated with postoperative analgetic metacam for three days and monitored daily. One mouse injected with the recombinant Aβ preparation died four days after injection.

Histology

On day 21 the injected mice were perfused transcardially with ice cold PBS, followed by ice cold 4 % paraformaldehyde in PBS. The brain was removed and immersed in fixative overnight at 4 ⁰C and finally equilibrated in a cryoprotectant solution of 30 % sucrose/PBS at 4 ⁰C. Coronal sections (30 μm thick) were cut on a sliding microtome with freezing stage. Antigen detection was conducted on free-floating sections spaced 240 μm apart, from positions AP 1.9 mm to AP -3.5 mm (from the bregma), starting with incubation in a blocking solution (5 % donkey serum/5 % horse serum/0.25 % Triton X-100/PBS) for 1 h at room temperature, followed by primary antibody incubation overnight at 4 ⁰C on a shaking platform. The sections were washed in PBS, and incubated with donkey anti-rabbit Cy2- and donkey anti-mouse Cy3-conjugated secondary antibodies (1 :200 and 1 :250 respectively) for 2 h at room temperature. Immunofluorescence-stained sections were mounted on glass slides subbed in chrome gelatin, and coverslipped with PVA-DABCO coverslipping solution. Primary antibodies for immunostaining included rabbit anti-amyloid β-peptide antibody (1 :300, Zymed, Invitrogen) and ATlOO (1 :300; Pierce; Ser212/Thr214). For Gallyas silver staining the sections were pre-mounted, dried overnight and stained according to the standard protocol, dehydrated in a series of ethanol and xylene incubations, and coverslipped with Eukitt (Electron Microscopy Sciences, Fort Washington, PA).

Statistics All statistical analysis was performed using SPSS 14.0 (SPSS Inc., Chicago, IL). Neurotoxicity effects were analyzed by two-way ANOVA (time point x treatment) followed by Fisher's LSD post-hoc comparisons. Histological scores were analyzed by Mann- Whitney U test comparing recombinant Aβl-42- and synthetic Aβl-42-injected mice. Values were accepted as significant if P < 0.05.

Thioflavin T assays in neurobasal medium

880 μl neurobasal medium with B-27 and N-2 supplements (GIBCO, Invitrogen) were mixed with 20 μl of 2 mM thioflavin T, HCl for neutralization of the Aβ stock solution and NaCl to a final concentration of 10 mM. The assays were performed as the thioflavin T assays described.

Further purification of synthetic Aβl-42 Synthetic Aβl-42 was subjected to RP-HPLC analysis as described above (100 μl of a 50 μM solution per run). The main peak containing Aβl-42 was collected, lyophilized and aliquoted as described for purified recombinant Aβl-42. Aggregation assays were performed as described.

Optical analysis of recombinant and further purified synthetic Aβl-42

Optical analysis via GC-MS was performed by CAT. GmbH & Co. Chromatographie und Analysentechnik KG (Germany). The approximate fraction of synthetic Aβl-42 peptides containing exclusively L-amino acids (L) can be calculated with the obtained D-enantiomer contents (0.1 % D-methionine, 0.14 % D-arginine, 1.01 % D-histidine; see Table 1) and the number of these amino acids in Aβl-42 (1 methionine, 1 arginine, 3 histidine): L=0.9990*0.9986*0.98903=0.9650.

