WO1994029441A1 - Protein which catalyses peptide refolding and uses thereof - Google Patents

Abstract

A protein, that is characterised in catalysing protein or peptide functional refolding processes and having the amino acid sequence as follows: Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Ile Met Ile Ser Phe Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Val Gln Lys Lys; or a fragment or a homologous sequence thereof that maintain the ability to catalyse protein or peptide said functinal refolding processes.

Description

PROTEIN WHICH CATALYSES PEPTIDE REFOLDING AND USES THEREOF.

DESCRIPTION

The invention relates to proteins, that are able to catalyse the refolding process of protein molecules, to a process to obtain such proteins and to uses thereof. More specifically the invention concerns proteins catalysing the folding process of proteins or peptides, i.e., by providing the correct tertiary or quaternary structure and by restoring the biologically active conformation, to be used for the production of recombinant proteins through biotechnology techniques, for pharmaceutical, cosmetic or foodstuff industry.

A protein is not biologically active when its native conformation is lost. The native conformation, which represents the most thermodynamically stable protein structure, shall take place in the polypeptide chain during the synthesis thereof on the ribosome chain, or immediately thereafter.

It was found (Gething, M. and Sambrook, Y., Nature, 355, 33-45, 1992) that this in vivo process is often catalysed by the so-called "chaperonin" proteins, that facilitate the folding process by binding to the new synthesised polypeptide chain. The chaperonin catalysing activity is ATP-dependent, being said triphosphate involved in the chaperonin release from the protein chain (Sadis, S. and Hightower, Biochemistry, 31 9406-9412, 1992).

Many proteins stabilise their own active conformation through disulfide bonds, that are formed by oxidising two cysteine residues, that are present into the same polypeptide chain, or among different chains. Different enzymes are known to be able to catalyse the disulfide bond of polypeptide chains. A typical enzyme is the disulfide iso erase protein (PDI) (Freed an, R.B. and al., Biochem. Soc. Sy p., 55, 167- 192, 1989) . PDI, as isolated from different vertebrate tissues, is an homodimer, having a molecular weight of 2x57,000 Da and a strong acidic isoelectric point (pi). This protein is largely present in the hepatic tissues, being app. 0.4% of all extractable proteins. A number of proteins could represent the PDI substrate, being PDI able to catalyse either the formation, or the reduction or the isomerization of disulfide bonds, according to the redox conditions in the reaction. The PDI enzymatic activity is dosed by following the scrambled ribonuclease A (srRNAse) renaturation process. srRNAse is a ribonuclease, containing disulfide bonds in not correct sites. The enzyme could be obtained by chemically oxidising a ribonuclease, previously reduced in the presence of high urea concentration, thus preventing the polypeptide chain to be arranged in the most thermodynamically favourable active conformation, which is stabilised by specific intramolecular disulfide bonds. Recently a role played by PDI in many protein modification processes was suggested (Obata, T. and al., J. Biol. Che ., 263, 782- 785, 1988; Wetterau, J. and al., J. Biol.Chem., 265, 9800-9807, 1990; Koivu, J. and al., J. Biol. Chem., 262, 6447-6449, 1987; Geetha-Habib, M. and al., Cell, 54, 1053-1060, 1988) .

The PDI primary structure is characterised in having two homologous regions, similar to the active site of thioredoxin (TH) . TH is characterised in having two cysteine residues in close proximity, thus creating a dithiol/disulfide redox site. TH is a small acidic protein, having a molecular weight of app. 12,000 Da, that could be isolated from prokaryotes, yeasts, plants and mammalian cells (Holmgren, A., J. Biol. Chem., 264, 13963-13966, 1989) . TH is a multifunctional protein, catalysing the disulfide reduction, as well as other redox processes. It was found (Pigiet, V.P. and Schuster, B.J., Proc. Natl. Acad. Sci. USA, 83, 7643- 7646, 1986) that TH is able to renaturate both the srRNAse, and a denaturated and reduced ribonuclease (rdRNAse) . This could suggest that TH could play a role in the in vivo folding of cell proteins. Recently it was found (Lundstrom, J. & Holmgreen, J. Biol.Chem., 265, 9114-9120, 1990) that PDI acts as a thioredoxin reductase substrate, as TH, and has a TH similar activity, therefore further confirming the functional and structural similarities of these proteins.

