JP2003512029A - Methods for altering aggregation of polypeptides - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for designing a modified polypeptide having an altered propensity to aggregate compared to an unmodified polypeptide. Such methods analyze the amino acid sequence of a predetermined polypeptide to determine the propensity of a polypeptide to form a local structure; the modified polypeptide determines the propensity to form a local structure, and the unmodified polypeptide forms a local structure. Determining whether the modified polypeptide has an altered tendency to aggregate in a denatured state relative to the unmodified polypeptide. Thereafter, a selected modified polypeptide having an altered aggregation tendency is generated. The invention also provides a method of producing a modified polypeptide having an altered propensity to aggregate as compared to an unmodified polypeptide, the method comprising: Introducing at least one amino acid modification into the predetermined polypeptide sequence to have an altered propensity to form a local structure at and optionally (ii) recovering the modified polypeptide and / or optionally (iii) )) Causing the modified polypeptide to form an aggregate.

Description

Detailed Description of the Invention

INTRODUCTION The present invention relates to mutant polypeptides with an altered propensity to form aggregates, particularly fibrils, and uses for such polypeptides.

BACKGROUND OF THE INVENTION Amyloid fibrils are highly aligned protein aggregates associated with a range of diseases including Alzheimer's disease, type II diabetes, systemic amyloidosis and infectious spongiform encephalopathy (Tan 1994; Ghetti 1996; Kelly 199
7; Cohen 1998). The fibers can be observed with an electron microscope and have a diameter of 7
To 12 nm, typically about 10 nm, as long unbranched structures. When examined by Fourier Transform Infrared (FT-IR) spectroscopy and X-ray fiber diffraction, amyloid fibrils show a crossed β diffraction pattern, suggesting a high β structure content.

We have previously discovered that proteins unrelated to amyloid disease are found to convert to a fibrillar structure under mildly denaturing conditions in vitro, exhibiting the same characteristics as disease-associated fibrils. It was therefore concluded that the ability to form fibrils is a common property of all polypeptide chains (Guijarro, 1998; Chiti, 1999).
.

The formation of aggregates by proteins, in particular fibrils, can have advantages or disadvantages depending on the role or application of the protein. Aggregation can be achieved, for example, as a plastic, in electronics, for catalysis, or especially when the released protein is pharmaceutically active,
It may be advantageous for use of the aggregate as a sustained release form of the protein.
However, the formation of aggregates is considered inconvenient for a number of reasons, for example as a symptom or cause of amyloid disease, or when used at desired concentrations or conditions for physiological activity, therapeutic administration or industrial applications. To be Therefore, it is highly desirable to modify a protein so that its propensity for aggregation is compatible with its intended role or application.

Aggregation occurs when proteins in their native (globular) state are denatured (breaking intramolecular bonds), exposing their polypeptide backbone and hydrophobic side chains. This provides the opportunity for hydrogen bonds and other interactions to form between denatured protein molecules (formation of intermolecular bonds), leading to the formation of aggregates. Amyloid disease destabilizes the natural state (Chiti, 1999) and thus promotes aggregation (Dobson, 199).
9) Connect to the conditions. In particular, lysozyme (Booth 1997; Canet
1999), immunoglobulin light chain (Hurle, 1994) and transthyretin (McCutchen, 1995) amyloidosis-related mutant proteins have amino acid sequence changes that cause changes in the overall stability of the protein. It is shown.

The activity and regulation of wild-type proteins may depend on the stability of the wild-type native state. Proteins have evolved to work optimally under physiological conditions. Under such conditions, the native state is only marginally stable as compared to the denatured state, thus the wild-type protein
It exists in dynamic equilibrium as a population of proteins with native, partially denatured and fully denatured states (Dobson, 1999). Proteins with altered natural stability can disrupt this equilibrium and thus exhibit abnormal activity or regulation relative to the wild-type protein.

DISCLOSURE OF THE INVENTION In such a background, the inventor has surprisingly discovered that the aggregation tendency of a polypeptide can be modified without substantially changing the natural state global folding. This is a modified polypeptide with the dual advantages of (i) tailored aggregation (as appropriate for the intended role or application) and (ii) wild-type-like native state stability, activity and regulation. Means that can be generated.

The present invention thus provides a method of designing a modified polypeptide with a desired altered propensity to aggregate as compared to an unmodified polypeptide, which method comprises analyzing a predetermined amino acid sequence of the polypeptide. Determining the propensity of the polypeptide to form the local structure; comparing the propensity of the modified polypeptide to form the local structure with the propensity of the unmodified polypeptide to form the local structure; thereby the modified polypeptide to the unmodified polypeptide Determining whether the modified polypeptide has an altered propensity to aggregate in the denatured state as compared to, and producing a selected modified polypeptide having the desired altered propensity to aggregate.

The invention also provides a method of producing a modified polypeptide having an altered propensity to aggregate as compared to an unmodified polypeptide, which method comprises: (i) comparing the modified polypeptide to an unmodified polypeptide; At least one amino acid modification into the predetermined polypeptide sequence such that it has an altered propensity to form local structures in the denatured state, and optionally (ii) recovering the modified polypeptide and / or optionally (Iii) allowing the modified polypeptide to form aggregates.

The present invention also provides a method of producing a polypeptide having an altered propensity to aggregate, particularly an altered propensity to form non-covalent aggregates, which method comprises: Has an altered propensity to form a local structure as compared to a peptide, and (ii) exhibits a gross native fold whose native state is substantially equal to that of the unmodified polypeptide, and optionally (iii) the modified polypeptide And / or optionally (iv) introducing amino acid sequence modifications into the modified polypeptide so that the modified polypeptide forms aggregates.

The invention also provides the following: a method of producing a polynucleotide encoding a polypeptide of the invention, its complement, or a fragment of any sequence thereof, comprising: (a) Modifying the polynucleotide encoding the unmodified polypeptide such that the modified nucleotide sequence encodes the modified polypeptide of the invention, or (b) synthesizing the polynucleotide encoding the modified polypeptide of the invention. A method that includes one of.

A method for producing a vector for use in expressing a polypeptide of the invention or cloning a polynucleotide of the invention, comprising inserting the polynucleotide of the invention into the vector.

-A method of producing a cell, cell culture or multicellular organism comprising, or transformed with, a polynucleotide of the invention or a vector of the invention, comprising: (a) Transforming cells with a polynucleotide or vector of the invention,
And optionally (b) culturing the cells obtained by (a) under conditions that allow selective growth of the transformants, and optionally (c) conditions suitable for producing a multicellular organism. A method comprising culturing the cells obtained according to (a) or (b) below.

A method of producing a polypeptide of the invention, comprising: a) maintaining the cells, culture or organism of the invention under conditions suitable for producing the polypeptide of the invention, and A method comprising either isolating the polypeptide or b) chemically synthesizing the polypeptide of the invention.

A method for the production of aggregates, which comprises contacting the polypeptides of the invention under conditions which allow nucleation.

-A polypeptide, polynucleotide, vector, cell, cell culture, organism or aggregate of the invention.

To alter the aggregation tendency of the polypeptide such that the denatured state of the polypeptide has an altered propensity to form a local structure compared to the unmodified polypeptide, and the native state of the unmodified polypeptide is Use of a modification of a polypeptide amino acid sequence to have a global native fold substantially equivalent to that of the case.

-A polypeptide of the invention, a polynucleotide of the invention, a vector of the invention, a cell, a cell culture or organism of the invention, or an aggregate of the invention, and a pharmaceutical composition comprising a pharmaceutically acceptable carrier. .

A polypeptide of the invention, a polynucleotide of the invention, a vector of the invention, a cell of the invention, for use in a method of treatment or diagnosis of the human or animal body.
A cell culture or organism, or an aggregate of the invention.

-A polypeptide of the invention, a polynucleotide of the invention, a vector of the invention, a cell of the invention, a cell culture or organism, or of the invention in the manufacture of a medicament for use in the treatment of amyloid or related diseases. Use of agglomerates of.

From an aggregate of the invention for use in a method of treatment or diagnosis of the human or animal body, wherein the release rate of the polypeptide from the aggregate is free of the polypeptide of the invention. Aggregates that are altered as compared to the release rate of the unmodified polypeptide of.

-The use of the aggregates of the invention as an in vitro sustained release form of a polypeptide of the invention, wherein the release rate of the polypeptide from the aggregate does not comprise the polypeptide of the invention. Use of aggregates that are altered relative to the release rate of the unmodified polypeptide of.

-A method of treating a disease in a patient, comprising administering an agent of the invention.

[0024]   -A polypeptide or aggregate obtainable by the method of the invention.

A method for assessing the tendency of a polypeptide to form aggregates in a denatured state, the method comprising:
Analyzing the amino acid sequence of a polypeptide to determine the propensity of a polypeptide to form a local structure; compare the propensity of the polypeptide to form a local structure with the propensity of a predetermined polypeptide sequence to form a local structure And, thereby, determining whether the polypeptide has an altered propensity to aggregate in a denatured state as compared to a predetermined polypeptide.

A method of designing a polypeptide with a desired aggregation tendency, which comprises analyzing the amino acid sequence of the polypeptide to determine the propensity of the polypeptide to form local structures; introducing modifications to the amino acid sequence. And assessing the effect of the modification on the propensity of the polypeptide to form local structures; and thereby designing the modified polypeptide with the desired propensity for aggregate formation.

To an amyloid or agglutination-related disorder, comprising identifying the amino acid sequence of the polypeptide expressed in the individual associated with the amyloid or agglutinative disorder and assessing the propensity for aggregation of the polypeptide according to the methods of the invention. A method of assessing individual risk or sensitivity.

(Brief Description of Drawings) FIG. -Heat denaturation analysis of WT-ADA2h and M1, M2 and DM variants at pH 3.0. (A) WT heating (white circle), WT cooling (white square), DM heating and cooling (black circle, heating and cooling data overlap). The WT protein was monitored at 214 nm and the DM variant was monitored at 222 nm. (B-e) 25 ° C (white circle), 95
Far-UV CD spectra of WT-ADA2h and the mutant at 25 ° C. (white square) and after cooling at 25 ° C. (black circle). (B) WT; (c) DM; (d) M1; (e) M2. (F
) FTI of WT-ADA2h sample before heat denaturation (solid line) and after heat denaturation (dashed line)
Amide I band in the R spectrum. The second derivative of the spectrum is 1622 and 1649 cm ⁻¹ for the sample before heating and 1615 and 168 after heating.
A peak centered at 5 cm ^-1 is shown. The band at 1615 cm ^-1 suggests β-strand, while the band at 1685 cm ^-1 is generally amide I due to antiparallel interchain interactions.
It leads to division within the band (Fabian, 1993a; Krimm and Ban
dekar, 1986).

Residual helix content was calculated from CD at 95 ° C. as described in “Experimental Materials and Methods” and the following results were obtained: WT retained 45% of the native helix structure (
Spectra were collected in ultra-diluted samples that were subjected to very rapid temperature changes to prevent aggregation during data collection), M1 63%, M2 61%, and DM 6
Hold 8%. Therefore, the residual helical structure at 95 ° C for DM is about 50% more than the amount retained by WT.

FIG. -Urea reconstitution of WT-ADA2h and DM mutants at pH 3.0. (A)
Far-UV CD spectrum of WT-ADA2h. (B) Far-UV C of DM-ADA2h
D spectrum. Circles indicate samples prepared under initial conditions. Squares represent the polypeptide after incubating a sample of 4M urea and then diluting in 1M urea.

FIG. AGAG carried out over the entire sequence of PI3-SH3 to identify regions of the protein with a higher endogenous propensity to adopt a helical conformation
IR prediction.

FIG. -PI3-SH3 sequence. Secondary structural elements (s = strand, 3 = 310 helix) are shown after structural analysis by the DSSP package. Also shown is a region with an intrinsic helical propensity of over 2% (according to AGADIR prediction).

FIG. -AGADIR predictions representing the intrinsic helical propensity exhibited by wild type and E17R / D23R mutant sequences.

FIG. -Human PrP helix C residue 198 assessed using AGADIR-
227 Prediction of helical propensity. Wild type sequence and mutant peptide E200K, R
Includes those corresponding to 208H and V210I.

FIG. 7. -Far-UV CD spectra of four helix C peptides at pH 7.0. The buffer solutions were sodium phosphate 20 mM and β-mercaptoethanol 20 mM.

FIG. 8. -TFE titration of four helix C peptides monitored by measuring ellipticity at -222 nm. In each case, the buffer was 20 mM sodium phosphate, 20 mM β-mercaptoethanol.

FIG. 9. -Far-UV CD spectrum of helix C peptide at pH 2.0. The buffer was 20 mM phosphate, equilibrated at pH 2.0 with the addition of HCl and 20 mM TCEP. A) Peptide concentration 20 μM. B) Peptide concentration 60 μM.

FIG. -Far-UV CD spectrum of helix C peptide at pH 2.0 after 2 months incubation at room temperature. The peptide concentration was 60 μM.

FIG. 11. -ThT fluorescence (A), Congo red absorbance (B) and far-UV CD (
Time course of wild-type AcP aggregation monitored by C). Under conditions that promote aggregation (25% vol / vol TFE, 50 mM acetate buffer, pH 5.5, 2
(5 ° C) AcP was incubated and aliquots were taken at regular time intervals as described in the "Experimental Materials and Methods" section. The three approaches yield very similar velocity trajectories, and their analysis gives the same velocity values within experimental error.

FIG. 12. -Time course of ThT fluorescence increase upon aggregation for some representative mutants. The mutants presented are I75V (black circles) (panel A), V20A (white circles) and Y98Q (black circles) (panel B), E29D (white circles) and A30G (black circles) (panel C). Throughout the data the solid line is the best fit to a single exponential function. The best fit to the wild type data is represented by the dashed line in each panel and is shown for comparison. The experimental conditions are as described in FIG.

[0041]   Figure 13. -A bar showing changes in aggregation rate resulting from mutations of residues at various positions.
rough. Each bar represents a variant with an amino acid substitution at the position shown on the x-axis. y
The velocity data reported on the axis are for the mutant and for the wild-type protein.
Expressed as the natural logarithm of the ratio between the speeds of. A value of zero indicates that the mutant is wild type
It means to aggregate at the same rate as the protein. Values above and below zero are
It means the rate of aggregation of mutants that is faster and slower than the wild-type protein, respectively. Each
The error bars shown in the drawing for the mutant of * are * [2δv_mut)^Two+ (2δv _wt )^Two], Where δv_mutAnd δv_wtAre 1n (v _mut ) And 1n (v_wt) Standard error (standard error measures standard deviation)
Defined as the square root of the number divided by the mean). This operation is 1n (ν_mut/
ν_wtAs there is a 95% confidence that the actual value of) falls within the experimental error reported,
v_mutAnd v_wtIt is possible to widen the experimental error of (Taylor, 198).
2). Therefore, values with experimental error ranges higher or lower than zero are relative to the wild type.
Represents a significant rate change in the mutant.

(Detailed Description of the Invention) The present invention analyzes the amino acid sequence of a polypeptide to determine the propensity of a polypeptide to form a local structure, and predetermines the propensity of the polypeptide to form a local structure. A polypeptide forming an aggregate, comprising comparing the polypeptide with a propensity to form a local structure, thereby determining whether the polypeptide has an altered propensity to aggregate as compared to a predetermined polypeptide. On how to assess the tendency of. The present invention therefore relates to a method of predicting the propensity of a variant polypeptide to aggregate, particularly when denatured or partially denatured, as compared to a wild-type polypeptide.