Expression and purification of TEV protease

E. coli BL21(DE3)RIL cells bearing pRK793_TEV were grown in DYT medium (16 g/1 tryptone, 10 g/1 yeast extract, 5 g/1 NaCl) containing 0.1 mg/ml ampicillin and 30 μg/ml chloramphenicol at 30 °C until an OD_6O0 of 0.5 was obtained prior to induction of protein expression with 1 mM IPTG. After 4 h the cells were harvested by centrifugation (2 660 g, 10 min, 4 °C; yield: 7.3 g fresh cells per liter of culture) and resuspended in lysis buffer at 4 °C (50 mM HEPES-NaOH pH 7.5, 1 M NaCl, 10 % (v/v) glycerol, 10 mM imidazol; 3 ml/g of cells), followed by freezing in liquid nitrogen and storage at -20 ⁰C. The cells were lysed by sonication for 10 min on ice; the cell debris was removed by centrifugation (31 200 g, 30 min, 4 ⁰C). All purification steps were performed at 4 °C. The supernatant was loaded on an 18 ml Ni²⁺-NTA superflow column (Qiagen) equilibrated in buffer Bl (50 mM HEPES-NaOH pH 7.5, 800 mM NaCl, 10 % (v/v) glycerol, 10 mM imidazol) at a flow rate of 3 ml/min. After washing with buffer Bl until baseline, the column was washed with 3 CV of buffer Al (Bl with 50 mM NaCl). Elution of the TEV protease was performed by application of 100 % buffer B2 (Al with 400 mM imidazol). The collected protein was subsequently applied on a HiTrap SP HP 5 ml cation exchange column equilibrated in buffer Al at a flow rate of 2-3 ml/min. After washing with buffer Al until baseline, the TEV protease was eluted with a gradient of 0-100 % of buffer Bl for 15 CV. The fractions containing the TEV protease were collected and concentrated to 20 mg/ml in an Amicon Ultra centrifugation tube (MWCO 10 000). Finally the protein was applied on a Superdex 75 26/60 Prep, grade gel filtration column equilibrated in GF buffer (50 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 10 % (v/v) glycerol) at a flow rate of 1-2 ml/min. The pooled fractions containing the TEV protease at a concentration of 2.3 mg/ml were frozen in liquid nitrogen followed by storage at -80 °C. The exact concentration of the protein was determined via absorbance at 280 nm (extinction coefficient at 280 nm: 32 290 M^"1 cm^'1, MW: 28.6 kDa). 26 mg of TEV protease per liter of culture were obtained.

Example 1: Recombinant production of highly pure Aβl-42

A fusion construct was applied with an N-terminal His₆-tag, followed by a soluble fusion partner consisting of 19 repeats of the tetra-peptide NANP⁶ (SEQ ID NO: 3), and an enzymatical cleavage site preceding the Aβ sequence (SEQ ID NO: 1 and 2, respectively) and enabling separation of authentic Aβ from the fusion partner (Fig. Ia). The cleavage site was chosen to be the specific recognition sequence for TEV protease: ENLYPQG (SEQ ID NO: 4). TEV protease cleaves the peptide chain in front of the final glycine, which remains at the N-terminus of the C-terminal fusion partner. It has been described that high cleavage efficiencies after substitution of the glycine by other proteinogenic amino acids are retained in most cases. Upon substitution by aspartate, which is the first amino acid of Aβ, the efficiency was found to be approximately 90 %¹². Therefore, the glycine was deleted and the remaining part of the recognition sequence ENLYFQ (SEQ ID NO: 5) was inserted between the hydrophilic polypeptide and Aβ to obtain authentic Aβ after cleavage. The sequence of Aβ can be modified by point mutations or by deleting codons leading to shorter Aβ variants. It was started with human Aβl-42 as it is considered the key player in initiation of AD. Additionally, it is more amyloidogenic than Aβl-40 and therefore its purification is expected to be more complicated. To verify the applicability of point mutations the wild-type sequence (wt) and Aβl-42 containing the arctic mutation (E22G) were applied, which has been found to cause early-onset familiar AD (FAD) due to its increased amyloidogenicity. The fusion constructs are under the control of a T7 promoter, which enables initiation of protein production by addition of IPTG in the E. coli strain BL21(DE3). Upon expression the protein accumulates in inclusion bodies which can be solubilized by addition of 6 M GdmCl. After purification of the solubilized fusion protein via Ni²⁺-NTA affinity chromatography under denaturing conditions yields were received in the range of 100 mg fusion protein per liter of culture (Fig. Ib). The fusion proteins were further purified via RP-HPLC to obtain highly pure protein in aqueous ACN containing 0.1 % TFA, which can be lyophilized and applied to enzymatic cleavage (Fig. Ic).

Cleavage of 100 μM fusion protein with 5 μM TEV protease was performed at 4 ⁰C overnight. This concentration ratio is critical for the cleavage efficiency. A temperature below the optimum for TEV protease cleavage (30 °C) is applied, because the aggregation of the fusion protein and the generated Aβ is more decelerated at decreased temperature than the activity of the TEV protease, which is described to be retained at 4 °C¹³. Additionally, the risk for modification such as oxidation of methionine is reduced.