A third enzyme class, involved in SH redox processes, includes the sulphydryl oxidase class. This class consists of enzymes, which do not catalyse the disulfide bond interchanges, as PDI does. The enzyme sulphydryl oxidase, as isolated from bovine milk, was described (Janolino, V.G. and Swaigood, H.E., Arch. Bioc. Biophys.,258, 265-271,1987). This enzyme is a metalloglycoprotein, that is able to catalyse the oxidation of thiol groups of low molecular weight compounds, such as glutathione, cysteine, 2- mercaptoethanol, dithiothreitol, and of reduced proteins. Another soluble sulphydryl oxidase, that is able to catalyse the disulfide binding formation, was isolated from the male mammalian reproduction system (Ostrowski, M.C. and Kistler, .S., Biochemistry, 19, 2639-2645, 1980) . The same authors of the instant invention have found an enzymatic activity in prokaryotes for the first time, which is able to catalyse redox processes of SH residues (DBFE: disulfide bond-forming enzyme), thus allowing the renaturation of a protein model system, such as rdRNAse, (Guagliardi, A., Cerchia, L., De Rosa, M., Rossi, M. and Bartolucci, S., FEBS Lett., 303, 27-30, 1992) . The authors have purified a protein, having such catalysing properties, from the cytosol of the extreme thermoacidophilic archaeobacterium Sulfolobus solfataricus, strain MT4. However, the isolated enzymes up to the instant invention are substantially mesophilic enzymes and, as such, are therefore unstable, even for short times, under moderate temperature and special process conditions (not perfectly aqueous medium, presence of kaotropic agents, etc.).

The authors have now identified and characterised a very stable new enzyme, isolated from the archaeobacterium thermoacidophilic Sulfolobus solfataricus, strain DSM 1617 (Germany), that is able to catalyse the protein folding process through a mechanism which is different from enzymatic mechanisms known in the previous art. Actually, this enzyme is not only able to catalyse a protein folding by an ATP- dependent mechanism, but also to stabilise the structure by disulfide bond-forming, when sulphydryl residues are present. Therefore such enzyme shows two different catalysing activities, that are both critical in carrying out a correct protein refolding process.

Due to its own peculiarities, the enzyme according to the invention may be used advantageously in a number of industry fields, and particularly in drug, cosmetic and food industry. The enzyme of the invention have many applications for the production on industrial scale of recombinant proteins, wherein the formation of inclusion bodies affects negatively the process yield. As a matter of fact, due a not proper secondary structure, recombinant proteins often lack their own biologic properties and are present as quite insoluble bodies. By treating inclusion bodies with the enzyme of the invention, high yields in the production of enzymes through recombinant techniques are obtained. Also on industrial scale, the use of the enzyme of the invention allows to regenerate immobilised enzymes, which lost their catalysing activity because of a not correct folding. The characteristics of the enzyme of the invention are interesting for the use in therapy, e.g. of Alzheimer's disease which is caused by a not correct protein folding. In the cosmetic industry, the enzyme of the invention allows to act on skin ageing and hair treatments. Finally in the food industry, the enzyme of the invention could be used satisfactorily in improving the rheologic characteristics of wheat flour mixtures, associated in optimising the folding and the sulphide bond intermolecular cross-linking mechanisms in the mixture preparation, baking and cooking steps. Therefore it is an object of the invention a protein, characterised in catalysing either protein or peptide functional refolding processes; and in comprising the amino acid sequence as follows and as specified in SEQ ID No.l: Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp lie Ser Lys lie Lys Lys Val Trp Arg Val Gly Lys lie Met lie Ser Phe Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu Lys Asp Ala Pro Lys Glu Leu Leu Gin Met Leu Val Gin Lys Lys; or a fragment or an homologous sequence thereof, having the ability to catalyse said either protein or peptide functional refolding process.

In a preferred embodiment, said protein is purified from bacteria, preferably from the Sulfolobus genus, more preferably from Sulfolobus solfataricus species, and most preferably from DSM 1617 strain.

Alternatively, said protein is obtainable through a chemical synthesis or recombinant techniques.

It is a further object of the invention the use of the protein according to this invention for protein and peptide preparation processes according to recombinant DNA technique; or for the refolding process of immobilised enzymes.