The method of the present invention is used in the rational design of polypeptides with altered aggregation propensity, for example to design and produce modified polypeptides with higher or lower aggregation propensity as compared to unmodified proteins. be able to. The method of the present invention can also be used to compare the aggregation propensity of the first and second polypeptides based on their propensity to form local structures, and the production propensity based on the aggregation propensity selected. It can be used to select one or the second polypeptide. The modifications in the polypeptide that are the subject of design are described below.

A. Polypeptides of the Invention The invention provides modified polypeptides that have an altered propensity for aggregation. As used herein, polypeptide preferably relates to any polypeptide molecule and fragments thereof of at least 41 amino acids in length. Preferably the polypeptide fragment is at least 45 amino acids in length, more preferably at least 50, 70, 9
It is 0, 100 or more amino acids long. Even more preferably, the fragment has at most 20, 10 or 5 amino acids deleted from the original sequence. Most preferably, the polypeptide fragment retains the biological activity of the protein from which it is derived. In this regard, retention of biological activity refers to that of a binding protein in which the fragment exhibits substantially the same desired properties as the wild-type protein, e.g., a fragment of the enzyme typically retains substantially the same enzymatic activity. The fragments typically retain substantially the same binding specificity,
And that fragments of the antigenic protein typically retain substantially the same antigenic properties. Any polypeptide can be modified. Typically, the polypeptide will occur naturally, but may be non-unnatural in sequence, such as those found in mutant organisms or when recombinantly produced by transgenic or in vitro techniques. . Alternatively, the polypeptide may be synthetic, for example produced entirely by chemical processes.

B. Modifications The present invention is particularly directed to methods of producing polypeptides with altered propensity to aggregate, particularly those that tend to form non-covalent or non-crosslinked aggregates. Aggregates can be amorphous / non-specific aggregates such as fibers or inclusions. In a preferred aspect, the present invention provides methods that include analysis of the secondary structure of a polypeptide aimed at identifying modifications that have an altered propensity for aggregation. Therefore, the identification of desirable modifications as described below can form part of the design process of the present invention. In particular, such a method comprises, as a first step, analysis of the secondary structure of the polypeptide and identification of modifications that influence the propensity of the polypeptide to form local structures, and then production of the polypeptide thus modified. Can be included.

Modifications made to the polypeptides of the present invention result in changes in the primary structure, ie, the sequence of amino acids within the polypeptide backbone. Modifications result in altered or partially denatured forms of the polypeptide having an altered propensity to form local structures, particularly alpha-helical structures, or to form intermolecular interactions, as compared to the unmodified polypeptide. The inference about this change is that disruption of the polypeptide's native state is essential for the formation of aggregates, but once denaturation occurs, the tendency for aggregation is an intrinsic property of the denaturation state, namely denaturation. Or the tendency to bind to other modified polypeptides. Therefore, the propensity of a polypeptide to aggregate can be altered by altering the propensity of the denatured or partially denatured state to form intramolecular rather than intermolecular bonds. The inventor changes intramolecular or intermolecular bonds by changing the formation of an α-helical structure by changing the formation of a local structure, particularly compared with the tendency of forming a β-pleated sheet structure in a denatured state. It has been shown that the balance of propensity to form or the balance of interactions such as hydrophobic or electrostatic interactions can be altered, and this leads to an altered propensity to form fibril-like aggregates .

Accordingly, the modifications of the present invention, in the denatured or partially denatured state, alter primary sequences with altered propensity to form regions of local secondary structure, such as α-helix and / or β-pleated sheet secondary structure. Yields a polypeptide with. In one embodiment, a given modification results in a polypeptide that has a higher propensity to form α-helical secondary structure and thus a lower propensity to form aggregates (such as fibrils) as compared to an unmodified polypeptide. Occurs. In another embodiment, the modifications provided are relative to the unmodified peptide
It results in a polypeptide that has a low propensity to form α-helical secondary structure and thus a high propensity to form aggregates (like fibrils). Therefore, the polypeptide of the present invention may be the polypeptide obtained by this method of modification. Furthermore, the aggregate obtained from the polypeptide of the present invention may be the aggregate of the present invention.

Preferably, the aggregates formed according to the invention, or those with a low tendency to be formed by the polypeptides of the invention, are non-covalent or non-crosslinked aggregates. In particular, covalent aggregates may be formed by disulfide bridges formed between two cysteine residues present within the polypeptide monomer. Polypeptides may contain intramolecular disulfide cysteine bonds, although it is possible to create such disulfide bonds during the production of the protein or after denaturation of the protein. The present invention relates to aggregates that are predominantly non-covalent aggregates, that is, aggregates that are not necessarily formed through disulfide bonds, but through taking a high β structure content.

In some embodiments, it will be particularly desirable to reduce the tendency of the polypeptide of interest to form aggregates in the denatured or partially denatured state.

B1. Selection of Amino Acid Residues for Modification The choice of amino acid residue for modification depends on the desired outcome of the modification, and the method used by one of skill in the art to select the residue to be changed in a given situation You will be familiar with.

If it is desired to reduce the tendency of the polypeptide to form fibril-like aggregates, the denatured polypeptide has an α-helical secondary structure, either a native-like or a non-natural-like α-helical structure. The amino acid sequence should be modified to increase its propensity to form. This can be achieved in two mutually compatible ways. First, we identify the regions that form the α-helix secondary structure in the natural state (the method for identifying the α-helix secondary structure is described below), It can be modified to include. Second, regions that do not form α-helix secondary structure in the native state can be identified and modified to include amino acids that enhance the propensity of the α-helix forming denatured state.

Substitution preferably results in a mutation. However, the mutation may also be by other means, for example, a deletion of one or more amino acids, such as 1, 2, 3 or 4 amino acids, a transposition, and / or 1, 2, 3 or 4 It can also be generated by the insertion of one or more amino acids, such as amino acids. As highlighted above, preferably the altered tendency of the polypeptide to form aggregates is an altered tendency to form non-covalent aggregates. Thus, in a preferred aspect of the present invention, modifications to the amino acid sequence do not include modifications of cysteine residues involved in the formation of disulfide bonds that lead to the formation of covalent aggregates. Preferably, the amino acid residue selected for modification is not a cysteine residue. In one aspect of the invention, the substitutions or modifications do not include the removal of negative charges such as the conservative substitution of Asp to Asn or Glu to Gln.

As outlined below, amino acid modifications preferably lead to a polypeptide exhibiting a gross native folding or structural topology that is substantially equivalent to that of the unmodified polypeptide. Preferably, the modified polypeptide exhibits an overall stability in the native state that is substantially equal to the overall stability of the unmodified polypeptide in the native state.

Thus, it is preferred that the structural characteristics similar to those exhibited by the wild-type protein be retained in the mutant polypeptide. Such structural features may include the number of secondary structural elements and / or their position in the protein sequence, the content of secondary structure or the amount of each secondary structural element in its native state, and the final tertiary structure of the polypeptide. Includes original tertiary structure (ie, the arrangement in space of secondary structure elements and their interactions).

The degree to which these structural features are retained in the mutant polypeptides of the present invention can be assessed by standard biophysical and / or biochemical techniques. In particular, one or more of the following measuring methods can be implemented. In order to analyze the secondary structure of the full-length protein, several combinations of the following assay methods such as 2, 3, 4, 5 are used.
Or a combination of 6 is used: (1) Circular dichroism spectra in the near-ultraviolet region and the far-ultraviolet region in the presence and absence of a chemical modifier and / or at various temperatures; ) Fluorescent signals from aromatic residues in the presence and absence of chemical denaturants and / or at different temperatures, and fluorescent signals originating from specific probes; (3) Fourier Transform Red of native and denatured proteins. External spectrum; (4) One-dimensional and / or multi-dimensional nuclear magnetic resonance; (5) X-ray diffraction analysis; (6) Analytical ultracentrifugation to determine the size of native protein, and / or size exclusion chromatography. , And / or light scattering and / or NMR pulse field gradient experiments.

Furthermore, analysis of native polypeptides and fragments of polypeptides of the invention can be carried out using the techniques described above.

Preferably, the modified polypeptide retains at least one of its biological functions, such as enzymatic activity, ligand binding or other biological activity, intact. However, the substitutions need not necessarily be conservative modifications and / or charge substitutions. Amino acid substitutions are those that affect the propensity to secondary structure of the polypeptide sequence. For example, when the change occurs at a position three-dimensionally distant from the active site of the protein (
That is, non-conservative changes, such as substitutions, will not destroy the properties of the polypeptide if the region selected for mutation in the design process is separated from the active site). On the other hand, if the design of the mutant requires changes in regions of the protein that are close to or directly associated with the active site, then conservative changes may be considered more appropriate, and preferably non-conservative. You should try first before considering any changes.

The design of mutations that enhance local interactions such as α-helix propensity can be carried out as follows: (i) The target site for mutagenesis is preferably such that the side chain is within the α-helix. Those in the α-helix that do not interact with residues that are not included and that are in sufficient solvent contact that are not necessary for activity are selected. Its purpose is to ensure that the protein packing is maintained, that the mutation does not eliminate mid- or long-range interactions, and that the activity of the protein is retained. It is preferable to select residues that are sufficiently solvent accessible for mutation.

(Ii) Mutations are designed to increase alpha helix propensity as dictated by helix / coil transition algorithms adapted to heteropolypeptides such as AGADIR (Munoz and Serrano, 1997; M).
unoz and Serrano, 1994; Munoz, 1994a; Munoz,
1994b). The algorithm is modified to predict stability for a particular sequence in the alpha helix conformation rather than helix content for peptides. The template sequence used is typically an X-ray α on each side to prevent end effects.
Corresponds to helix plus approximately 4 residues, eg 4, 5 or 6. Sequences that differ in residues at the target position for mutagenesis can be evaluated by the program. Those skilled in the art will recognize that mutations that reduce local interactions such as α-helix propensity can also be designed by this method by selecting residues whose side chains interact with residues not included in the α-helix. Will do. Similar techniques apply to other local structures such as β
It can also be applied to pleated sheets.

If it is desired to increase the tendency of the polypeptide to form fibril-like aggregates, the amino acid sequence should be modified to reduce the propensity of the modified polypeptide to form α-helical secondary structure. is there. This may be accomplished by selecting regions that naturally form α-helix secondary structure and modifying them to contain amino acids that reduce the propensity of the α-helix to reform the α-helix.

Similarly, the propensity of the polypeptide to form local structures relative to the unmodified polypeptide is altered, leading to an altered propensity to aggregate, particularly an altered propensity to form non-covalent aggregates, and intermolecular interactions. Modifications to the wild type amino acid sequence could be made that alter the formation of In particular, as Roseman, 1998 stated,
One or more amino acid residues can be replaced with another residue of low hydrophobicity. Alternatively, the substitution may lead to a reduced intrinsic propensity to form a β structure, as defined for example by the method of Chou and Fasman, 1978 or Street and Mayo, 1999. Such substitutions reduce the tendency of the polypeptide to form aggregates. On the other hand, substitutions that increase hydrophobicity or increase the propensity to form β structures increase the propensity of polypeptides to form aggregates, particularly non-covalent aggregates. The technique for predicting the propensity to form a β-sheet structure can also be implemented using computer-run algorithm modeling.

The selection of target sites for mutagenesis can be performed, for example, by identifying regions of the polypeptide that have an endogenous helical propensity. For example, non-helical polypeptides could be examined using appropriate modeling software such as AGADIR to identify sequences with an endogenous helical propensity. Mutagenesis can be used to increase the endogenous helical propensity and thus reduce the aggregation propensity, even if the helical propensity to form is low.

Alternatively, a set of mutants is prepared for the selected polypeptides,
Make conservative substitutions. The rate of aggregation can then be monitored to identify regions within the polypeptide that play a more important role in aggregation. Appropriate mutations within these regions can then be identified to reduce their propensity to form hydrophobic or alpha helix structures, eg, to reduce their propensity to form non-covalent aggregates. .

In an alternative aspect of the invention, a sudden increase in hydrophobicity, a decrease in the tendency to form α-helices, or a tendency to form a β-sheet structure in order to increase aggregate formation. Carry out mutation B2. Prediction of Secondary Structure Techniques for predicting regions of secondary structure, such as α-helical secondary structure, from the amino acid sequence of a polypeptide are known in the art, typically AGADIR (
Munoz and Serrano, 1997) and Zimm-Bragg and Lift.
Modeling with a computer-run algorithm such as son-Roig formalism is used. These approaches are becoming increasingly reliable, as the understanding of α-helical secondary structure is virtually complete (Munoz and Serrano, 1997).

The mutations or modifications of the present invention are selected such that their fold, structural topology and preferably overall stability in nature are substantially equal to the unmodified polypeptide. That is, for example, the native folding of the polypeptide, particularly the formation of α-helices or β-sheets associated with the native polypeptide, is substantially unchanged compared to the unmodified polypeptide. Thus, for example, mutant polypeptides can be produced that retain at least one of the biological activities of the protein and are subjected to normal folding. In particular, the modified polypeptide will retain the biological function of the wild-type protein, such as enzymatic activity, ligand binding or antigen production. Furthermore, the preferably folded mutant or modified polypeptide retains essentially the same overall stability as the unmodified polypeptide, such that the modified polypeptide does not exhibit a greater tendency for denaturation as compared to the unmodified polypeptide. To do. Preferably, the modified polypeptide exhibits an altered propensity to form aggregates under denaturation. Therefore, when the polypeptides of the present invention are subjected to denaturing conditions, the tendency for aggregation is preferably altered compared to wild type. In a preferred embodiment, the modified polypeptides according to the present invention have a reduced tendency to aggregate compared to wild type.

C. Further Modification of the Amino Acid Sequence The amino acid sequence of the polypeptide can be further modified for any reason. Typically the modification will aid purification by the addition of a histidine residue or a T7 label, or will aid in identification or purification, or will be removed from the cell if the polypeptide does not naturally contain such a sequence due to the addition of a signal sequence. It is believed to help promote their secretion of. Sequence modifications may change the activity of the polypeptide, for example by changing the amino acids that contribute to the activity of the active site, the ligand binding site or the regulatory region. Polypeptides can be modified by fusion with other amino acid sequences. The fusion may thus result in additional amino acid sequences in one or both terminal regions of the polypeptide, ie the amino-terminal or carboxy-terminal regions, or in the internal region of the polypeptide. The polypeptide is
It can be modified additionally or alternatively by deletion. Deletions may remove sequences from one or both terminal regions of the polypeptide, i.e. from the amino-terminal or carboxy-terminal regions, or may remove sequences from one or more internal regions of the polypeptide. Modifications by deletions and additions can therefore encompass substitutions in which any number of amino acid residues can be replaced by a different amino acid, either individually or as contiguous sequences. Addition and / or deletion modifications typically include two or more amino acids, eg, one amino acid, typically 5, 10, 20, 50, 100 or more amino acids. .

D. Generation of Mutant Polypeptides Bearing Modified Amino Acid Sequences Polypeptides designed to include the changes as defined above can be generated by any method of polypeptide synthesis known in the art.
Typically, the polypeptides of the present invention are produced by chemical synthesis or by recombinant in vitro or in vivo expression.

Polypeptides can be chemically synthesized using a variety of solid phase techniques (eg, Roberge et al., 1995), and can also be used, eg, ABI 431 A Peptide.
Automated synthesis may also be performed using the Synthesizer (Perkin Elmer).