After cleavage, the reaction mixture is applied to RP-HPLC (Fig. Id). The highly hydrophobic Aβ can easily be separated from the other components of the cleavage reaction. The yield of Aβl-42 wt and arctic was 21.5 mg and 20.7 mg per liter of culture, respectively. To analyze the purity of the preparations, they were subjected to MALDI-MS (Fig. Ie, see Fig. 6a,b for higher m/z range). The observed masses correspond to the calculated molecular weights of the authentic Aβl-42 variants; practically no impurities or modifications such as the frequently observed oxidation of M35 could be detected. The purity of recombinant Aβl- 42 wt was compared to synthetic preparations, which are commercially available and have been declared to be 97.1 % pure. First, recombinant and synthetic Aβl-42 were analyzed via analytical RP-HPLC (Fig. If). The elution profile of recombinant A/?l-42 shows a single peal- containing the protein, whereas the synthetic peptide contains a variety of impurities causing several minor peaks with similar retention times as authentic Aβl-42. The MALDI-MS spectra of synthetic preparations were measured (Fig. Ig) and again, a variety of impurities with similar masses as the authentic peptide were found, indicated by the presence of minor peaks. Batch to batch differences of synthetic preparations of Aβ have been described. Therefore, the mass spectrum of the synthetic preparation was compared to the spectrum of another batch, which had been measured three years in advance. It was found that the older batch contained even higher amounts of impurities, although it had been declared to be 98.4 % pure.

Example 2: Recombinant Aβl-42 aggregates faster than synthetic Aβl-42

After the production procedure for highly pure recombinant Aβl-42 could be established, it was intended to investigate its aggregation in comparison to commonly used synthetic Aβl- 42. Thioflavin T assays were performed to follow the aggregation kinetics. After an initial lag phase and a phase of rapid growth, the fluorescence maximum was observed in stirred reactions with recombinant Aβl-42 after 25 min and after 75 min with the synthetic preparation (Fig. 2a). Under quiescent conditions the reactions proceed considerably slower, which can be explained by the present shearing force in stirred assays that leads to disruption of fibrils and more free growing ends. Again, recombinant Aβl-42 aggregates faster than the synthetic peptide (Fig. 2b). Thioflavin T assays with different batches of recombinant Aβl-42 showed that there is no batch-to-batch variation considering the aggregation behavior (Fig. Ic). To follow the fibrillization kinetics of Aβ by monitoring the decrease in the soluble Aβ concentration, samples from aggregation assays were applied without thioflavin T to ultracentrifugation and the supernatants were analyzed via RP-HPLC (Fig. 2c,d). The peak area of Aβl-42 in the elution profiles corresponds to the remaining amount of soluble Aβ. The decrease of soluble Aβl-42 is associated with the increase of fluorescence intensity in thioflavin T assays (compare Fig. 2a,b). No remaining soluble Aβ could be detected shortly before the fluorescence maximum in thioflavin T assays was observed. Again, recombinant Aβl-42 aggregates considerably faster than the synthetic peptide and stirred reactions proceed more rapidly than aggregation under quiescent conditions. It has been supposed that only monomers of Aβ with β-structure can enter the pathway of aggregation¹⁴. In many studies carried out in vitro the initial conformation of Aβ is not investigated. To analyze the secondary structure in the stock solutions of Aβ and in aggregation reactions, far-UV circular dichroism spectra was measured. The stock solutions in 10 mM NaOH, which are applied for aggregation studies within 24 h after solubilization, exhibit random coil structure during this period of time (Fig. 2e). Immediately after dilution into aggregation buffer, the random coil structure is preserved, whereas at the end of the reaction β-sheet structure is adopted (Fig. 2f).

The concentration of Aβl-42 is determined by absorbance. As the synthetic preparations contain impurities, the concentration of the authentic peptide is overestimated. The rate of aggregation is supposed to depend on the peptide concentration; consequently the lower concentration of authentic Aβ in synthetic preparations is a potential cause for decelerated aggregation. The concentration of recombinant Aβl-42 was adjusted to the authentic concentration of synthetic Aβl-42 by reducing it to 82.1 % (determined via RP-HPLC analysis), and performed aggregation assays (Fig. 7). The aggregation of recombinant Aβl-42 was not decelerated upon reduction of the concentration, suggesting that the impurities in synthetic preparations decelerate the aggregation reaction rather than the overestimated concentration. To verify this assumption, the synthetic preparation was further purified via RP-HPLC (Fig. 8). Although some impurities remain after this additional purification procedure, the aggregation reaction is clearly accelerated. However, it did not recover the rate of recombinant Aβl-42. To investigate this further, the HPLC-purified, synthetic Aβl-42 peptide was analyzed for racemization, as certain amino acids, in particular cysteine and histidine, show a low tendency for conversion to D-amino acids during FMOC peptide synthesis.