A further use of the enzyme of the invention consists of preparing pharmaceutical compositions, preferably to be used in the Alzheimer's disease therapy; or, alternatively in products of the food industry; or, alternatively in cosmetic compositions.

The invention will described in the following by way of some illustrating, but not limiting, examples.

EXAMPLE 1 - PROCEDURE TO EXTRACT AND PURIFY THE PROTEIN a) PROTEIN EXTRACTION

Cells of Sulfolobus solfataricus, strain DSM 1617 were grown, as described by Ammendola et al., Biochemistry, 31, 12514, 1992. 100 g of bacteria, suspended in 40 ml of 20 mM of Tris-HCl, pH 8, containing 0.2 M of NaCl and 5% of glycerol, were lysated through instantaneous depressurization using a Parr's bomb. The homogenate, clarified by a 160,000 g centrifugation at 4°C, contained app. 5 g of proteins. b) PURIFICATION ON DEAE SEPHAROSE FAST FLOW (Pharmacia)

400 mg of homogenate, exhaustively dialysed in Spectra/Por dialysis tubes with a cut-off of 6,000, with 100 volumes of 10 mM Tris-HCl, pH 8,4 (A buffer), were then charged on a 2.5 x 20 cm DEAE Sepharose Fast Flow (Pharmacia) column, balanced with the A buffer at 4°C and eluted with A buffer at a flow rate of 40 ml/h. The active fraction (50 mg) was concentrated by a vacuum centrifugation and exhaustively dialysed against the A buffer. c) PURIFICATION ON FPLC Mono Q

Fractions of 20 mg of protein were purified on a 0.5 x 5 cm Mono Q column (Pharmacia), balanced with A buffer and eluted with the same buffer at a flow rate of 1 ml/min. The active fraction (6 mg) was concentrated by a vacuum centrifugation and exhaustively dialysed against the A buffer. d) PURIFICATION ON 75 FPLC SUPERDEX

Aliquots of 6 mg of the protein were purified on a 2.6 x 60 cm Superdex column (Pharmacia), balanced with A buffer, and eluted with the same buffer, containing 0.2 M of NaCl, at a flow rate of 2 ml/min. The active fraction (0.3 mg) was concentrated by a vacuum centrifugation and exhaustively dialysed with the A buffer. e) ELECTROPHORESIS ON POLYACRYLAMIDE GEL OF THE PURIFIED PROTEIN

The electrophoresis analysis of the sample obtained from the gel filtration was carried out at pH 4.5, as described by Reisfeld and al. , Nature, 195,

281, 1962, and showed only one protein band, thus indicating the homogeneity of the obtained protein. f) ELECTROPHORESIS ON SDS-POLYACRYLAMIDE GEL

In order to determine the enzyme molecular weight, an electrophoresis on a SDS polyacrylamide gel was carried out, as described by Laemmli, Nature, 227, 680, 1970. Stacking gel at 5% concentration and separating gel at 15% concentration were used. In order to define the molecular weight, a calibration kit (Sigma) was used within the range from 16.9 to 2.5 kDA, and the proteins were detected using a Silver staining kit (Sigma) . The electrophoresis showed only one band, having a molecular weight of 6200 + 300 Da. EXAMPLE 2 - DETERMINATION OF THE AMINO ACID SEQUENCE The a ino acid sequence, from the amino terminal, was defined by using a mod. 477A, Applied Biosystems Sequencer, together with a mod. 120A Analyser with phenylthiohydantoin, according to Manufacturer's instructions. The sequencing process was carried out for 63 cycles, thus providing identifiable residues and defining the whole protein sequence, as follows:

Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp lie Ser Lys lie Lys Lys Val Trp Arg Val Gly Lys lie Met lie Ser Phe Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu Lys Asp Ala Pro Lys Glu Leu Leu Gin Met Leu Val Gin Lys Lys.

The NH2 amino acid terminus is an alanine and the COOH terminus is a lysine; furthermore the protein does not comprise cysteine residues, has only one tryptofan residue and is substantially basic. The protein comprises 63 amino acids, having a predictable molecular weight of 7,000 Da, sufficiently near to the 6,200 Da value, as obtained from the electrophoresis test on SDS-polyacrylamide gel. Lysines at 4 and 6 positions are monomethylated, as resulted from the retention times of their corresponding phenylthiohydantoin derivatives. Six residues, from glycine-36 to glycine-41, represented likely the interaction site with ATP, in conformity with the results on other ATP interacting proteins (Saraste, M., Sibbald, P.R. and Wittinghofe, A., Trends in Biochem. Sci., 15, 430-434, 1990). The whole sequence of the protein of the invention did show no evident homology level with chaperonins.