Recombinant in vivo or in vitro production of a polypeptide can be accomplished by expression of a polynucleotide containing the sequence encoding the polypeptide of the present invention.

D. 1. Polynucleotides A further embodiment of the invention provides a polynucleotide comprising a sequence encoding a polypeptide of the invention. Polynucleotide sequences can be designed in the context of the preferred codon-usage for the particular organism in which the polynucleotide is expressed, with reference to the degeneracy of the genetic code. The polynucleotide is
It may be DNA or RNA containing the polynucleotide of the present invention and its complement, and may be either single-stranded or double-stranded. Thus, they basically consist of DNA or RNA encoding the amino acid sequence of the invention.

The polynucleotide may include synthetic or modified nucleotides therein. Many different types of modifications to polynucleotides are known in the art. It is to be understood that for the purposes of the present invention, the polynucleotides described herein may be modified by any method available in the art. Such modifications are
For example, it may be carried out to change the in vivo activity or longevity of the polynucleotide of the present invention.

The polynucleotides of the invention are used to make primers, eg for use in PCR (polymerase chain reaction) or alternative amplification reactions (eg to facilitate amplification or site-directed mutagenesis). it can. Such primers and other fragments are at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length and as used herein are also encompassed within the term polynucleotide of the invention. To be done.

Polynucleotides such as DNA polynucleotides and primers according to the present invention and their unmodified forms can be produced recombinantly, synthetically, or by any means available to those of skill in the art. They can also be cloned by standard techniques. That is, the polynucleotide can be cloned into any vector available in the art. The polynucleotide is typically provided in isolated and / or purified form.

In general, short polynucleotides of the invention, eg, primers, are produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence, one nucleotide at a time. Methods for doing this using automated techniques are readily available in the art.

Longer polynucleotides of the present invention and in unmodified form can be produced by combining short polynucleotides by standard techniques, such as ligation. They can also be produced using recombinant means, such as PCR cloning techniques. This involves making a pair of primers (eg, about 15-30 nucleotides) to the region of the gene to be cloned and contacting such primers with the target polynucleotide. For unmodified forms, the target polynucleotide used is typically genomic DNA (typically to allow cloning of the entire gene, including introns and promoter regions) or mRNA or cDNA prepared therefrom. Obtained from cells in morphology. For the production of the polynucleotide of the present invention, a small amount of polynucleotide produced by any means can be used as a target polynucleotide in a PCR amplification reaction. Amplification is carried out under suitable conditions to bring about selective amplification.
After amplification of the desired region, the amplified fragments can be separated (eg, by purifying the reaction mixture on an agarose gel) and recovered. Primers can be designed to contain appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into an appropriate cloning vector.

The techniques described herein are generally well known in the art, but in particular Sambro
See ok et al., 1989.

The polynucleotides of the present invention are obtained by site-directed mutagenesis of polynucleotides containing unmodified sequences. This technique can be implemented in any way. Typically such a procedure can be performed by PCR. Such an approach typically involves the addition of a change in a nucleotide residue suitable to produce the desired change,
Includes the use of primers containing nucleotide sequences that are nearly identical to the regions of unmodified sequence for which mutations are desired. This primer is then used in a PCR amplification reaction to introduce the desired change in the nucleotide sequence of the PCR product. The desired site for site-directed mutagenesis is typically within the coding sequence of the gene, and thus PCR
The product can be a truncated form of the target polynucleotide. Full-length modified polynucleotides of the invention can be produced by combining the amplification product with other polynucleotides that have unmodified or modified sequences (produced by some method). The combination can be performed by any method known in the art, for example, ligation. This approach may also be useful if, for example, silent codon changes are required to optimize codon preference for the particular host cell in which the polynucleotide sequence is being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to further alter or modify the properties or functions of the polypeptide encoded by the polynucleotide.

The modified polynucleotides produced can be tested for the desired sequence by their sequencing. This is done, for example, by contacting a sample containing the putative modified polynucleotide with a probe containing the polynucleotide or primer of the present invention as a target under hybridization conditions and sequenced by, for example, the Sanger dideoxy chain termination method (see Sambrook et al., 1989). Can be carried out by determining

Such methods generally extend a primer by the synthesis of a strand complementary to the target polynucleotide in the presence of a suitable reagent, which may include one or more of the A, C, G or T / U residues. This includes causing the chain elongation and the termination reaction to selectively stop the elongation reaction, separating the elongated products by size, and determining the sequence of nucleotides at which the selective termination has occurred. Suitable reagents include DNA polymerase enzymes, deoxynucleotides dATP, dCTP, dGTP and dTTP, buffers and ATP. Dideoxynucleotides are used for selective termination.

D. 2. Vectors The polynucleotides of the invention can be incorporated into any vector available in the art. The vector of the invention therefore consists essentially of the polynucleotide of the invention. Usually the vector is a recombinant, replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Therefore, in a further embodiment, the invention provides for the introduction of a polynucleotide of the invention into a replicable vector, introducing such vector into a compatible host cell, and growing the host cell under conditions which result in replication of the vector. , Providing methods of making the polynucleotides of the invention. The vector can be recovered from the host cell. Suitable host cells are described below in connection with expression vectors.

D. 2.1. Expression Vector Preferably, the polynucleotide of the invention in the vector is operably linked to control sequences capable of effecting the expression of the coding sequence by the host cell, ie the vector is an expression vector. Such expression vectors can be used to express the polypeptides of the invention.

The phrase “operably linked” refers to the proximal relationships in which the components being described are capable of functioning in their intended manner. A control sequence "operably linked" to the coding sequence is ligated so that expression of the coding sequence occurs under conditions compatible with the control sequences.

Such a vector can be transformed into a suitable host cell as described above to effect expression of the polypeptide or polypeptide fragment of the invention. Accordingly, in a further aspect the present invention provides a process for preparing a polypeptide or polypeptide fragment according to the invention, said process under conditions for providing expression of the polypeptide or fragment as described above. Culturing a host cell transformed or transfected with such an expression vector and recovering the expressed polypeptide or fragment.

The vector can be, for example, a plasmid, virus or phage vector, with an origin of replication, optionally a promoter for the expression of the polynucleotide of interest, and optionally a regulator of such promoter. The vector may include one or more selectable marker genes, such as the ampicillin resistance gene for bacterial plasmids, the neomycin resistance gene for mammalian vectors, or the kanamycin resistance gene for plant vectors. The vector can be used in vitro, for example for the production of RNA or for transfecting or transforming host cells. Vectors can also be adapted for use in vitro, for example in methods of gene therapy.

A further embodiment of the invention provides a host cell transformed or transfected with a vector for replication and expression of a polynucleotide of the invention.
Such cells have been selected to be compatible with the vector, such as bacteria, yeast,
It can be a plant, insect, or mammal.

D. 3. Expression in Host Cells The expression vector of the present invention can be used for calcium phosphate precipitation, DEAE-dextran transfection, electroporation, particle bombardment or Ag.
It can be introduced into host cells using the R. bacterium tumefaciens mediated method. Expression from the host cell can be transient. Stable host cell transformation may be achieved by integrating the polynucleotide of the present invention or a fragment thereof into the host cell genome. Typically the transformed genome is nuclear,
Transformation of other genomes may also be desirable, such as the mitochondrial genome of eukaryotic cells or the plastid genome of plant cells. Instead, a stable transformation can be achieved using an autonomously replicating vector. The expression vector can include a selectable marker and / or such a selectable marker can be cotransformed with the expression vector to select for stable transformed cells.

Suitable cells include cells in which the vectors described above can be expressed. These are E.
microbial cells such as bacteria such as E. coli, CHO cells, COS7 cells, P388 cells, mammalian cells such as HepG2 cells, KB cells, EL4 cells or Hela cells, insect cells, yeast or plant cells such as Saccharomyces , Typically containing cells of crop plants such as wheat, corn or rapeseed. Baculovirus or vaccinia expression systems may also be used.

Cell culture is performed under standard conditions. Commercially available media for cell culture are widely available and can be used according to the manufacturer's instructions.

D. 4. Recovery The polypeptides and aggregates of the invention expressed in host cells can be recovered by any technique known in the art. This can lead to the separation and purification of the polypeptide or aggregate. In some cases, polypeptides may aggregate within host cells. Aggregates typically lyse host cells, isolate the aggregates by centrifugation, wash the isolated aggregates, and optionally dissociate the aggregates to denature individual polypeptides. Can be separated by. Typically the isolated or purified polypeptide is at least 10% to 100%, more preferably at least 40% or 50%, more preferably at least 60% or 70% of the dry mass of the polypeptide present in the sample, Even more preferably at least 8
It accounts for 0%, 90%, and 95%. Most preferably, the purified polypeptide comprises at least 99% of the dry mass of polypeptide present in the sample.

E. Aggregates Aggregates of the invention may include non-naturally occurring polypeptides. The polypeptide is
For example, a glycosylated polypeptide or a polypeptide containing a functional group such as a modified amino acid residue, a pharmaceutically active compound, a metal or a thiol group capable of binding to one or more reactants. , Can be a chemically modified polypeptide.

Preferably the aggregates formed are fibrils. The fibers of the present invention are preferably long and / or linear and / or unbranched. Most preferably they are long, linear and unbranched. The diameter of the fibers is generally 1 to 20 nm, preferably 5 to 15 nm, more preferably 7 to 12 nm. Fiber diameter can be varied by selecting the appropriate polypeptide.

The fibers of the present invention can include hollow cores that are considered useful in a variety of applications.

E. 1. Aggregate Generation The aggregates of the present invention may be generated by any method known in the art.
Aggregation typically occurs by nucleation. Nucleation occurs when intermolecular bonds are formed between polypeptides in partially or fully denatured states. The process of nucleation can thus occur in any situation by contacting the polypeptide under suitable conditions. Suitable conditions for nucleation are any conditions capable of producing a partially or fully denatured polypeptide molecule, but such conditions promote denaturation to the extent that intermolecular bonds are prevented from forming. must not. Optimal nucleation conditions are different for each polypeptide. Important parameters for nucleation typically include variations in solvent, polypeptide concentration, salt, ligand, temperature and pH. One of ordinary skill in the art would be able to determine the appropriate conditions for a given polypeptide. Typically nucleation is preferably at a 3-6-7M concentration,
For example, by incubating in urea at 4-6M, preferably about 4-5M, preferably for at least 1 hour, then preferably diluting to at most 1M, and preferably followed by further incubation for at least 1 hour. it can.

In some cases, it may be advantageous to change the optimal nucleation conditions for a given polypeptide. For example, where aggregation is inconvenient, it may be desirable to produce a polypeptide that is more resistant to aggregate formation. Therefore, in order to overcome unfavorable aggregation, it is possible to use the polypeptides of the invention which have a lower propensity to form local structures, preferably α-helical secondary structures, as compared to polypeptides bearing unmodified sequences. . The polypeptides of the invention may be produced under any conditions, such as to improve the rate of formation of aggregates such as fibrils, or to allow production of unmodified polypeptides under conditions unsuitable for nucleation. It can be adapted to enhance aggregation.

E. 2. Aggregate formation measurements Aggregate formation must be measured to confirm optimal nucleation and aggregate formation conditions, or to determine if amino acid sequence modifications had the expected effects on aggregation propensity. . Aggregate formation, such as fibril formation, can be measured by any technique known in the art. Typically used techniques are circular dichroism, sedimentation analysis, thioflavin T and Congo red binding assays, polypeptidase resistance assays, Fourier transform infrared spectroscopy, electron microscopy and X, as exemplified below. Includes line diffraction.

F. Uses and Examples of Polypeptides of the Invention F. 1. Uses of the Polypeptides of the Invention The polypeptides of the invention may be used in any application. Typically, it will be used in medical and industrial applications where aggregate formation, such as fibrosis, is convenient or inconvenient.

F. 1.1 Medical and Industrial Uses of the Polypeptides of the Invention Polypeptides made for use in medical or industrial applications typically experience a range of conditions. During the separation, modification, storage, administration or use, in the reaction vessel (for synthetic method) or intracellular, extracellular or in vitro (for recombinant expression method)
The conditions experienced by the engineered polypeptides, such as those in, may promote unfavorable nucleation, formation of aggregates such as fibrils, or may be inappropriate for desired aggregate formation.

The polypeptides of the present invention can therefore be used to overcome the adverse production of aggregates such as fibrils. For example, the polypeptides of the present invention may sometimes occur during synthetic or recombinant production, or may be necessary for therapeutic benefit or industrial applicability,
It can be used to prevent aggregation at high polypeptide concentrations. The polypeptides of the invention are used in conditions where the unmodified polypeptide cannot be used, such as conditions where solvent selection, temperature, pH, or salinity promote undesired aggregate formation of the polypeptide bearing the unmodified sequence. You can. Any polypeptide can be used in this way.
Examples of pharmaceutically useful polypeptides include calcitonin, insulin and cytokinins. For example, the polypeptides of the invention having a low tendency to aggregate could be used in industrial processes such as powder detergents, or in pharmaceutical or diagnostic processes.

The polypeptides of the present invention may also be used in applications where nucleation and aggregation may be beneficial. For example, fibril-like aggregates produced by recombinantly expressed polypeptides can form inclusion bodies, which are typically in the form of polypeptides that can be conveniently separated by centrifugation. Polypeptides of the invention having a higher propensity to form fibril-like aggregates in intracellular, extracellular or in vitro conditions can be produced, thereby facilitating separation. Aggregates have many potential applications, but their formation can be a slow process. The polypeptides of the present invention can be used to improve the rate of formation of fibril-like aggregates, thereby increasing the efficiency of production. For example, it may be advantageous to attach a polypeptide of the invention to a molecule, eg, a pharmaceutically active molecule, for use in delivery delivery of a drug. In one embodiment of this, it may be desirable for the modified polypeptides to form fibril-like aggregates, eg for use as a sustained release mechanism (see F.1.2 below). Polypeptides of unmodified sequence can form aggregates only under harsh conditions that are undesirable for the binding molecule.
Therefore, the polypeptides of the invention will render the binding molecule substantially unaffected,
That is, it can be adapted to aggregate under suitable mild conditions so that the molecule can perform its required function upon release, thus providing a suitable vehicle for the binding molecule.

F. 1.2 Use of Fibers as a Sustained Release Mechanism Once formed, fiber-like aggregates can dissociate to release polypeptide molecules. This can be particularly useful, for example, if such aggregates can be introduced as a sustained release mechanism. This is considered to be useful when the released polypeptide is useful for some application, such as itself as a therapeutic agent. Such polypeptides include pharmaceutically or therapeutically active polypeptides such as hormones, vaccines, polypeptides suitable for the treatment of cancer or involved in blood coagulation or fibrinolytic events, and in industrial processes such as biological powders. It may include polypeptides useful in detergents and the like.

Aggregate component polypeptides are also believed to be useful as "vehicles" or "carriers" for sustained release mechanisms of other molecules. For example, a molecule that binds to a polypeptide within an aggregate can be inactive until the polypeptide molecule to which it binds dissociates from the aggregate. After release of the polypeptide from the aggregate, the molecule can remain bound to the released polypeptide or the binding can be broken by passive or active means. Molecules that can be attached to the polypeptides of the invention by covalent or some other type of linkage can be used. The molecule is considered to be useful for any application such as a medicament for use in the treatment of the human or animal body, or for use in the manufacture of a medicament for use in treating a disease such as cancer.