Table 1 : Analysis of optical purity via GC-MS .

Further purified synthetic Aβ1-

Amino acid 42j D-«nantiomer content (%)^a alanins <0.1 valine <0.1 i sole uci ne <0.1 leucine <0.1 serine <0.1 aspartate and asparagine <0.1 methionine 0.1 phenylalanine <0.1 glutamate and glutarruπe <0.1 tyrosine <0.1 lysine <0.1 arginine 0.14 histidine 1.01

Absolute error < H- 0.1 %; analysis according to GMP; reference: recombinant Aβ1-42

As summarized in Table 1 above, the racemate analysis after complete hydrolysis of the purified, synthetic Aβl-42 indeed revealed a content of 1.01% D-histidine, 0.14% D-arginine, and 0.1% D-methionine relative to racemate-free recombinant Aβl-42. As one methionine, one arginine, and three histidine residues are contained in the Aβl-42 sequence, this translates into approximately 3.5 % of the synthetic molecules possessing at least one D-amino acid, which cannot be separated for the all-L-form by conventional, preparative reversed-phase HPLC. These impurities could inhibit fibril growth because incorporation of a peptide with a D-amino acid would either require dissociation form the growing end of a fibril for further fibril growth, or even completely inhibit fibril growth. This explanation is in agreement with a previous study showing that synthetic Aβ variants containing D- instead of L-aspartate show slower aggregation kinetics compared to the synthetic wild type peptide .

Samples were prepared for electron microscopy from stirred and quiescent aggregation assays at the beginning of the reaction, at the time point where the fluorescence intensity maximum in thioflavin T assays had been observed and after incubation overnight (Fig. 3). At the beginning of the reaction networks of protofibril-like structures were observed. At the fluorescence maxima, mature fibrils were present, and after incubation overnight the fibrils seem to form bundles. The fibrils formed under quiescent conditions are longer than those formed in stirred reactions. After incubation overnight the length of fibrils in stirred assays seems to be reduced, whereas in quiescent reactions the long fibrils entangle. Fibrils formed from recombinant Aβl-42 exhibited homogenous morphology. In contrast, preparations with synthetic peptide contain globular particles and the fibrils exhibit a comparably heterogeneous morphology.

Example 3: Recombinant Aβl-42 is more toxic to primary neuronal cultures

LDH release induced by Aβ preparations or vehicle after 24 and 72 h-incubation was quantified (Fig. 4a). Overall, concentrations of 74 nM and 0.74 μM had no effect on LDH release. However, addition of 7.4 μM monomelic recombinant Aβl-42 led to increased LDH release in comparison to monomelic synthetic Aβl-42 after 72h-incubation (Fisher's LSD, P=O.003), that was confirmed with three independent experiments. This led to the emergence of a significant time point x treatment interaction [F(2,30)=3.967, P=O.03], as well as significant main effects of treatment [F(2,30)=6.357, P=0.005] and time point [F(l,30)=20.735, PO.001]. The direct comparison of different aggregation states of 7.4 μM Aβ (Fig. 11 a) in a single experiment, suggested that the monomelic form, compared with the preformed fibrils and the preparation at the half of the maximum thioflavin T fluorescence amplitude, of the recombinant Aβl-42, induced significantly higher LDH release (Fisher's LSD, P=0.001 and P=0.002, respectively).

Activation of apoptosis evaluated by TUNEL assay was significantly higher with Aβl-42 as compared to synthetic Aβl-42 after 24 h (Fisher's LSD, P<0.001). After 72 h, almost all the cells treated with Aβ were TUNEL positive, and therefore, recombinant did not differ from the synthetic peptide (Fig. 4b). The loss of neuronal loss judged by MAP -2 immunostaining, was concentration-dependent and more prominent in cultures incubated with recombinant Aβl-42 . The difference in neuronal loss, cell rounding and neurite retraction was most pronounced at 0.74 μM monomeric Aβl-42. Additionally, there was a detectable difference in the light microscopic pattern of Aβ immunoreactivity, wherein recombinant Aβl-42 had a plaque-resembling appearance, even when added as monomeric to neuronal cultures (Fig. 10). The assessment of neurotoxicity was paralleled by RP-HPLC analysis of soluble Aβ content in the collected medium samples (Fig. 11). The amount of soluble Aβ decreases over time, when monomeric Aβ is added to the cell culture. As expected, soluble recombinant Aβ decreases faster than synthetic Aβ.