Recently the isolation of a protein was described from a different strain of Sulfolobus solfataricus, defined as MT-4, and having the amino acid sequence of the enzyme of the invention, the only reported biological activity of which having been the ability thereof to catalyse RNA hydrolysis (Fusi, P., Tedeschi, G., Aliverti, A., Ronchi, S., Tortora, P. and Guerritore, A., EJB, 211, 305-310, 1993). On the contrary, such enzymatic activity is not shown by the protein from the DSM 1617 strain of the Sulfolobus solfataricus according to this invention. EXAMPLE 3 - ENZYMATIC REOXIDATION AND RENATURATION OF A REDUCED AND DENATURATED RIBONUCLEASE

The renaturation activity of the enzyme of the invention was tested using as a substrate a reduced and denaturated ribonuclease (rdRNAse) . The rdRNAse was firstly prepared from 20 mg of bovine seminal ribonuclease (RNAse) , dissolved in 0.2 M of Tris-HCl, pH 9.0, containing 6 M of guanidine chloride and 0.28 M of 2-mercaptoethanol. The mixture, under a nitrogen flow, was corked and incubated in the dark, overnight at 37°C. The reactants were removed by a molecule removing chromatography procedure on a G-25 Superfine, 1 x 36.5 cm, Sephadex column, eluted with 0.01 M of degassified HC1 under nitrogen flow, at a flow rate of 12 ml/min. rdRNAse containing fractions were collected, submitted to a nitrogen flow and maintained at - 20°C. The yield was 50%, based on amount of the start material. The whole RNAse reduction (7.4 SH mol/1 RNAse mol) was tested, using the Ellman's reaction, as described in Ellman, G.L., Arch. Biochem. Biophys., 82, 70-77, 1954.

The reoxydation and the reactivation of rdRNAse were carried out in presence of the enzyme of the invention. The rdRNAse reoxidation was tested through defining the thiol group residual concentration by using the Ellman's method. The SH group titration was carried out by reacting them with a solution of 0.1 mM of DTNB, in 0.1 M sodium phosphate, pH 7.0, and 10 mM of EDTA. The absorption at 412 nm of this solution was measured each 5-10 minutes and the thiol group concentration was calculated on the basis of a value of the thiobenzoate anion extinction factor 13,600 cm^-1M^_1 A typical dosage of the reoxidation rdRNAse process, catalysed by the enzyme of the invention, is described here below. 50 mM of a sodium phosphate solution, pH 8.0, containing 15 μM of rdRNAse, and the enzyme of the invention, were incubated in a sealed glass vial at 30, 45 or 55°C. The same mixture, but containing a buffer instead of the enzyme of the invention, acted as a control, measuring the spontaneous reoxidation of sulphydryl groups. The concentration of sulphydryl groups was measured at desired time intervals by the Ellman's method on the sample fractions, picked up with stirring from the incubated mixtures. While a spontaneous reoxidation process after 6 hours reached a peak not higher then 33%, a 6 hour interval was sufficient to reoxidize 90% of rdRNAse, when the enzyme of the invention was present.

The reactivation of rdRNAse catalysed by the enzyme of the invention, was carried out by testing the ribonuclease activity recovery, using the Kunitz's method, as described in Kunitz, M., J. Biol. Chem., 164, 563, 1946, through the absorption decrease at 300 nm by a solution of 0.5 mg/ml of RNA in 50 mM of sodium acetate, pH 5.2 (final volume 1 ml) at 30°C (readings vs. air readings) . The ribonuclease activity was dosed moreover through the absorption increase at 260 nm of a solution, containing 100 μg of RNA in Tris-HCl, 50 mM, pH 7.8, 25 mM of KC1, 5mM MgCl₂ (final volume 1 ml) at 30°C (reading versus air readings) . A standard operating procedure is described here below. Two identical mixtures (the control and the mixture containing the enzyme of the invention) , as those described to carry out the rdRNAse reoxidation test, were incubated at 30°C. At desired time intervals, the ribonuclease activity was dosed on fractions picked up from each mixture. The renaturation percentages in both control (i.e., free from the enzyme of the invention) and catalyst containing mixtures were calculated in respect of the catalysis rate of a native RNAse solution under the same test conditions. While the spontaneous renaturation process in a 6 hour interval reached a peak not higher than 4%, the presence of the enzyme of the invention during the same time interval was sufficient to provide the 40% of the rdRNAse renaturation. EXAMPLE 4 - SCRAMBLED RNAse ENZYMATIC REOXIDATION