F. 1.5 Antibodies The present invention also provides monoclonal or polyclonal antibodies specific for the polypeptides and / or aggregates of the present invention. Typically, they are able to distinguish between modified and unmodified polypeptides of the invention. Such monoclonal antibodies can be prepared by conventional hybridoma technology using the polypeptides of the invention as an immunogen. Polyclonal antibodies can also be prepared by conventional means, including inoculating a host animal, such as a rat or rabbit, with a polypeptide of the invention and collecting immune serum. A polypeptide can be haptenized against another polypeptide for use as an immunogen in an animal or human so that such antibodies can be generated. For the present invention,
The term "antibody" includes antibody fragments such as Fv, F (ab ') and F (ab') 2 fragments as well as single chain antibodies. Furthermore, antibodies and fragments thereof are for example EP-A-2.
As described in 39400, humanized antibodies (humanised ant)
ibody).

The antibody can be used to detect a polypeptide of the invention present in a biological sample by a method comprising the following: (a) providing the antibody of the invention, (b) antibody- The biological sample is incubated with the antibody under conditions that allow formation of the antigen complex, and (c) determine whether an antibody-antigen complex containing the antibody has been formed.

Suitable samples include extracts or cell types from any tissue. Typically the tissue is the brain, liver, kidney, spleen, heart or lymphatic system.

The antibodies of the invention can be bound to a support and / or packaged in a kit in a suitable container with suitable reagents, controls, instructions and the like.

F. 1.6 Examples of Polypeptides of the Invention Some preferred polypeptides of the invention are growth factors such as cytokines and neurotrophic factors. Cytokines are preferred, interleukins, especially IL
-4 is particularly preferred. Other interleukins such as IL-6 and IL-12 may also be used in accordance with the present invention.

F. 2. Uses of the polynucleotide of the present invention F. 2.1 Diagnostics The polynucleotides and primers of the invention can be used to investigate polymorphisms in genes that lead to altered propensity for the products of those polypeptides to aggregate, particularly as fibrils. Testing individuals with disease states such as Alzheimer's disease, type II diabetes, systemic amyloidosis, infectious spongiform encephalopathy, Huntington's disease, Parkinson's disease, cystic fibrosis, emphysema etc. for genetic polymorphisms Yes, this can be examined for correlation with disease state. Therefore, the polynucleotides or primers of the invention may be conveniently packaged in the form of a test kit in a suitable container. In such a kit, the probe may be bound to the support, if the assay format for which the kit is designed requires binding of the probe to the support. The kit also includes suitable reagents for processing the sample to be probed and hybridizing the probe to nucleic acids in the sample, control reagents, instructions,
And so on.

F. 2.2 Gene Therapy The polynucleotide of the present invention can be inserted into an appropriate polynucleotide vector and used for gene therapy. Therefore, the polynucleotides of the present invention also include suitable vectors for gene therapy. Vectors suitable for gene therapy can be of any type, but are typically viruses. Preferred viral vectors include polynucleotide sequences derived from retroviruses, lentiviruses, adenoviruses and adeno-associated viruses. Gene therapy is in vivo (
In vivo) or ex vivo cells can be performed, the latter optionally being transplanted into the host organism. Such a technique allows expression of the polynucleotide of the invention in all cell types, preferably somatic cells. Preferred polynucleotides for expression in gene therapy are those encoding polypeptides responsible for amyloid fibrils and prion-related diseases, more preferably Alzheimer's disease, type II diabetes,
Systemic amyloidosis, scrapie, infectious spongiform encephalopathy, Parkinson's disease,
Including those encoding a polypeptide that causes Huntington's disease, cystic fibrosis, emphysema, or the like.

F. 2.3 Generation of Transgenic Organisms The host cells of the invention (see above) are used to produce a stably transformed first generation and subsequent progeny that contain at least one polynucleotide of the invention. it can. Propagation of one transformed cell by techniques known in the art generally allows stable transgenic cultures to be generated. Commercially available media for cell culture are widely available and can be used according to the manufacturer's instructions. These can then be used for mass production of the expressed polypeptides of the invention. Alternatively, the transformed cells of the invention can be used to produce transgenic multicellular organisms when derived from multicellular organisms. Although any multicellular organism can be produced, plants and animals are preferred. More preferably, transgenic animals other than humans are produced. Particularly preferred animals include cows, sheep, goats, horses, pigs, chickens, turkeys and deer. Most preferred transgenic animals include those transformed with a polynucleotide encoding a polypeptide of the invention associated with amyloidogenic disease or prion diseases such as scrapie or bovine spongiform encephalopathy. The transgenic organisms of the invention can therefore be bred to provide stable transgenic progeny. Therefore, it is considered possible to develop an animal strain having stable resistance to such diseases by producing the transgenic animal of the present invention.

F. 2.4 Administration Any of the above-mentioned polypeptides, polynucleotides, vectors, cells, cell cultures, organisms, antibodies or aggregates, in any form or in combination with any of the other agents mentioned above, may be Included in the term "agent." An effective non-toxic amount of such an agent can be administered to a human or non-human patient in need thereof. Some of the non-human species considered to be preferred are described in F. Listed in 2.3. The condition of patients affected by the disease can therefore be improved by the administration of such agents. The agent may be administered prophylactically to an individual who does not have the disease to prevent the individual from developing the disease.

The invention thus provides an agent for use in a method of treating the human or animal body by treatment. The present invention provides the use of an agent in the manufacture of a medicament for treating a disease. The present invention thus provides a method of treating an individual, comprising administering the agent to the individual.

Agents are typically administered by standard techniques for administration, such as injection.

The same or different agents of the invention can be administered, typically after the initial administration of the agent. In one embodiment, the subject is administered one, two, three or more separate doses with at least 12 hours, one day, two days, seven days, 14 between each.
Leave daily, monthly or longer intervals.

The agent may take the form of a pharmaceutical composition containing the agent and a pharmaceutically acceptable carrier or diluent. Suitable carriers and diluents include isotonic saline solutions, such as phosphate buffered saline. Typically the composition is formulated for parenteral, intravenous, intramuscular, subcutaneous, transdermal, intradermal, oral, intranasal, vaginal or rectal administration.

The vaccination dose can be determined according to various parameters, in particular according to the substance used, the age, weight and condition of the patient to be treated, the route of administration and the required regimen. The physician will be able to determine the required route of administration and dosage for any particular patient. Suitable doses, however, may be 10 μg to 10 g of agent, eg 100 μg to 1 g. These values are believed to be the total amount administered in the complete treatment regimen, or to represent each separate dose within the regimen.

When the polynucleotide is the agent, a transfection agent may also be administered to enhance uptake of the polynucleotide by the cell. Examples of suitable transfection agents include cationic agents such as calcium phosphate and DE.
AE-dextran) and lipofectants (eg Lipofectam ™ and Transfectam ™).

When the agent is a polynucleotide in the form of a viral vector, the amount of virus administered is 10 ⁴ to 10 ¹² pfu, preferably 10 ⁷ to 10 ¹⁰ pf.
u (eg in the case of adenovirus vectors), more preferably about 10 ⁸ pfu for herpesvirus vectors. Pox virus vector (
Vaccinia virus) may also be used, typically in any of the doses described above. When injected, typically 1 to 2 ml of virus will be administered in a suitable pharmaceutically acceptable carrier or diluent.

As outlined above, analysis of the endogenous propensity of a polypeptide to form local structures has been demonstrated in the design of modified polypeptides with altered propensity to form aggregates, particularly non-covalent aggregates. Can be used. The same analysis can also be used to assess the propensity of a polypeptide to form non-covalent aggregates. Such an analysis
Not only can it be used in the design of polypeptides with altered aggregation propensity, but also to analyze the general tendency of polypeptides to aggregate, for example whether such polypeptides are suitable for production in a particular recombinant system under selective conditions. It can also be used to check if. For example, if analysis of the propensity of a polypeptide to form a local structure reveals that the polypeptide of interest has a high propensity to form an α-helical structure, the polypeptide is aggregated when expressed in vitro. Can be evaluated. Such an assay can also be used to select a polypeptide having a desired or selected aggregation propensity between a first and a second polypeptide, eg, a known variant protein.

Analysis of the propensity of polypeptides to form local structures such as α-helical structures also
It may also be used in the diagnosis or assessment of the likelihood that an individual will suffer from a disease associated with the formation of amyloid fibrils or aggregates. For example, analysis can be performed for proteins known to be associated with amyloidosis, or associated with the formation of aggregates in disease states such as amyloidosis or Alzheimer's disease. A comparison of the propensity of polypeptides to form aggregates, and a comparison of the propensities for polypeptides found in non-diseased and diseased individuals, indicates that the individual is subsequently associated with amyloidosis or other diseases associated with aggregate formation. It is thought to provide an index of whether the risk of contracting is high. In particular, protein amino acid sequences associated with amyloid disease could be identified for an individual. If a variant polypeptide that exhibits a low propensity to form an alpha helix is identified, then such an individual may be considered highly susceptible or at increased risk for amyloid or aggregate-related disease.
Such individuals could then be treated to reduce their tendency to form aggregates. Instead, it was confirmed that the variant polypeptides expressed by that individual had a high propensity to form aggregates, thereby, for example, as sufferers of CJD or Alzheimer's disease associated with aggregate or amyloid formation. Such variant polypeptides could be analyzed as part of a diagnosis to aid in the diagnosis of the individual.

Examples Examples 1 to 4 describe the inventors' tests for interleukin 4 (IL-4), a pleiotropic cytokine produced primarily by type 2 helper T lymphocytes (TH2). In this study, two possible strategies for improving the refolding yield of recombinant IL-4 are presented based on the elimination of surface hydrophobic residues and local stabilization of secondary structural elements. Helix / coil transition algorithm,
AGADIR (Munoz and Serrano, 1997; Lacroix, 19
98) rational stabilization of α-helices with heat and GdnHCl denaturation
It has been shown to increase the stability of L-4 but, more importantly, to double the in vitro refolding yield of recombinant IL-4 protein.

Example 1: Mutation Design Three different differences using AGADIR1s-2 (Lacroix, 1998) to increase the average helix content to 20% and increase the stability of the alpha helix by approximately 1.8 Kcal / mol. Designed kinds of mutations. The N-capping box motif (T-XX-XQ) present in the wild-type protein has a better corresponding motif (S-X-).
X-E). Better helix-forming factor (Chakrabarty)
And Baldwin, 1995; Munoz, 1994d) the helical sequence: Gl.
It was introduced into n71 and Phe73, mutated to Ala and H74 replaced with Asn. The reason why Asn is preferable to Ala in displacing H74 is that when another Ala is introduced at this position, a hydrophobic cluster is generated at the N-terminus containing 5 Ala residues, which reduces the solubility of the protein. , Because it may cause aggregation. Finally, two optimal electrostatic pairs (E-X-X-R and E-
X-X-X-H) was introduced. The mutant carrying the stabilized helix C is designated herein as IL-4BChelix.

Example 2 Structural Characterization of Peptides To demonstrate that the predictions made by the algorithm are correct, two peptides corresponding to the wild-type and mutant sequences of the C-helix were synthesized. Circular dichroism and NMR analysis show a clear increase in the helical content of the mutant peptide. A with respect to helix content of wild type (IL-4WT) and mutant peptides
The values predicted by GADIR1s-2 are in agreement with the experimental results.

Example 3 Thermodynamic and Structural Characterization of Proteins Two IL-4 mutants, IL-4W91S and IL-4BChelix, were overexpressed and purified to homogeneity to show NMR spectra similar to wild-type protein. It was To investigate the effect of the designed mutations on protein stability, equilibrium heat and GdnHCl denaturation tests were performed. IL-4 is a highly stable protein that cannot be denatured at temperatures in the range 0-100 ° C. Therefore, complete denaturation (u
n folding) transitions, thermal denaturation experiments were performed in the presence of 2M GdnHCl. Under these conditions, an overall transition can be seen, but the end of the curve corresponding to the fully denatured state is missing. Interestingly, WT proteins undergo cold denaturation below 20 ° C. Higher GdnH
At Cl concentrations, such proteins are completely denatured at high temperatures, but do not fold well at low temperatures. As a result, it is not possible to obtain accurate thermodynamic data from these curves, but the temperature at which half the protein denatures (Tm) can be deduced. Regarding the Tm value, IL-4W91S was the most stable protein, followed by IL-4BCheli.
x and IL-4WT.

Reliable thermodynamic data can be obtained from the GdnHCl denaturation curve. Consistent with the temperature denaturation test, we found that IL-4W91S was the most stable protein (W
1.4 kcal / mol more stable than T), then IL-4BChelix
(It is more stable at 0.5 kcal / mol than WT).

Example 4 Refolding Yield and Activity Assay Two experiments were performed to determine the refolding yield of IL-4WT and mutant proteins. Proteins were overexpressed in 1 L shake flasks as described in "Experimental Materials and Methods". In two more experiments, IL-4WT and two mutant proteins were isolated from cells obtained after low cell density fermentations at 10 L and 100 L scale (see “Experimental Materials and Methods”). In these four experiments, the refolding yield for IL-4BChelix was IL-4W.
It is approximately twice that of T, while the Kd for IL-4Rα remains the same as the wild-type protein. Preliminary data on the oxidative folding of IL-4 are found in the accepted IL-
It is suggested that the increased refolding yield of 4BChelix results from the destabilization of non-natural isoforms that accumulate during the refolding of wild-type protein. On the other hand, replacement of Trp91 with Ser does not improve protein refolding.

Discussion of Examples 1 to 4 This study aimed to improve the refolding yield of recombinant IL-4 without buffering the binding to IL-4Rα. The main goal has been to design mutants that retain their biological activity while refolding in higher yields than WT. Such IL-4 mutants could be used as a scaffold to introduce other mutations. This strategy would make large-scale production of IL-4 and IL-4 antagonist mutants economically more efficient, for example.

Thermodynamic Analysis of Oxidized Proteins The stability of two mutants of the oxidized form of IL-4 as well as their binding to IL-4Rα was measured and compared to the wild type protein. 4.3k for IL-4WT
A denaturation free energy of cal / mol was obtained.

Removal of solvent-contacted hydrophobic residues should stabilize the target protein through a “reverse hydrophobic action” (Pakula and Sauer, 1990). In the example presented, substitution of Trp91 by Ser results in a large stabilizing effect (1.4
kcal / mol), induces a 8 ° C. shift in the Tm of the wild-type protein. The observed stabilization results from an increase in the m value. This increase in m-value is believed to result from destabilization of the folding intermediate, the denatured state, or both (see Shortle, 1996 for a comprehensive review). Ashes the Phe residue (F14) in contact with the solvent
A similar effect was observed in the chemotaxis protein, CheY (Munoz, 1994d) when substituted by n (an increase in ΔG of 1.4 kcal / mol and an increase in m value from 1.6 to 1.9). . In this case, the change in gradient was due to the relative destabilization of the folding intermediate, which could only be detected dynamically. The equilibrium denaturation curve for IL-4 was fitted assuming a three-state model. C
No significant improvement in curve fitting was detected, as in the case of heY. Therefore, at present we cannot attribute the change in m-value to the destabilization of the equilibrium folding intermediates present in the folding of the oxidase.