Thiofiavin T assays in non-conditioned medium revealed that aggregation of 7.4 μM recombinant and synthetic Aβl-42 proceeds within time ranges of several hours under these conditions. Recombinant Aβl-42 aggregated again faster than the synthetic preparation. The indicator phenol red does not significantly change the rate of aggregation (Fig. 9).

Example 4: Recombinant Aβl-42 accelerates formation of neurofibrillary tangles in

P301L mice

Synthetic Aβl-42 fibrils injected into the brains of P301L mutant tau transgenic mice caused increases in the numbers of neurofibrillary tangles (NFTs) in cell bodies within the amygdala from where neurons project to the injection sites¹⁵. To investigate the effect of recombinant Aβl-42 in this in vivo model, and to compare its effects with the synthetic peptide, the P301L mice were injected with synthetic and recombinant fibrils, and the number of NFTs and Ser212/Thr214 hyperphosphorylated neuronal perykaria in the brains were analyzed 21 days after the injections. The presence of the injected peptide was confirmed by immunohistochemistry (Fig. 12a). The recombinant Aβl-42 fibrils significantly accelerated NFT formation in P301L tau transgenic mice in comparison to synthetic fibrils-injected mice (median: 28 and 18, respectively, Fig. 5a, Fig. 12b). Hyperphosphorylation of the Ser212/Thr214 tau epitope, as detected by ATlOO antibody, was significantly higher in recombinant Aβl-42- than in synthetic Aβ 1-42 -injected mice (median: 39 and 22.5, respectively, Fig. 5b, Fig. 12c).

Conclusions

Despite the high level of acceptance general consensus about the causative role of Aβ in Alzheimer's disease, the aggregation process that is critical for the peptide bioactivity and disease pathogenesis still remains controversial. Previous studies were performed with synthetic Aβ containing highly unnatural impurities that, according to the results obtained in accordance with the present invention, change both the aggregation kinetics and bioactivity properties. The detected features of the synthetic peptide and batch-to-batch variability in impurity content could also explain the irreproducibility and diversity of results obtained within and among different laboratories. Hitherto published methods for recombinant production of Aβ⁴' ^6"11' ²⁰' ²¹ produce modified Aβ (oxidation of methionine, presence of point mutations or persisting presence of fusion proteins), are inefficient and complex or focus on shorter fragments of Aβ. Moreover, the analysis of the product in most cases is confined to the purity via gel electrophoresis and the fibrillization propensity via thioflavin T fluorescence, hi accordance with the present invention a novel strategy for Aβl-42 production in standard E. coli expression system has been developed. The cost-effective production of the peptide on the commercial scale is certainly the first advantage of the method. Second, the obtained material is highly pure, devoid of contaminating peptides and other side reaction products that are, as demonstrated, critical for aggregation kinetics deceleration. Third, the method secures batch-to-batch reproducible conditions and preservation of the peptide in monomelic state that are prerequisite for in vitro Aβ aggregation studies. Furthermore, the production process can be applied for shorter Aβ variants, such as Aβl-40, and variants with point mutations, for instance those known to cause FAD. An important property of the recombinant peptide was its enhanced neurotoxicity, demonstrated in primary neurons and in the P301L tau transgenic mouse model. Under some experimental conditions; synthetic preparations of Aβ 1-42 did not show toxic effects at all in contrast to recombinant Aβl-42. The toxicity of the monomelic preparation, at first sight in discrepancy with the current concept about the pathogenic role of oligomeric assemblies¹⁶, could be explained with the formation of low molecular weight Aβ oligomers during the cell treatment, as detected by immunoblotting. Therefore, the recombinant Aβl-42 oligomers can mediate the detrimental biological effects as the naturally secreted low-n soluble Aβ assemblies¹⁷, ¹⁸.