The activity of the enzyme of the invention was defined by using scrambled RNAse (srRNAse) as substrate. srRNAse was prepared as described here below. In a typical procedure, 2 mg/ml of rdRNAse, prepared as in the Example 3, were diluted to 0.5 ml/mg with HC1, 0.1 M, and added (by weigh) with guanidine chloride, 4M, and pH was regulated to 8.5 with Tris-HCl, 1 M. The solution was allowed to be exposed to the air in the dark for 4 days at environment temperature. The salt was removed through a chromatography on Sephadex G-25 Superfine, as previously described; the protein peak was concentrated on SAVANT and maintained at - 20°C. The yield was 100%.

The rdRNAse reoxidation reaction, catalysed by the enzyme of the invention, was monitored by the Ellman's reaction, according to an experimental protocol as in Example 3. The reoxidized srRNAse did show no ribonuclease activity. While the spontaneous reoxidation process after 6 hours reached a peak not higher than 3%, in presence of the enzyme the same time interval was sufficient to reoxidize 30% of srRNAse.

EXAMPLE 5 - ENZYMATIC RENATURATION OF ALCOHOL DEHYDROGENASE EITHER FROM HORSE LIVER OR FROM S^ SOLFATARICUS The activity of the enzyme of the invention was defined by using a denaturated alcohol dehydrogenase, isolated from horse liver (HLADH) as well as from Sulfolobus solfataricus (SsADH) , as substrate. In a typical procedure, ADH was fully denaturated by incubating 6 mg of both HLADH and SsADH enzymes, in 3 M guanidine chloride, for 12 hours at 37°C. The enzymatic activity was dosed for HLADH at 35°C and for SsADH at 60°C respectively in an assay mixture of barbital buffer 25 mM, pH 8.0, NAD 2 mM and benzilic acid 5mM; then the reduced co-enzyme disappearance at 340 nm was monitored with a DSM-100 Spectrophotometer, having a cell equipped with a thermostat. The renaturation of both enzymes was determined in the presence or in the absence of the enzyme of the invention, by diluting 30 μg of each denaturated enzyme in guanidine chloride with 6 ml of sodium phosphate buffer 0.1 M, pH 8.0, zinc chloride 5 μM. The renaturation was monitored while maintaining the HLADH solution at 25°C and the SsADH solution at 50°C. The renaturation percentage was calculated in respect of the value that an identical enzyme amount would have shown before the renaturation process. 2 μg of the enzyme of the invention were added to each 30 μg fractions of the denatured enzyme, either in absence and in presence of ATP 0.5 mM of ATP, in the renaturation test. HLADH and SsADH, under the spontaneous renaturation conditions, as previously described, recovered no more than 18% and 25% of their catalysing ability, respectively, while the addition of the enzyme of the invention, as well as an ATP addition in the renaturation mixture, increased the above percentages up to 50% and 65%, respectively. The renaturation process was less effective when ATP was absent.