As far as the stabilization of the helix is concerned, increased protein stability is observed (and in some cases it is possible to produce thermostable proteins) (Villegas, 1
996), but the increase in stability has been shown to be less than expected. This is due to the simultaneous stabilization of the denatured state under natural conditions. Therefore, IL-4
The results obtained in are in line with expectations. The mutation introduced in the C-helix stabilized the protein to a lesser extent (0.5 kcal / mol)
Tm induces a small shift (6 ° C). In this case, the stabilizing effect is primarily, GdnGCl concentration required to denature half of the protein, comes from elevated _{(GdnGC1) 1/2.}

Improved Refolding Yield for Reduced IL-4 Protein Although two of the mutants were found to have similar low refolding yields of IL-4W91S as the wild type protein, There is a significant improvement (about 2-fold) with IL-4BChelix. This change in refolding yield was observed in four different experiments in which the bacteria were grown in flasks, 10 L and 100 L fermentors, and is therefore not the result of specific expression conditions but a general improvement in the refolding process of the reduced protein. Reflects. HPLC analysis of the refolding process for the three proteins shows that in the case of the IL-4BChelix mutant, there is no major peak corresponding to the non-proliferating folding intermediate. This is believed to suggest that stabilization of α-helix C relatively destabilizes the folding intermediate leading to the formation of unnatural disulfide bridges.

The rational design strategy described in this study was useful for optimizing the sequence of this helix in a variety of polypeptides, especially cytokines, due to its foldability in vitro and in heterologous hosts. Means could be provided. The ability to produce large quantities of cytokines also makes it possible to isotopically label members of a family whose three-dimensional structure is not yet known at ¹⁵ N, and especially ¹³ C, at a reasonable cost (Markley and Kainosho, 1993). Will enable. The labeling strategy will also allow examination of the dynamic properties of these proteins, which will reveal the flexibility of functional epitopes and the mechanism used for receptor binding.

Biological Implications Biochemical and structural tests are often hampered by the limited amount of protein available. The ability to produce large amounts of protein is also the first prerequisite for developing clinically important proteins into commercial drugs. IL-4 is one of the major players in allergic reactions, and protein antagonists produced by mutating the residues involved in binding to the low affinity component of the dimeric receptor system are highly therapeutic. Have a profit. However, like most cytokines, IL-4 and its variants refold in vitro only at very low yields, making the production of such proteins tedious and costly. There is.

A two-fold improvement in IL-4 in vitro refolding yield was brought about by using a rational design to enhance the helical properties of the C-helix. This stabilization suggests that optimization of the alpha helix results in increased protein stability and accelerated folding reactions. We propose that stabilization of the α-helix within the protein is a general strategy for improving refolding yield in vitro.

Experimental Methods of Examples 1 to 4 AGADIR Design of Helix Stabilizing Mutations. The design of mutations that enhance helix properties was carried out based on the following principles: i) The target site for mutagenesis does not interact with residues whose side chains are not included in the α-helix and Those in the α-helix are selected that are not necessary and are in sufficient solvent contact. The intent is to maintain the packing of the protein, to ensure that the mutation does not eliminate mid- or long-range interactions, and that the activity of the protein is retained. ii) mutations are described in AGADIR (Munoz and Serrano, 1997; Lacroix, 1
Designed to enhance alpha-helical properties as suggested by helix / coil transition algorithms for heteropolypeptides, such as 998). Basically the algorithm is modified to predict stability for a particular sequence within the alpha helix conformation, rather than the helix content for peptides.
The template sequence used corresponds to the X-ray alpha helix plus four residues on each side to prevent terminal effects. Several sequences are evaluated programmatically that differ in the residues at the positions targeted for mutagenesis.

Cloning. The IL-4 gene is based on the previously described plasmid R15prC.
Obtained by PCR from 109 / IL4 (Kruse, 1991). NocI and BamHI restriction sites were introduced at the 5 ′ and 3 ′ ends, respectively, using the following oligonucleotides: Oligo 5 ′: CTG GAG ACT GCC ATG GAT CAC AA
G TGC GAT; oligo 3 ′: A CGC GGC TCC TTA TC
A GCT CGA ACA. Genes encoding wild-type IL-4 and mutant proteins were inserted into PBAT4 (Peranen, 1996). Nc at the 5'end
By introducing the oI site, wild-type IL-4 having an additional amino acid (Asp) at the N-terminus and a mutant protein were expressed.

Site-directed mutagenesis. Mutants IL-4W91S and IL-4BCheli
x was obtained by PCR using oligo 5'and oligo 3'as flanking sequences and the following mutagenic primers (Ho, 1989): IL-4W91.
Sa: AGG AAC CTC AGT GGC CTG GCG GGC T
TG. IL-4W91Sb: CAA GCC CGC CAG GCC ACT
GAG GTT CCT. IL-4BC Helixa: CTG GGT GC
G AGT GCA GCA GAA GCA AAC AGG CAC AA
G C. IL-4BChelixb: G CTT GTG CCT GTT T
GC TTC TGC TGC ACT CGC ACC CAG.

Protein expression. For shake flask fermentation, Escherichia col
i (E. coli) AD494 (DE3) (Derman, 1993) was transformed with the plasmid. A 1 L flask containing LB medium was inoculated with a single colony and incubated at 37 ° C on a shaker. After OD ₆₀₀ reached 0.5, IPTG
Was added to give a final concentration of 0.16 mM. Cultures were incubated overnight and cells were harvested by centrifugation.

Fermentations in 10 and 100 L bioreactors were performed to produce higher amounts of protein. For these fermentations the plasmid was transformed into E. coli W3110 (
It was transformed into DE3). Complete medium (30g / L soybean peptone, 20g
/ L yeast extract, 20 g / L glycerol, 5 g / l KH ₂ PO ₄ , 1 g / l
It was grown in MgSO ₄ , 100 mg / l ampicillin) at 37 ° C. to an OD _{600 of} 3.0. At this stage, 0.4 mM IPTG was added to induce the expression of the recombinant protein. After a 4 hour incubation period, cells were harvested by centrifugation.

[0139]   Protein purification. Suspend the collected cells in 25 mM Tris-HCl pH 8.0
did. Lysozyme (1 mg / g cell dry weight) was added to disrupt the cells enzymatically,
Incubated for 30 minutes at room temperature. The released inclusion bodies are centrifuged at 8000g.
And collected (30 minutes). The pellet was added with 0.1 M Tris-HCl pH8 /
Suspended in 1 mM EDTA / 0.1% amphoteric detergent and centrifuged (8000 g, 15
For 4 minutes) and washed 4 times. Washed inclusion bodies with 8M GnHCl / 0.1m Tr
It was dissolved in is-HCl, pH 9. Kella and Rurella (1985) and
And Kella (1988), an excess of sulfite and tetrathiol on the SH group.
Add onate and add S-SO_ThreeThe group was chemically modified. 5 volumes of 50 mM Na_TwoHPO _Four , PH 7, 1 mM EDTA, 0.4 mM L-cysteine, 0.6 mM L
-200 to 300 mg / L protein by transverse ultrafiltration against arginine
Refolding was performed at the concentration. Trifluoroacetic acid-acetonitrile linear gradient
In-process by RP4-HPLC on Vydac C4 resin
Analysis was performed.

Peptide synthesis. Peptides were synthesized on polyoxyethylene-polystyrene graft resin in a continuous flow apparatus. The peptide chain is constructed by Fmoc chemistry (Ca
It was performed using in situ activation of amino acid building blocks with rpino, 19729 and PyBOP (Coste, 1990). Peptides were purified by reverse phase HPLC and characterized by laser desorption mass spectrometry (MALDI).

Circular dichroism. 278K Jasco-71 in 2mm path cuvette
The far UV CD spectrum of the peptide was recorded on a 0 instrument. The measurement was performed every 0.1 nm with a reaction time of 2 seconds, a band width of 1 nm, and a scan speed of 50 nm / min. 28
The peptide concentration calculated from the absorbance at 0 nm is (Pace, 1995) 25 μM.
Met. Taking into account the length of the peptides were calculated helical percentage from the mean residue ellipticity at 222nm according to the following:% helical = 100100θ ^abs _222nm /(39500(1~2.57/n) equation in one formula, n is the number of residues in the peptide, 100θ ^abs _222nm is 222n
Ellipticity of peptide in m.

Thermal denaturation. By monitoring the change in CD signal at 222 nm over a temperature range of 6 to 95 ° C. in a 2 mm path cuvette, the Jasco-7
Thermal denaturation was measured with 10 instruments. The measurement was carried out with a reaction time of 2 seconds, a band width of 1 nm, and a temperature gradient of 50 ° C./hour, increasing by 0.5 °. The peptide concentration calculated from the absorbance at 280 nm was 10 μM. Measurements 25mM _Na 2 _HPO 4/2
Performed in M GdnHCl, pH 6.5.

Chemical denaturation. GdnHCl denaturation and regeneration experiments were performed with 50 mM Na ₂ HPO ₄ , pH.
Carried out in 7.0 at 25 ° C. The protein concentrations used were IL-4 wild type and IL-4
It was 7 μM for W91S and 5 μM for IL-4BChelix. The denaturant and protein were mixed using the Jasco automatic titration system. Protein denaturation and refolding was monitored by following the change in CD signal at 222 nm. The ellipticity readings were normalized to the denatured fraction using the following standard equation: _Funf = ([theta]-[theta] _N ) / ([theta] _{D- [} theta] _N ) Equation 2 where [theta] is the denaturant. Is the ellipticity value at a specific concentration of, and θ _N and θ _D represent the ellipticity of completely natural and fully denatured species at each denaturant concentration, and were calculated from linear regression of baseline values before and after denaturation. . Assuming a two-state model, the equilibrium constant for denaturation at each denaturant concentration can be calculated using the following equation: K _D = (F _N −F) (F−F _U ). Where F _N and F _U represent the fractions of fully native and fully denatured protein obtained by linear fitting of the baseline values before and after the transition region, respectively. GdnHCl
It has been experimentally found that the free energy of denaturation in the presence of γ is linearly correlated with the concentration of denaturant) Pace, 1986): ΔG _D = ΔG _D ^{H 2 O} −m [GdnHCl] Eq. The constant m reflects the cooperative phenomenon of transition and is believed to correlate to the difference in solvent-contacted surface between the native and denatured states. When you put all these dependencies into account, change is ellipticity as a function of the concentration of denaturant, can be adapted to the following _{equation: F unf = F N + a} [GdnHCl] + {( F _U + b [GdnHCl]) {
exp ((m [GdnHCl] -ΔG _D ^H2O ) / RT) / (1 + exp ((m
[GdnHCl] −ΔG _D ^{H 2 O} ) / RT))}} Equation 5 where the dependence of the intrinsic ellipticity on the denaturant concentration in both the native and denatured state is:
They are considered in the terms a [GdnHCl] and b [GdnHCl], respectively (linear approximation (Santoro and Bolen, 1988)).

NMR spectroscopy. NMR samples of IL-4 and mutant proteins were prepared using lyophilized proteins with 45 mM sodium deuterium acetate (NaOAcd ₄ ) pH 5.
It was dissolved in ₃ , 10% D ₂ O, 0.1 mM TSP, 0.2% NaN ₃ to prepare a protein concentration of 500 μM. The spectrum was recorded at 303K. C-helix wild-type and mutant peptides were loaded with 25 mM Na ₂ HPO ₄ pH 6.0.
Analyzed at a peptide concentration of 1.5 mM in 295 K in 10% D ₂ O and 0.1 mM TSP, 0.2% NaN ₃ . All spectra are Bruker DRX 5
Obtained on a 00 and 600 spectrometer. Two-dimensional (2D) TOCSY (total correlation spectroscopy) and NOESY (nuclear Overhauser weighted spectroscopy) spectra were recorded using standard pulse sequences and phase cycling.

Binding assay. BIAcore 2000 (Pharmacia Biose
The binding affinity was measured by surface plasmon resonance using a plasma sensor. The recombinant extracellular domain of the receptor α chain ([C182A, Q206C] IL4-BP) was incorporated into the biosensor CM5 at 1500-2 as described by Shen (1996).
The density was fixed to 000 pg / mm ² . HPS buffer (10 mM Hepes,
Ligand binding was analyzed at 25 ° C. by perfusion of pH 7.4 / 150 mM NaCl / 3.4 mM EDTA / 0.005% surfactant P20) plus 0.5 M NaCl at a flow rate of 50 μl / min.

Examples 5 and 6 Examples 5 and 6 describe the inventors' study of an 81 residue polypeptide, ADA2h, the activation domain of human procarboxypeptidase A2. This domain has two α helices packed against a four-stranded β sheet (
Catsus, 1995; Garcia Saez, 1997; Aloy, 1
998). It has been found that such proteins fold in two states at neutral pH through a compact transition state and have some secondary structure and a trace hydrophobic core (Villegas, 1995; Villegas, 1998). The inventor has used the wild-type polypeptide (WT-ADA2h) as one of (M1-ADA2h
And helix 2 in M2-ADA2h) or both α-helices (DM-ADA2h) were compared with three mutants (Villegas, 1997) in which several α-helices were stabilized by some amino acid substitutions. The stabilizing mutations on the solvent-contacted surface of the ADA2hα helix are: α helix 1 (
_{M1-ADA2h): N 2 5} / K, Q28 / E, Q32 / K, E33 / K (Vi
llegas, 1996), α helix 2 (M2-ADA2h): Q60 / E
, V64 / A, S68 / A, Q69 / H (Villegas, 1996). The double mutant (DM-ADA2h) contains substitutions corresponding to both M1 and M2 (V
illegas, 1997).

Example 5 Comparison of Aggregation Tendency in Wild Type and Mutant Polypeptides Three mutants M1, M2 and DM at pH 3.0 show fully reversible denaturing transitions (10 μM-3 mM concentration range). At), at high temperature, a CD spectrum typical of denatured polypeptides is shown (FIGS. 1a & 1c-1e). The WT polypeptide, however, undergoes a non-reversible transition (FIG. 1a). The CD spectra after cooling to 95 ° C. and 25 ° C. show that the polypeptide has transformed into a conformation with a broad β-sheet structure (FIG. 1b). Analytical centrifugation analysis
It shows that the samples are highly aggregated (40% of the samples showed SW _{, 20 of} 35S, thus suggesting Mr above 10 ⁶ Da, which averages 100 mer.
Corresponding to the above aggregates). The conversion of α-helical structure to β-sheet for WT-ADA2h polypeptide upon heat denaturation is confirmed by FTIR spectroscopy. Prior to heating, the band of amide I shows two major components at 1622 and 1649 cm ⁻¹ , respectively (FIG. 1f), which are ascribed to β-sheet and α-helical structures (Surewickz, 1993), respectively. Consistent with the natural state of. After incubating the samples at 90 ° C., two new bands centered at 1615 and 1685 cm ⁻¹ respectively appear in place of the first band (FIG. 1f). This pattern usually leads to aggregated species with β-pleated structure (F
laser, 1992; Gasset, 1992; Fabian, 1993b).
. The band at 1615 cm ^-1 represents a β-sheet, while the band at 1685 cm ^-1 is associated with cleavage within amide I by antiparallel interchain interactions (Krimm and Ba.
nddes, 1986; Fabian, 1993a). The polypeptide after heating also showed a significant shift in the Congo red absorbance spectrum from 486 to 500 nm with increasing absorbance, showing a thioflavin-T bond (Table I). All of these properties are consistent with the formation of amyloid deposits by the WT-ADA2h polypeptide.