Together, the recombinant peptide of the present invention provides an indispensable tool for in vitro and in vivo studies aimed at elucidation of the mechanisms underlying AD, as well as for screening of the Aβ targeting compounds. References

I . Hardy, J. & Selkoe, D. J. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297, 353-6 (2002). 2. Finder, V. H. & Glockshuber, R. Amyloid-beta aggregation. Neurodegener Dis 4, 13- 27 (2007).

3. Zagorski, M. G. et al. Methodological and chemical factors affecting amyloid beta peptide amyloidogenicity. Methods Enzymol 309, 189-204 (1999).

4. Dobeli, H. et al. A biotechnological method provides access to aggregation competent monomeric Alzheimer's 1-42 residue amyloid peptide. Biotechnology (N Y) 13, 988-

93 (1995). US Patent Nr. 5,750,374 bzw. EP 0 641 861 → granted

5. Soto, C, Castano, E. M., Kumar, R. A., Beavis, R. C. & Frangione, B. Fibrillogenesis of synthetic amyloid-beta peptides is dependent on their initial secondary structure. Neurosci Lett 200, 105-8 (1995). 6. Luhrs, T. et al. 3D structure of Alzheimer's amyloid-beta(l-42) fibrils. Proc Natl Acad

Sci U S A 102, 17342-7 (2005). 7. Caine, J., Volitakis, I., Cherny, R., Varghese, J. & Macreadie, I. Abeta produced as a fusion to maltose binding protein can be readily purified and stably associates with copper and zinc. Protein Pept Lett 14, 83-6 (2007). 8. Subramanian, S. & Shree, A. N. Expression, purification and characterization of a synthetic gene encoding human amyloid beta (Abetal-42) in Escherichia coli. Indian J

Biochem Biophys 44, 71-5 (2007).

9. Sharpe, S., Yau, W. M. & Tycko, R. Expression and purification of a recombinant peptide from the Alzheimer's beta-amyloid protein for solid-state NMR. Protein Expr Purif 42, 200-10 (2005).

10. Wiesehan, K., Funke, S. A., Fries, M. & Willbold, D. Purification of recombinantly expressed and cytotoxic human amyloid-beta peptide 1-42. J Chromatogr B Analyt Technol Biomed Life Sci 856, 229-33 (2007). WO 02081505

I I . Thapa, A. et al. Purification of inclusion body-forming peptides and proteins in soluble form by fusion to Escherichia coli thermostable proteins. Biotechniques 44,

787-96 (2008).

12. Kapust, R. B., Tozser, J., Copeland, T. D. & Waugh, D. S. The Pl' specificity of tobacco etch virus protease. Biochem Biophys Res Commun 294, 949-55 (2002).

13. Nallamsetty, S. et al. Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro. Protein Expr Purif 38, 108-15 (2004).

14. Lazo, N. D., Grant, M. A., Condron, M. C, Rigby, A. C. & Teplow, D. B. On the nucleation of amyloid beta-protein monomer folding. Protein Sci 14, 1581-96 (2005).

15. Gotz, J., Chen, F., van Dorpe, J. & Nitsch, R. M. Formation of neurofibrillary tangles in P3011 tau transgenic mice induced by Abeta 42 fibrils. Science 293, 1491-5 (2001). 16. Haass, C. & Selkoe, D. J. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nat Rev MoI Cell Biol 8, 101-12 (2007).

17. Lesne, S. et al. A specific amyloid-beta protein assembly in the brain impairs memory. Nature 440, 352-7 (2006).

18. Shankar, G. M. et al. Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med 14, 837-42 (2008).

19. Kapust, R. B. et al. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng 14, 993- 1000 (2001).

20. Zhang, L. et al. Expression, purification, and characterization of recombinant human beta-amyloid42 peptide in Escherichia coli. Protein Expr Purif (2008). 21. Macao, B. et al. Recombinant amyloid beta-peptide production by coexpression with an affibody ligand. BMC Biotechnol 8, 82 (2008).

22. Tomiyama, T. et al. Racemization of Asp23 residue affects the aggregation properties of Alzheimer amyloid beta protein analogues. J Biol Chem 269, 10205-8 (1994).