EXAMPLE 6 - RENATURATION OF SEPHAROSE IMMOBILISED SsADH The activity of the enzyme of the invention was defined using Sepharose immobilised and denaturated SsADH. SsADH was immobilised by covalent binding to CNBr activated Sepharose 4B at 4°C into a 100 mg/ml of a matrix in phosphate buffer 0.1 M, pH 8, according to the technique, as described by R. Axen, J. Porath and S. Ernback, Nature, 214, 1302, 1962. 5.0 mg of SsADH were allowed to react overnight with 10 ml of activated Sepharose; the remaining reactive groups were blocked with ethanolamine 0.1 M, and the sample fully washed out with a phosphate buffer 0.05 M, pH 7.5. The amount of the covalently bound enzyme resulted 3.1 mg/10 ml of Sepharose. The enzyme activity, as measured after the mixture centrifugation carried out according to the previously described technique, was 1.2 units/ml of gel. Two resin samples were centrifuged and suspended in 1 ml of guanidine hydrate chloride 2.4 M and maintained overnight at 37°C. No activity resulted after 16 hours, therefore the enzyme was denaturated. The suspensions were then centrifuged and washed three times with a 1 ml of phosphate buffer 0,1 M, pH 8.0. Both resin samples were suspended in 1 ml of renaturation phosphate buffer 0.1 M, pH 8.0, and ZnCl₂ 5 μM, and to one sample was added 6 μg of the enzyme of the invention, ATP and Mg⁺⁺ to a final concentration of 0.5 mM. The sample containing the enzyme of the invention, as well as the control, were maintained at 50°C, and 0.1 ml fractions were picked up at desired intervals in order to measure SsADH activity. After 2 hours, the immobilised SsADH recovered only 17% of its original activity when the enzyme of the invention was absent, whilst in the same interval and in presence of the enzyme of the invention, 78% was recovered. EXAMPLE 7 - INCLUSION BODIES

Inclusion bodies, that are produced further to overexpressing SsADH in E.coli, were isolated through a cell lysis with lysozyme, a lysate centrifugation at 6,000g for 5 minutes, followed by a further centrifugation at 15,000 g for 30 minutes. The pellet was dissolved in 3 M of guanidine chloride for 5 hours at 37°C to a 10 mg/ml concentration. Then the solution was diluted to 33 μg/ml in a sodium phosphate buffer 0.1 M, pH 8.0, zinc chloride 0.5 μM, in presence of 5 μg/ml of the enzyme of the invention. This mixture was incubated for 1 hour at 40°C, and 80% of the activity was recovered, on the basis of the known specific SsADH native activity. When the renaturation process was carried out in absence of the enzyme of the invention, the activity recovery resulted 23% only. EXAMPLE 8 - ENZYME KINETICS PARAMETERS

The enzyme showed a 2 hour stability when incubated at 80°C with an optimised pH of 7.2 in phosphate buffer 0.1 M, being the optimised temperature dependent upon the nature of the protein substrate. Many protein renaturations were obtained within the temperature range from 30 to 70°C.

This invention was described with reference to some preferred embodiments; it is understood that any variation or modification could be made by anyone skilled in the art without lost of scope of protection as defined by the following claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Consiglio Nazionale delle Ricerche

(B) STREET: P.le Aldo Moro, 7

(C) CITY: Rome

(E) COUNTRY: Italy (F) POSTAL CODE (ZIP) : 00185

(ii) TITLE OF INVENTION: Protein that are able to cat either protein or peptide functional refolding; process for production and uses thereof.

(iii) NUMBER OF SEQUENCES: 1

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (E

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 63 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Sulfolobus solfataricus (B) STRAIN: DSM 1617 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp li 1 5 10 15

Ser Lys lie Lys Lys Val Trp Arg Val Gly Lys lie Met lie Ser Ph 20 25 30

Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Gl , 35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gin Met Leu Val Gin Lys Lys 50 55 60

Claims

1. Protein characterised in catalysing the protein and peptide functional refolding process and comprising the amino acid sequence as shown in SEQ ID No. 1; or a fragment, or an homologous sequence thereof, that maintains the ability to catalyse said functional refolding process on proteins and peptides.

2. Protein according to claim 1 characterised in having been purified from bacteria.

3. Protein according to claim 2 wherein said bacteria are from the Sulfolobus genus.

4. Protein according to claim 3 wherein said bacteria are from the Sulfolobus solfataricus species.

5. Protein according to claim 4 wherein said bacteria are from the DSM 1617 strain.

6. Protein according to claim 1 characterised in being obtainable by chemical synthesis or recombinant techniques.

7. Use of the protein according to any of previous claims for protein or peptide preparation processes using recombinant DNA techniques.

8. Use of the protein according to claims from 1 to 6 for the immobilised enzyme functional refolding process.

9. Use of the protein according to claims from

1 to 6 for the preparation of pharmaceutical compositions, for the Alzheimer's disease therapy.

10. Use of the protein according to claims from 1 to 6 in foodstuff industry.

11. Use of the protein according to claim from

1 to 6 in cosmetic composition industry.