WT and D after incubation in the presence of a range of urea concentrations (4 to 7M)
The behavior of the M polypeptide was also investigated. At 7M urea, the CD spectra of both polypeptides show highly denatured species (decreased ellipticity at 222 nm). WT in 7M urea
When both samples, DM and ADA2h, were rapidly diluted to a concentration of 1 M urea, the samples folded to their native state. However, the WT polypeptide
Incubation in M urea for 1 hour, followed by dilution in 1 M urea and incubation for 1 hour resulted in a CD spectrum suggesting a broad β-sheet structure (FIG. 2a).
). No such behavior was observed for the DM polypeptide (Fig. 2b), WT
Restored its native spectrum after the same successive equilibration steps that promoted β-sheet structure expression in ADA2h. Consistent with this, it was found that DM-ADA2h was completely digested by pepsin after successive equilibration steps in urea as well as after heat denaturation. However, the WT polypeptide showed low sensitivity to digestion in samples in which increased β-sheet structure was detected (Table 2). actually,
There is a clear correlation between the transition to β structure and increased resistance to proteolysis revealed by CD and FT-IR.

Example 6: Aggregate Analysis Analysis of aggregated WT polypeptides by electron microscopy revealed long, unbranched, narrow (
Diameter 30-100Å), showing clear evidence of apparently very soft fibers. They typically form a tight network of twisted structures. The sample observed after a long incubation of 90 hours at 90 ° C. is characterized by longer and more regular fibers than after a short incubation of 1 hour and more clearly shows the twist of a ribbon-like pattern at irregular intervals. . The structural changes that result from longer incubations at elevated temperatures can be explained by the gradual rearrangement of the protofilaments that make up the fibers. Fibers have a low concentration of 20 μM WT-AD
It can be observed in the aggregates formed from the solution containing A2h. WT-AD subjected to chemical denaturation of fibers with similar characteristics as shown by short incubation time
It was also observed in the A2h sample, which was consistent with the development of resistance to proteolysis under similar conditions. As expected, no fibers could be detected from any of the DM mutant samples subjected to similar conditions of heat and chemical denaturation.

Fibers from heat-denatured samples of WT-ADA2h were also characterized by X-ray fiber diffraction and found to have different crossed β-ray diffraction patterns characteristic of amyloid fibrils (Blake and Serpell, 1996; Sunde). , 1997)
. Reflexes are observed at 4.7 Å (corresponding to interchain distance in the direction of the fiber axis) and 9.3 Å (corresponding to β-sheet distance in the direction perpendicular to the fiber axis). A clear anisotropy was observed in sharp 9.3 Å diffraction (the equatorial axis is the vertical axis in the display). Another weak reflection was observed at 3.1 Å, probably due to overtones, with anisotropy and acute angle of 9.3 Å reflection. 4 with no apparent anisotropy
The second weak reflection is observed at 3.8Å. This type of reflex has been previously observed in studies of amyloid fibrils from various sources (Sunde, 199).
7).

ADA2h is therefore an example of a polypeptide that can aggregate in the form of amyloid fibrils and that is not associated with any known disease. However, it should be noted that mutations in helices 1 and 2 of ADA2h substantially reduce the propensity for aggregation of this polypeptide. This cannot be correlated with the overall stability of the native state when aggregation is initiated under conditions where both WT and the mutant polypeptide are at least partially denatured. A low propensity to aggregate has been shown to be associated with a high propensity for α-helices of modified polypeptides. Stabilization of the α-helical structure promotes local intrastrand interactions compared to interstrand interactions in the denatured state and thus prevents polypeptide aggregation.

This study is therefore more than just an overall global folding
It has been suggested that mutations that stabilize regions of the secondary structure of A2h inhibit its aggregation into amyloid fibrils. Apparently, disruption of the native state of the protein is essential for the formation of ordered aggregates. Therefore, it is quite reasonable that there is a strong correlation between mutations that destabilize protein structure and its tendency to form fibrils. However, the propensity of the aggregation process to occur should also depend on the intrinsic properties of the denatured state resulting from the disruption of the natural fold. Mutations that alter the properties of the denatured state are not expected to simply correlate with the structure, as well as the stability, of the native protein, as in the case of the mutations presented here. This conclusion suggests an explanation for the existence of disease-related amyloidogenic mutants that simply do not result in decreased protein stability.

It is well established that mutations to native proteins can enhance amyloidogenic behavior. For the first time, the results regarding ADA2h show that the natural function of proteins is
It has been shown that modifications that maintain structure, and even stability, can reduce native protein aggregation into amyloid fibrils.

Gene therapy could be used to incorporate modified proteins with low aggregation propensity into the organism or to produce transgenic animal strains that are resistant to prion diseases. Furthermore, polypeptides of industrial or medical importance, such as insulin and calcitonin, but with a high propensity to aggregate (Sluz
ky; 1992; Arvinte, 1993; Moriarty, 1998) can be constructed to a lower aggregation propensity by altering their secondary structural propensity, perhaps using an approach similar to that used for the ADA2h protein. Will.

[0155]

[Table 1]

[0156]

[Table 2]

Experimental Materials and Methods for Examples 5 and 6 Expression and purification of ADA2h. WT-ADA2h and its stabilized mutants were expressed and purified as previously reported (Villegas, 1996).
Villegas, 1997; Villegas, 1998). All recombinant proteins were examined by MALDI-TOF-MS and found to have the molecular weight expected from the sequence.

Circular dichroism. CD spectra of 20, 80, 160 and 200 μM polypeptide samples in 50 mM sodium phosphate (pH 7.0) or 25 mM glycine (pH 3.0) were measured at 278, 2 in a 2.0 or 0.2 mm quartz cuvette.
Recorded using a JASCO-710 spectropolarimeter at 98 and 368 ° K. Fifty
The measurements for 30 scans recorded in nm / min were averaged. The heat-induced denaturation of 20 μM protein samples was monitored at a heating rate of 50 ° C./h in the temperature range of 278 to 368 K by following their ellipticity at 222 nm or 214 nm. Residual helical structure calculations at 95 ° C were performed by measuring [Θ] at 220 nm (Chen et al., 1974). To prevent artifacts due to protein aggregation, collect the spectrum of the wild-type protein at a concentration of 5 μM and
The sample was heated at a rate of 150 ° C./hour in the range of.

Sedimentation analysis. 20 and 200 μ in pH 3.0 buffer containing 25 mM glycine
For M polypeptide samples, sedimentation experiments were performed in a 3000 g Beckman XLA analytical ultracentrifuge. Samples at 5-95 ° C at 50 ° C rate
It was heated to 95 ° C. and then left at 95 ° C. for 10 minutes. A 200 μM polypeptide sample in 50 mM sodium phosphate, pH 7.0 was used as a negative control.

Thioflavin-T and Congo red binding assay. WT-ADA2h and D
Samples of M-ADA2h (concentrations in the range 20-500 μM) were incubated at 90 ° C. for 30 minutes prior to assay. 2.5 mM Thioflavin-T stock solution was freshly prepared in 10 mM potassium phosphate, 150 mM NaCl, pH 7.0,
It was passed through a 0.2 μm filter before use. Typically 10 μl of sample is added to 6
Diluted in reaction buffer (10 mM potassium phosphate, 150 mM NaCl, pH 7.0) containing 5 μM Thioflavin-T (final volume 1 ml). The sample was pipetted out of the pipe several times to facilitate dispersion of the fibers and disrupt large aggregates. In a Perkin-Elmer LS 50B luminescence spectrometer, an excitation wavelength of 440 nm (slit width 5 nm) and 482 nm (slit width 10 n
Data was collected using the emission wavelength of m). Fluorescence values were examined after 3 to 5 minutes to ensure that thermal equilibrium was achieved. Samples were continuously agitated to prevent signal oscillation due to the presence of large fiber aggregates, and signals were averaged over 60 seconds to enhance the signal to noise ratio. Klunk et al. (Klunk, 1989a; Kl)
Congo red binding assay was performed according to unk, 1989b). A 10 μl aliquot of the sample containing 5 μM Congo Red (1 ml final volume)
Diluted in 0 mM potassium phosphate, 150 mM NaCl, pH 7.0. The Congo red solution was prepared just prior to its use and passed through a 0.2 μm filter. The absorption spectra of the samples in the reaction solution along with the negative controls (dye without polypeptide and polypeptide sample without dye) were collected to subtract the signal associated with dye absorption and the scatter contribution to the signal.

Proteinase resistance. WT and DM- in 25 mM Glycine (pH 3.0)
A 20 μM sample of ADA2h was incubated with 4M urea, then diluted and dialyzed to a urea concentration of 1M and then incubated with proteinase. A sample in the same buffer was also subjected to proteolysis after incubation at 95 ° C for 10 minutes. These samples, along with untreated samples of the same polypeptide,
Two different ADA2h: pepsin ratios (100: 1 and 40 in the presence of pepsin
Digested with 0: 1) for 2 hours at 20 ° C. and 15 minutes at 0 ° C., respectively. The digested sample was then subjected to a Vydac C4 column (214TP54, particle size 5 μm, pore size 300Å, 1.0 × 25c) with a linear gradient of 10 to 52% acetonitrile.
It was analyzed by RP-HPLC in m). Detection was performed at 214 nm on a Waters 994 model.

[0162]   Fourier transform infrared spectroscopy. Bio-Rad with liquid nitrogen cooled MCT detector
  Record the infrared spectrum on an FT S 175C FT-IR spectrometer,_TwoOf gas
It was cleaned with a continuous flow. A 500 μM sample of WT-ADA2h^TwoH_TwoO, Gu
Lysine 25 mM, p^TwoPrepared in H3.0 (using electrode reading for isotope effect
Corrected) at 25 ° C before and after incubating the sample for 30 minutes at 90 ° C.
The spectra were collected at. Polypeptide solution with 12 μM Mylar spacer
Separated pair of CaF_TwoI placed it between the windows. 2 cm for each sample ^-1 256 interferogram (interferogram) with a spectral resolution of
rams) were collected. Spectra of buffer without polypeptide under the same conditions
Toll was collected and subtracted from the spectrum of the polypeptide sample. Different space
A second derivative of the amide I band spectrum was constructed to study the wavenumbers of the Kutru component.
Made

Electron microscopy. Samples were applied to Formvar coated nickel grids (400 mesh), negatively stained with 2% uranyl acetate (w / v) and viewed on a JEOL JEM1010 transmission electron microscope operating at 80 kV.

X-ray diffraction. The fiber suspension was added to a Microcon-100 ultrafiltration tube (Amicon
) To remove salts and buffers that could interfere with X-ray measurements. Samples were prepared with air dried salt-free ADA2h-WT fiber samples between two wax-filled capillary ends. The capillaries were slowly separated while drying to facilitate orientation of the fibers along the elongation axis. A small stalk of fibers protruding from one end of the capillary was obtained. Samples were aligned by X-ray flux and placed on a 180 or 300 MAR-Research image plate (MA
R Research, Hamburg, were collected 20 to 30 minutes diffraction image in Cu K _a rotating anode having a Germany). IPDISP and Mar
Images were analyzed using View software.

Example 7: A new form of PI3-S that is unable to aggregate into amyloid fibrils
Rational Design of H3 We have previously studied the domain, PI3-, which was previously studied in the rational design of new polypeptide variants in order to construct non-helical proteins with modified aggregation properties.
The SH3 domain (SH3 domain of bovine phosphatidylinositol-3′-kinase) was selected. There are two main motivations for investigating this protein:
1) its native structure is all β (two β-sheets of triple-stranded and double-stranded, each placed in a β-barrel feature) and 2) amyloidogenic properties of the domain and PI3-
Important data are available on the structure of amyloid fibrils formed by SH3.

In order to determine which residues of the protein should be modified, predictions were made with AGADIR software (Munoz and Serrano, 1997; Lacroix et al., 1998) to identify specific sequence regions with a constant endogenous helical propensity. Identified. The analysis yielded three major regions of constant propensity (although propensities were much lower in all three) (FIG. 3).

The first region was selected subject to a higher endogenous propensity to facilitate the design of new variants with the required characteristics.

The second requirement for design was to identify residues in the regions described above where the side chains were in good solvent contact and were not directly involved in the β backbone (FIG. 4). Several proposed mutants that meet these requirements were analyzed using AGADIR software. We focused in the first step on those containing 1, 2 and 3 substitutions in order to minimize significant effects on the natural overall structure and / or stability. With the same goal in mind, the introduction of highly hydrophobic residues was avoided as much as possible. After performing several series of predictions by AGADIR, mutants with two point substitutions at residues 17 and 23, which appear to have a much more stable endogenous helical propensity, were selected (Figure 4). The mutants selected were therefore E17R, D23R, in each case the substitution of natural glutamic acid at position 14 and the substitution of aspartic acid at position 23 with arginine. Predictions for the intrinsic helical propensity of the modified fragment show an increase for the 17R / 23R mutant when compared to the wild-type protein (Figure 5).
.

Mutants were processed according to standard recombinant DNA protocols to express the protein. Similar batches of wild-type protein were expressed and co-purified under the same conditions so that the results obtained could be compared to the modified protein.

A sample of protein at a concentration of 5 mg / mL was incubated at pH 2.0 and 37 ° C. under standard conditions for amyloid formation (Guijarro et al., 1998; Ji.
menez et al., 1999; Chamberlain et al., 2000). Under these conditions, formation of amyloid fibrils was expected for the wild-type protein during the first 3 or 4 days. Aliquots of both samples were analyzed after 4 weeks of incubation and analyzed by electron microscopy, thioflavin-T binding and ultracentrifugation.

Analysis of the sample by EM and thioflavin-T binding was performed using E17R / D23R
It was clearly suggested that there was no aggregation in the samples containing the mutants, whereas the samples prepared with the wild-type protein showed characteristic fibers (Table 3). Quantitation of the protein present in the supernatant fraction clearly confirms the absence of aggregation in the sample containing the mutant protein.

[0172]

[Table 3]

To exclude the relationship between protein behavior and its stability, denaturation profiles of both wild type and mutant proteins in the presence of guanidine were analyzed. The thermodynamic parameters obtained from such an analysis clearly indicate that the mutant protein is somewhat unstable as compared to the wild type (Table 4). Therefore, the effect on the aggregation properties of proteins does not come from stabilization of the native structure, but due to changes in the intrinsic tendency of the polypeptide chain. On the other hand, the unfolding pattern (especially the co-ordination coefficient m) suggests that the mutein is compact with a defined tertiary structure.

[0174]

[Table 4]

In conclusion, we have previously described ADA2h domain and IL- to prevent aggregation.
It was shown that it is possible to rationally construct non-helical proteins using the same methods used for the 4 proteins. Even though the mutant form exhibits lower thermodynamic stability (due to increased non-natural local propensity), it retains the major structural features of the native protein but lacks the aggregation ability of the original polypeptide. It is a protein. We can therefore conclude that the use of the same methods already used for ADA2h and IL-4 polypeptides has led to the successful design of new polypeptides that are unable to aggregate into amyloid fibrils. Moreover, the native helical structure in the original polypeptide does not appear to be necessary for the application of such a method.

Example 8 We investigated the helicity and aggregation properties of wild-type human prion helix-3 and four Creutzfeldt-Jakob disease-related mutants: E200K, R208H, V201I, Q217R.

Circular dichroism. The CD spectrum of the helix C peptide was recorded using a JASCO-720 spectropolarimeter. Sample concentration is 20 to 100 μM, 1.
Spectra were collected at 278 ° K in 0 mm or 0.1 mm quartz cuvettes. Measurements for 5 scans were averaged and recorded at 10 nm / min. 20m
M sodium phosphate, 20 mM β-mercaptoethanol pH 7.0 is added to neutral p
Used as a buffer for H measurement. Peptide with pH 2.0 in advance
And 20 mM phosphoric acid equilibrated with 20 mM TCEP to suspend the sample
Incubated at 2.0.