Claims

1. A method for the production and purification of a hydrophobic peptide, which method comprises the steps of:

(a) expressing a recombinant nucleic acid molecule encoding a fusion protein of the formula: A-B-C or C-B-A, wherein

(i) A is a hydrophilic polypeptide;

(ii) B comprises at least part of a protease cleavage site; and

(iii) C is a desired hydrophobic peptide;

(b) passing an aqueous solution containing the fusion protein recovered from step (a) through a hydrophobic matrix column, preferably an RP-HPLC column;

(c) subjecting the fusion protein obtained by step (b) to a solution containing said protease; and

(d) purifying the resulting desired hydrophobic peptide through a hydrophobic matrix column, preferably an RP-HPLC column.

2. The method of claim 1, wherein A comprises an affinity tag, preferably a HIS-tag, and the fusion protein is purified between steps (a) and (b) through affinity chromatography, preferably through an Ni -NTA agarose column.

3. The method of claim 1 or 2, wherein the hydrophilic polypeptide comprises a peptide sequence of the formula (NANP)x, wherein x is 10-40, preferably wherein x is 19 (SEQ ID NO: 3).

4. The method of any one of claims 1 to 3, wherein said protease is a nuclear inclusion protein a (NIa) protease, preferably wherein said cleavage site consists of the amino acid sequence ENLYFQD (SEQ ID NO: 6) and said NIa protease is derived from tobacco etch virus (TEV).

5. The method of any one of claims 1 to 4, wherein the desired hydrophobic peptide is beta-amyloid peptide (Aβ) selected from wild type Aβ_1-42 (SEQ ID NO: 1) or a variant or fragment thereof, for example wherein said variant comprises the arctic mutation E22G (SEQ ID NO: 2) and said fragment is Abeta^o.

6. The method of any one of claims 1 to 5, wherein the nucleic acid molecule is operatively linked to an expression control sequence, preferably of the T7 promoter, and wherein expression of the nucleic acid molecule is carried out in a host cell, preferably in E. coli.

7. Homogeneous monomelic recombinant hydrophobic peptide preparation with a purity of at least 99% obtainable by a method of any one of claims 1 to 6, preferably wherein said peptide is Aβ or a variant or fragment thereof.

8. The peptide preparation of claim 7, which is a pure, racemate-free preparation.

9. The recombinant hydrophobic peptide of claim 7 or 8, which is detectably labeled, preferably with a the detectable label selected from the group consisting of an enzyme, a radioisotope, a fluorophore, a heavy metal or at least one amino acid isotope, preferably wherein said isotope amino acid contains ²H, C or ¹⁵N.

10. A peptide aggregate comprising or essentially consisting of one or more recombinant hydrophobic peptides of any one of claims 7 to 9, preferably wherein the peptide aggregate possesses a fibril or protofibril-like structure.

11. A composition or kit comprising a recombinant hydrophobic peptide of any one of claims 7 to 9 or a peptide aggregate of claim 10, preferably wherein the composition is a pharmaceutical or diagnostic composition or a vaccine and said kit is diagnostic kit.

12. A kit useful for the preparation of a desired hydrophobic peptide, said kit comprising a recombinant hydrophobic peptide of any one of claims 7 to 9, a peptide aggregate of claim 10, a fusion protein as defined in any one of claims 1 to 6, a recombinant nucleic acid molecule encoding said fusion protein, an expression vector comprising said nucleic acid molecule, which is operatively linked to an expression control sequence, a host cell comprising said nucleic acid molecule or expression vector, preferably wherein the host cell is E. coli; and optionally a protease, preferably a protease as defined in claim 4.

13. Use of a recombinant hydrophobic peptide of any one of claims 7 to 9 or a peptide aggregate of claim 10 for the preparation of a composition, vaccine or kit for the prevention, amelioreation, treatment or diagnosis of a disease caused by toxic, misfolded and/or aggregated forms of a peptide, monitoring of the progression or therpapy of such a disease, in vitro or in vivo studies aiming at elucidation of the mechanisms underlying a disease, screening of peptide binding compounds, preferably antibodies, or for the screening of drugs, preferably drugs interfering with self- aggregation of peptides.

14. The use of claim 13, wherein said peptide is Aβ or a variant or fragment thereof and said disease is Alzheimer's disease.

15. The use of claim 13 or 14, wherein the use involves the detection of said peptide or a peptide aggregate comprising said peptide, preferably wherein detection is performed by MRI, NIR or PET.