Electron microscopy. Samples were applied to Formvar coated nickel grids (400 mesh), negatively stained with 2% uranyl acetate (w / v) and viewed on a JEOL JEM1010 transmission electron microscope operating at 80 kV.

Secondary structure propensity predictions were performed for all previously described PrP disease-related mutations in helix C. The protein is reported to retain its stability largely intact in all these mutants, except for the Q217R substitution. For the Q217R substitution, ΔΔG ⁰ _fold 8.9 ± 1.7 kJ
mol ⁻¹ has been reported (Liemann and Glockschuber 19
99). However, the helical propensity for the two mutants E200K and R208H is about half that expected for the wild-type protein, while mutant V2
The propensity of 10I and Q217R is somewhat higher (Table 5). Interestingly, these two mutations clearly correspond to solvent-contacting residues and therefore cannot participate in long-range interactions within the protein.

To further investigate the involvement of secondary structure propensity in explaining some of these CJD-related Prp mutants, a peptide spanning hPrP helix C and two predicted low secondary structure propensities CJK-related mutants, E200K and R208H
Was analyzed along with another variant corresponding to the mutation V210I (FIG. 6, Table 5). The peptide spans residues 198-227 of the hPrP sequence.

[0181]

[Table 5]

All peptides showed a generally disordered conformation when analyzed by CD when suspended in phosphate buffer pH 7.0 (FIG. 7). However, both peptides corresponding to the wild-type and V210I mutant sequences were
It shows a higher helical content than that detected in 00K and R208H (see also Table 5). TFE titration of the four peptides confirmed the same trend in helical propensity (Figure 8), demonstrating that the theoretical predictions made using AGADIR are correct (Figure 6).

It was recently reported that recombinant prion proteins can be converted to fibers in vitro by incubating the proteins under reducing conditions at low pH or under low pH and 1 M guanidine conditions. (Jackson,
Hosszu et al., 1999; Sweetnicki, Morillas et al., 20.
00). To investigate the effect of low pH on the conformation and properties of helix C, four peptide aliquots were added to pH 2 in the presence of TCEP as a reducing agent.
． Incubated at 0. The spectra of the four peptides showed variation with concentration, probably due to aggregation (Figures 9A-B), but below 20 μM their CD characteristics appeared to remain constant. At this concentration, the four peptides show similar trends as seen at pH 7.0. Nevertheless, wild type and V210I peptides show much higher helix content (FIG. 9A). In contrast, the far-UV CD spectra of mutants E200K and R208H also show low α-helical content when compared (Fig. 9A).

Samples of peptides at pH 7.0 and 2.0 were incubated for several weeks to assess the presence of aggregates. None of the peptides incubated at pH 7.0 showed changes in the far-ultraviolet spectrum (data not shown), whereas pH
All incubated at 2.0 showed a clear β-pleated CD profile centered at least 214 to 216 nm (FIG. 10). Samples were analyzed by EM to examine the nature of aggregates in all four peptides.
Although fiber structure is present in all peptide samples, some differences in fiber quality are observed among them. Thus, while both wild-type and V201I mutants exhibit highly flexible and disordered fibrils, E200K and R208H tend to form very long and linear helix structures, with a high degree of compaction. Suggest sex. Example 9: Strategies for identifying regions of protein sequences that are important in the process of aggregation and amyloid fibril formation. Mutants of proteins with conservative amino acid substitutions distributed throughout the protein sequence were generated. Mutation studies were performed on human muscle acyl phosphatase (AcP). AcP has a simple fold with a βαββαββ-topology,
It is a 98-residue protein that has been shown to be capable of forming amyloid fibrils in vitro without complex factors such as disulfide bridges or cofactors.

Mutation Selection 34 mutant proteins with one amino acid substitution distributed throughout the sequence of AcP were expressed and purified. As with other globular proteins, the conformational stability of AcP is very sensitive to amino acid substitutions, especially when the mutated residues are hidden within the hydrophobic core. We therefore replace each residue with another residue that has similar characteristics but results in a small cavity or insertion (eg Phe to Leu, Ile to Val, Ser to Thr or Glu to Asp. Conservative mutations were selected. The mutants produced were S5T, V9A, Y
11F, V13A, V17A, V20A, F22L, Y25A, E29D, A3
0G, I33V, G34A, V36A, V39A, T42A, G45A, V47
A, V51A, P54A, M61A, W64A, L65V, K67A, P71A
, I75V, I78S, E83D, I86V, S87T, L89A, Y91Q,
It was S92T, F94L and Y98Q. For this set of mutants,
The polypeptide chain was probed at every approximately three residues, and the involvement of various regions of the polypeptide chain in the aggregation process was evaluated.

Strategies for Measuring Aggregation Rates Amino acid substitutions that destabilize the native folding of globular proteins are thought to promote aggregation, as they result in the denaturation or occupation of partially folded conformations. From such a state, it seems that the aggregation process is started. For this reason, various AcP mutants have been shown to be completely denatured, and 25% (vol / vol) trifluoroethanol (TFE) was used as a condition under which aggregates were observed to express relatively slowly. The rate of aggregation of AcP in the presence of Aggregation tests under these conditions can eliminate the conformational stability contribution of the native protein to the aggregation process. Therefore, the effect of amino acid substitution on the intrinsic rate of polypeptide chain aggregation can be examined and compared directly.

25% (vol / vol) TFE in 50 mM acetate buffer, pH 5.5, 25
Aggregation was initiated by incubating a solution of AcP at a concentration of 0.4 mg / ml at 0 ° C for an extended period of time. Aliquots were taken from the samples at regular time intervals and used as described in the "Materials and Methods" section, Far Ultraviolet Circular Dichroism (CD) or optical test in the presence of Congo Red and Thioflavin T. The amount of protein aggregation was quantified. By the time the first experimental data is obtained after the addition of TFE, AcP is completely denatured. Within 2 hours, AcP can be seen as granules or very short fibers by electron microscopy, CD and Fourier transform infrared spectroscopy (FT-I).
R) has a broad β-sheet structure and is converted into relatively ordered aggregates with the ability to bind specific dyes such as Congo red and ThT. Figure II shows the time course of increased ThT fluorescence after initiation of aggregation, increased absorbance at 540 nm from Congo red and decreased ellipticity at 216 nm resulting from β-sheet structure for wild-type AcP. The time-dependent changes in all three spectroscopic parameters are very similar to each other. There is no detectable lag time resulting from the gradual nucleation process in the aggregation kinetics studied here and a direct measurement of velocity is possible. Aggregation rates measured as described in the "Experimental materials and methods" section are the same within experimental error for the three measurement sets;
The rates of AcP aggregation monitored by changes in ThT fluorescence, Congo red absorbance and CD ellipticity were 5.43 ± 0.30%, 5.74 ± 0.55% and 5.09 ± 0.79% per minute. (Percentages are calculated relative to the overall change in spectroscopic values from time zero to apparent equilibrium).

This procedure allows a relatively rapid analysis of aggregation behavior for a large number of samples, provided that the size of the aggregation is small enough to be reproducibly detected using spectroscopy. These direct experiments can therefore monitor the early stages of the aggregation process before the development of mature amyloid fibrils after a few days. However, it has been shown that such early aggregates are well-ordered, have many elements of the ultrastructure of amyloid fibrils, and that their formation correlates well with subsequent amyloid fibril formation. .

One issue that must be carefully considered when considering complex processes such as aggregation and amyloid formation is the reproducibility of kinetic behavior and kinetic parameters resulting from such experiments. To address this issue, ThT resulting from aggregation
The time course of fluorescence was measured 7 times for wild-type AcP. The resulting velocity signature is highly reproducible, yielding values of 5.43% and 0.79% / min for the average aggregation rate and its standard deviation, respectively. This number for standard deviation was used to assess the significance of the difference in aggregation rate measured for various mutants compared to wild-type protein.

Effect of Mutations on Aggregation Rate The time course of changes in ThT fluorescence induced by aggregation was measured for all 34 mutants of AcP. FIG. 12 shows the results obtained for various representative mutants compared to the results obtained with the wild-type protein (data for wild-type is shown in dashed lines in all three panels). Most mutants show similar aggregation rates to the wild-type protein within experimental error, as shown here for the I75V mutant (FIG. 12A). However, some mutants were found to aggregate significantly slower (Figure 12B) or faster (Figure 12C) than the wild-type protein. To confirm that these differences were reproducible and significant, mutants with different rates than wild-type AcP were analyzed at least twice.

FIG. 13 summarizes the results obtained for all AcP variants.
Each bar shows the aggregation rate of the AcP variant carrying the mutation at the position reported on the x-axis. Kinetic data are reported as the natural logarithm of the ratio of the numbers for the mutant and the wild-type protein (1n (ν _mut / ν _wt )). A value of zero means that there is no difference between the mutant and wild-type protein aggregation rates,
Numbers above and below zero correspond to faster and slower aggregation rates than wild type, respectively. The analysis of the propagation of experimental error for ν _mut and ν _wt, carried out according to the statistical principle, described in the footnote of FIG. 13, shows whether the difference between the mutant and wild-type protein velocities is significant. Will help you evaluate. AcP
It is clear from the figure that most of the mutants of E. coli aggregate at a rate equal to the wild-type protein within experimental error. This is true for all 22 mutations discussed here within the regions of sequences 1-15 and 32-86. In contrast, all six mutations in regions 16 to 31 (ie V17A, V20A, F22L, Y2
5A, E29D and A30G) and 5 of the 6 mutations within the C-terminal region 87-98 (S87T, L89A, Y91Q, S92T and Y98Q) lead to aggregation rates that differ significantly from those of the wild-type protein. The sensitivity of polypeptide chains to amino acid substitutions within these regions suggests that these extensions play a crucial role in the rate-determining step of AcP aggregation.

The tests presented here show that only the region of the sequence containing residues 16 to 31 and 87 to 98 is the early step in amyloid formation, ie the rate of conversion of the entire conformation to the initial aggregates present in the denatured state. Suggest that it is important in determining. Of course, the question arises as to what kind of information these results provide regarding the mechanism of AcP aggregation. The mutational approach used here provides information about regions of the protein that play a critical role in the process of aggregation, rather than identifying regions associated with the characteristic β-sheet structure or resulting aggregates. From spectroscopic studies performed with CD and FT-IR, it is clear that a significant proportion of AcP sequences are present in the β-sheet conformation within amyloid fibrils as well as within preformed aggregates. Regions other than those found to be important in the kinetic processes studied here are considered to be part of the β-sheet structure of aggregates and are less involved in the determination step for their expression.
The result therefore is that the regions of sequences 16 to 31 and 87 to 98 are of amyloidogenic species formed prior to intermolecular binding of various AcP molecules or self-assembly of the polypeptide chain during the growth of aggregates. It is suggested to be involved in stabilization. Such specific information about the rate-determining step of the process is important for the design of strategies aimed at delaying and inhibiting the process of amyloid formation.

Hydrophobicity and Beta Sheet Propensity Determine Aggregation Rate Analysis of specific amino acid substitutions that alter aggregation rate provides important details about the aggregation process. All mutations that delay aggregation (V17A, V20A,
F22L, Y25A, L89A, Y91Q and Y98Q) have low hydrophobicity (Ro
seman, 1988) and a low endogenous propensity to form β structures (Chou and Fasma, 1978 method or Street and Mayo,
Substitution of one residue for another with (as defined by any of the 1999 methods). Two accelerated mutations (S87Y and S92T
) Involves the addition of an extra methyl group at the position of the mutation, promoting β-sheet structure. The two accelerating mutations in regions 16-31 (E29D and A30G) alter the hydrophobicity of the protein to a lesser extent, but more dramatically, they are alpha of the polypeptide chain.
Significantly reduces helical propensity (Munoz and Serrano, 1994a, b
). Hydrophobicity and alpha-helical propensity within the key region therefore appear as determinants of the aggregation process for AcP. This means that the intermolecular binding of AcP molecules, which determines the rate of the aggregation process, makes use of both hydrophobic interactions and the formation of β-sheet structures.

Experimental Materials and Methods for Example 9 Mutant Generation and Purification Site-directed mutagenesis and purification of AcP mutants was performed as previously described (Taddei et al., 1996b). DNA sequencing and electrospray mass spectrometry were used to confirm that the desired mutation was present in each case. All proteins used in this test have a cysteine at position 21 replaced with a serine residue. For simplicity, proteins carrying only the C21S mutation are referred to as wild-type AcP and all those carrying the C21S substitution in addition to the mutation of interest are referred to as single point mutants. Thioflavin T, Congo Red and trifluoroethanol were all purchased from Sigma-Aldrich.

Aggregation kinetics demonstrated by Thioflavin T fluorescence AgP and its mutants were aggregated at 25% (vol / vol) TFE, 50 mM.
It was started by incubating the protein at a concentration of 0.4 mg / ml at 25 ° C. in acetate buffer, pH 5.5. A 62 μl aliquot of this solution, taken at regular time intervals, was mixed with 438 μl of 25 μM ThT, 25 mM phosphate buffer, pH 6.0 and the fluorescence was measured at 480 nm (using an excitation wavelength of 445 nm) to determine protein aggregates. Was monitored for expression. This test is based on an increase in ThT fluorescence upon binding to ordered aggregates (LeVine II
I, 1995). Fluorescence measurements were performed using a Shimadzu RF-5000 spectrofluorometer kept at a constant temperature of 25 ° C. Since the aggregation rate was found to be substantially dependent on the protein concentration, UV absorption using an ε ₂₈₀ value of 1.49 ml / mg / cm showed that various stock solutions of AcP variants were stored immediately before use. The protein concentration was investigated. Epsilon ₂₈₀ values for AcP mutants with tyrosine or tryptophan residues substituted with other residues were calculated as previously described (Grill and von Hippel, 1989).

Aggregation kinetics demonstrated by Congo red binding Expression of aggregates by wild-type AcP was also revealed by measuring the increase in Congo red absorbance at 540 nm (Klunk et al., 1989). The agglomeration process was initiated as described above. 62 μl aliquots were taken at regular time intervals and mixed with 438 μl of a solution containing 10 μM Congo Red, 150 mM NaCl, 5 mM phosphate buffer, pH 7.4. The absorbance of such mixtures was measured at 540 nm 2-3 minutes after equilibration.

Aggregation Kinetics Evidenced by CD Measuring the expression of aggregates over time using a Jasco J720 spectropolarimeter kept isothermally and a 1 mm path length cuvette to measure the ellipticity reduction at 216 nm. According to the conditions described above.

[0198]   Determination of aggregation rate   The test presented here consists of a comparative analysis of the aggregation processes of various AcP variants.
, It is important to quantify the aggregation rate in each case. We delayed nucleation
Immediately after, that is, at the stage where the aggregation rate is maximum, the aggregation rate (ν) is measured
. Regarding the aggregation rate of AcP, this corresponds to the time point when the first data is obtained
. The first data point of the ThT fluorescence vs. time plot is therefore the aggregation rate
A linear fit was used to measure the increase in fluorescence per minute (ΔF) at maximum.
Let This increase was converted to a percentage change in total fluorescence using the following formula:       ν = 100 · ΔF / (F_eq-F₀) In the formula, ν is the aggregation rate, and F_eqIs the final fluorescence obtained at the end of velocity observation, F ₀ Is the initial fluorescence extrapolated at time zero. The use of this equation is
Allows direct comparison of body aggregation rates (especially when initial or final fluorescence values differ
When). It also facilitates comparison of velocity measurements obtained for various spectroscopic probes.
It

[0199]

[Table 6]

[Brief description of drawings]

FIG. 1A: Thermal denaturation analysis of WT-ADA2h and M1, M2 and DM variants at pH 3.0. (A) WT heating (white circle), WT cooling (white square), DM heating and cooling (black circle, heating and cooling data overlap).

FIG. 1B: Thermal denaturation analysis of WT-ADA2h and M1, M2 and DM variants at pH 3.0. WT at 25 ° C (white circle), 95 ° C (white square) and 25 ° C (black circle) after cooling
-Far-UV CD spectra of ADA2h and mutants. WT

FIG. 1C: Thermal denaturation analysis of WT-ADA2h and M1, M2 and DM variants at pH 3.0. WT at 25 ° C (white circle), 95 ° C (white square) and 25 ° C (black circle) after cooling
-Far-UV CD spectra of ADA2h and mutants. DM

FIG. 1D: Thermal denaturation analysis of WT-ADA2h and M1, M2 and DM variants at pH 3.0. WT at 25 ° C (white circle), 95 ° C (white square) and 25 ° C (black circle) after cooling
-Far-UV CD spectra of ADA2h and mutants. M1

FIG. 1E: Thermal denaturation analysis of WT-ADA2h and M1, M2 and DM variants at pH 3.0. WT at 25 ° C (white circle), 95 ° C (white square) and 25 ° C (black circle) after cooling
-Far-UV CD spectra of ADA2h and mutants. M2

FIG. 1F: Thermal denaturation analysis of WT-ADA2h and M1, M2 and DM variants at pH 3.0. F of WT-ADA2h sample before heat denaturation (solid line) and after heat denaturation (dashed line)
Amide I band in TIR spectrum.

2A: Urea reconstitution of WT-ADA2h and DM mutants at pH 3.0, WT-
It is a far-ultraviolet CD spectrum of ADA2h.

FIG. 2B: Urea reconstitution of WT-ADA2h and DM mutants at pH 3.0, DM-
It is a far-ultraviolet CD spectrum of ADA2h.

FIG. 3: AGADIR predictions performed over the entire sequence of PI3-SH3 to identify regions of the protein with a higher endogenous propensity that adopt a helical conformation.

FIG. 4. PI3-SH3 sequence. Secondary structural elements (s = strand, 3 = 310 helix) are shown after structural analysis by the DSSP package.

FIG. 5 is an AGADIR prediction representing the intrinsic helical propensity exhibited by wild type and E17R / D23R mutant sequences.

FIG. 6: Prediction of the helical propensity of human PrP helix C residues 198-227 assessed using AGADIR.

[Figure 7]   4 is a far-UV CD spectrum of four helix C peptides at pH 7.0.

FIG. 8. TFE titration of four helix C peptides monitored by measuring ellipticity at 222 nm. In each case, the buffer was 20 mM sodium phosphate, 20 mM β-mercaptoethanol.

FIG. 9A is a far-UV CD spectrum of helix C peptide at pH 2.0 showing a peptide concentration of 20 μM.

FIG. 9B is a far-UV CD spectrum of helix C peptide at pH 2.0 showing a peptide concentration of 60 μM.

FIG. 10. Far-UV CD spectrum of helix C peptide at pH 2.0 after 2 months incubation at room temperature. The peptide concentration was 60 μM.

FIG. 11A   Time course of wild-type AcP aggregation monitored by ThT fluorescence.

FIG. 11B: Time course of aggregation of wild-type AcP monitored by Congo red absorbance.

FIG. 11C   Time course of aggregation of wild-type AcP monitored by far-UV CD.

FIG. 12A: Time course of ThT fluorescence increase upon aggregation for some representative mutants, I75V (filled circles) (panel A).

FIG. 12B: Time course of ThT fluorescence increase upon aggregation for some representative mutants, V20A (open circles) and Y98Q (black circles) (panel B).

FIG. 12C—Time course of ThT fluorescence increase upon aggregation for some representative mutants.
The mutants presented are E29D (white circles) and A30G (black circles) (panel C).

FIG. 13 is a bar graph showing changes in aggregation rate resulting from mutation of residues at various positions, each bar representing a variant with an amino acid substitution at the position shown on the x-axis.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 A61P 11/00 A61P 3/10 25/00 11/00 25/14 25/00 25/16 25/14 25/28 25/16 37/00 25/28 43/00 111 37/00 117 43/00 111 C07K 14/54 117 117 C12N 15/00 D C07K 14/54 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW) , EA (AM, AZ, BY, KG KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC , LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Srud, Hess England, Otsk Ford O.E.x 1.3・ Kew Tay, South Parks Road, New ・Mistory Laboratory, Oxkusford Center For Moreculer Sciences (72) Inventor Aviles, Francesque Spain, E-08193 Veziyaterra, Universitat Autonoma de Barcelona, I Institut Des Biolohia Huonumental, Unitat de Cienciés, Departamento de Biokimika y Biolohia Molecula (72) Inventor Dobson, Christopher Martin, England Tee, South Parks Road, New Chemistry Laboratory, Ottsk Ford Center For More Molecule Sciences (72) Inventor Serrano, Luis Germany, 69117 Heidelberg, Maijahoshi Utra -1, European-Moleculer Biology Laboratory F-term (reference) 4B024 AA01 AA11 BA26 CA04 DA02 EA04 GA11 HA01 HA12 4C084 AA01 AA02 AA06 AA07 AA13 BA02 BA08 BA44 BA50 CA18 DA16 DC50 NA14 ZA011 ZA091 ZA091 ZA021 ZA021ZA021 ZA021ZA ZC351 4C086 AA01 AA02 AA03 AA04 EA16 NA14 ZA01 ZA02 ZA15 ZA16 ZA59 ZB09 ZC02 ZC35 4C087 AA01 AA02 BB70 BC83 CA04 CA10 CA12 NA14 ZA01 ZA02 FA50 A20 FA50 CA50 A50 FA50 A40 FA50 A40 A50 A40 AA02 AA02

Claims (31)

[Claims] 1. A method for designing a modified polypeptide having an altered tendency to aggregate as compared to an unmodified polypeptide, comprising analyzing a predetermined amino acid sequence of the polypeptide to determine the local structure-forming polypeptide. Determining the propensity and comparing the propensity of the modified polypeptide to form a local structure with the propensity of the unmodified polypeptide to form a local structure, thereby allowing the modified polypeptide to aggregate in a denatured state relative to the unmodified polypeptide. A method as defined above, comprising determining whether it has an altered tendency and producing a selected modified polypeptide having an altered tendency to aggregate. 2. A method for producing a modified polypeptide having an altered tendency to aggregate as compared to an unmodified polypeptide, comprising: (i) providing the modified polypeptide with a local structure in a denatured state as compared with the unmodified polypeptide. At least one amino acid modification is introduced into the predetermined polypeptide sequence so as to have an altered propensity to form, and optionally (ii) recovering the modified polypeptide and / or optionally (iii) the modified polypeptide Forming an aggregate in the method. 3. The method of claim 1 or 2, wherein the native state of the modified polypeptide has a global folding that is substantially equal to the native state of the unmodified polypeptide. 4. The method of claim 1, 2 or 3 wherein the polypeptide has a reduced propensity to aggregate as compared to the unmodified polypeptide. 5. A type of local structure having an altered propensity to form a polypeptide,
The method according to any one of claims 1 to 4, which is an α-helical secondary structure. 6. Amino acid modifications of amino acids not required for the activity of the polypeptide, wherein the amino acid modification is (a) in a completely solvent exposed position and (b) does not interact with residues outside the local structure. The method according to claim 1, wherein the method is performed by substitution. 7. The method according to claim 1, wherein the aggregate is a non-covalent aggregate. 8. The aggregate of claims 1 to 7, wherein the aggregate is fibril.
The method according to any one of 1. 9. The unmodified polypeptide is a disease, preferably an amyloid disease,
Any of claims 1-8, which is associated with a cystic fibrosis or emphysema, more preferably an amyloid disease selected from Alzheimer's disease, type II diabetes, systemic amyloidosis, Parkinson's disease, Huntington's disease or infectious spongiform encephalopathy. The method described in paragraph 1. 10. The method according to any one of claims 1 to 9, wherein the polypeptide is a cytokine, preferably interleukin, more preferably interleukin-4. 11. A method for producing a polynucleotide encoding a modified polypeptide as defined in any one of claims 1 to 10, a complementary strand thereof, or a fragment of any sequence, which comprises: The amino acid sequence of the determined polypeptide sequence is analyzed to determine the propensity of the polypeptide to form the local structure, and the propensity of the modified polypeptide to form the local structure and the propensity of the unmodified polypeptide to form the local structure to be determined. The modified polypeptide is thereby compared to determine whether the modified polypeptide has an altered propensity to aggregate compared to the unmodified polypeptide, and the polynucleotide sequence encoding the unmodified polypeptide encodes the polynucleotide encoding the modified polypeptide. Identifying the modifications required to encode, and (a) the modified nucleotide sequence being any of claims 1-10. A polynucleotide encoding an unmodified polypeptide is modified to encode a modified polypeptide as defined in section, or (b) as defined in any one of claims 1-10. Any of the preceding methods comprising synthesizing a polynucleotide that encodes the modified polypeptide. 12. A method of producing a polynucleotide encoding a modified polypeptide as defined in any one of claims 1 to 11, a complementary strand thereof, or a fragment of any sequence thereof, comprising: (A) modify the polynucleotide encoding the unmodified polypeptide such that the modified nucleotide sequence encodes the modified polypeptide as defined in any one of claims 1 to 11, or b) Any of the above methods comprising synthesizing a polynucleotide encoding a modified polypeptide as defined in any one of claims 1-11. 13. Making a vector for expression of a modified polypeptide as defined in any one of claims 1 to 10 or cloning of a polynucleotide as defined in claim 11 or 12. A method according to claim 11 or 1.
Said method comprising inserting a polynucleotide as defined in 2 into a vector. 14. A cell, cell culture or poly containing a polynucleotide as defined in claim 11 or 12 or a vector as defined in claim 13 or transformed with such a polynucleotide or vector. A method for producing a cell organism, comprising: (a) a polynucleotide as defined in claim 11 or 12 or claim 1
Transforming the cells with a vector as defined in 3 and, optionally, (b) culturing the cells obtained by (a) under conditions allowing selective growth of the transformants, and optionally (C) culturing the cells obtained according to (a) or (b) under conditions suitable for producing a multicellular organism. 15. A method of producing a modified polypeptide as defined in any one of claims 1-10, comprising: a) a cell, culture or organism as defined in claim 14. Claims 1 to 1
0) keeping under suitable conditions to produce a modified polypeptide as defined in any one of 0 and isolating said polypeptide, or b) any one of claims 1-10. Any of the preceding methods comprising chemically synthesizing a modified polypeptide as defined in Section. 16. A method for the production of aggregates, comprising contacting a modified polypeptide as defined in any one of claims 1 to 10 under conditions allowing nucleation. 17. A modified polypeptide, polynucleotide, vector, cell, cell culture, organism or aggregate as defined in any one of claims 1-16. 18. An aggregate comprising a modified polypeptide as defined in any one of claims 1-10. 19. The aggregate according to claim 18, which has an altered tendency to dissociate as compared to an aggregate which does not comprise a modified polypeptide as defined in any one of claims 1-10. 20. To alter the aggregation propensity of a polypeptide so that the denatured state of the polypeptide has an altered propensity to form a local structure as compared to the unmodified polypeptide, and the native state is the unmodified polypeptide. Use of a modification of a polypeptide amino acid sequence so that it has a global folding that is substantially equal to. 21. A modified polypeptide as defined in any one of claims 1-10, 15 or 20 or a modified polypeptide according to claim 17, as defined in claim 11 or 12, or A polynucleotide according to claim 18, as defined in claim 13, or a vector according to claim 17, a cell as defined in claim 14 or a cell, cell culture or according to claim 17. 20. A pharmaceutical composition comprising an organism or an agent as defined in claim 16 or selected from the aggregates according to any one of claims 17 to 19 and a pharmaceutically acceptable carrier. 22. A modification as defined in any one of claims 1-10, 15 or 20 or according to claim 17 for use in a method of treatment or diagnosis of the human or animal body. A polypeptide, as defined in claim 11 or 12, or a polynucleotide according to claim 17, as defined in claim 13, or a vector according to claim 17, defined in claim 14. 20. A cell, cell culture or organism according to claim 17, or as defined in claim 16, or an aggregate according to any one of claims 17-19. 23. A modified polypeptide as defined in any one of claims 1 to 10, 15 or 20 or in claim 17 in the manufacture of a medicament for use in the treatment of amyloid disease. , As defined in claim 11 or 12, or the polynucleotide of claim 17, as defined in claim 13, or the vector of claim 17, as defined in claim 14. 20. A cell, cell culture or organism according to claim 17, or use of an aggregate as defined in claim 16 or according to any one of claims 17-19. 24. A polypeptide as defined in any one of claims 1-10, 15 or 19 or according to claim 17 for use in a method of treatment or diagnosis of the human or animal body. 20. An aggregate as defined in claim 16 or according to any one of claims 17 to 19 which is in a sustained release form of: The release rate of the unmodified polypeptide as defined in any one of paragraphs 1-10, 15 or 20 or from the polypeptide-free aggregate of claim 17 is altered as compared to the release rate. The aggregate. 25. Defined in claim 16 as defined in any one of claims 1-10, 15 or 20 or as an in vitro sustained release form of the polypeptide of claim 17. 20. Use of an aggregate as described above or in any one of claims 17 to 19 wherein the release rate of the polypeptide from the aggregate is 20.
18. An aggregate as defined in any one of claims 10, 15 or 20 or having an altered release rate relative to the release rate of the unmodified polypeptide from the polypeptide-free aggregate of claim 17. Use of. 26. A method of treating a disease in a patient in need thereof comprising administering to the patient an active agent as defined in claim 21. 27. A polypeptide or aggregate obtained by the method according to claim 1 or 2. 28. A method for evaluating the tendency of a polypeptide to form aggregates in a denatured state, comprising analyzing the amino acid sequence of the polypeptide to determine the propensity of the polypeptide to form a local structure, The tendency of the peptide to form a local structure is compared with the tendency of the predetermined polypeptide sequence to form a local structure, and thereby the polypeptide is aggregated in a denatured state as compared to the predetermined polypeptide. A method as described above comprising determining whether or not it has a tendency. 29. A method of designing a polypeptide having an altered tendency to aggregate, comprising analyzing the amino acid sequence of the polypeptide to determine the propensity of the polypeptide to form a local structure, and modifying the amino acid sequence. Said method comprising introducing and assessing the effect of the modification on the propensity of the polypeptide to form local structures, and thereby designing the modified polypeptide with an altered propensity to form aggregates. 30. A method of producing a polypeptide having a selected propensity to form aggregates, wherein the method of claim 28 or 29 is practiced to thereby form a selected or altered aggregate. Identifying said polypeptide for propensity for production and producing a selected polypeptide. 31. A method of assessing an individual's risk or susceptibility to an amyloid or aggregate-related disease, which comprises identifying the amino acid sequence of a polypeptide expressed in an individual associated with an amyloid or an aggregated disease. A method comprising assessing the tendency of a polypeptide to aggregate according to.