WO1994029441A1 - Proteine qui catalyse le repliement des chaines peptidiques et ses utilisations - Google Patents

Proteine qui catalyse le repliement des chaines peptidiques et ses utilisations Download PDF

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Publication number
WO1994029441A1
WO1994029441A1 PCT/IT1994/000084 IT9400084W WO9429441A1 WO 1994029441 A1 WO1994029441 A1 WO 1994029441A1 IT 9400084 W IT9400084 W IT 9400084W WO 9429441 A1 WO9429441 A1 WO 9429441A1
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WO
WIPO (PCT)
Prior art keywords
lys
protein
gly
glu
val
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PCT/IT1994/000084
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English (en)
Inventor
Mosè ROSSI
Mario De Rosa
Simonetta Bartolucci
Anna Maria Guagliardi
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Consiglio Nazionale Delle Ricerche
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Publication date
Application filed by Consiglio Nazionale Delle Ricerche filed Critical Consiglio Nazionale Delle Ricerche
Priority to AU69817/94A priority Critical patent/AU6981794A/en
Publication of WO1994029441A1 publication Critical patent/WO1994029441A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C07K1/1133—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by redox-reactions involving cystein/cystin side chains
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
    • C12Y108/01—Oxidoreductases acting on sulfur groups as donors (1.8) with NAD+ or NADP+ as acceptor (1.8.1)
    • C12Y108/01008—Protein-disulfide reductase (1.8.1.8), i.e. thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/04—Intramolecular oxidoreductases (5.3) transposing S-S bonds (5.3.4)
    • C12Y503/04001—Protein disulfide-isomerase (5.3.4.1), i.e. disufide bond-forming enzyme
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • PDI disulfide iso erase protein
  • srRNAse scrambled ribonuclease A 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.
  • PDI protein modification processes
  • 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.
  • TH is able to renaturate both the srRNAse, and a denaturated and reduced ribonuclease (rdRNAse) .
  • rdRNAse denaturated and reduced ribonuclease
  • This class consists of enzymes, which do not catalyse the disulfide bond interchanges, as PDI does.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • the enzyme of the invention allows to act on skin ageing and hair treatments.
  • 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.
  • 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.
  • said protein is purified from bacteria, preferably from the Sulfolobus genus, more preferably from Sulfolobus solfataricus species, and most preferably from DSM 1617 strain.
  • said protein is obtainable through a chemical synthesis or recombinant techniques.
  • 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 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.
  • rdRNAse reduced and denaturated ribonuclease
  • 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 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.
  • 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 ⁇ g of RNA in Tris-HCl, 50 mM, pH 7.8, 25 mM of KC1, 5mM MgCl 2 (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.
  • 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.
  • srRNAse scrambled RNAse
  • substrate RNAse
  • srRNAse scrambled RNAse
  • 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.
  • HLADH horse liver
  • SsADH Sulfolobus solfataricus
  • 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 ⁇ g of each denaturated enzyme in guanidine chloride with 6 ml of sodium phosphate buffer 0.1 M, pH 8.0, zinc chloride 5 ⁇ M.
  • 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 ⁇ g of the enzyme of the invention were added to each 30 ⁇ g 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.
  • 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.
  • 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 ZnCl 2 5 ⁇ M, and to one sample was added 6 ⁇ g 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.
  • 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 ⁇ g/ml in a sodium phosphate buffer 0.1 M, pH 8.0, zinc chloride 0.5 ⁇ M, in presence of 5 ⁇ g/ml of the enzyme of the invention.
  • 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.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
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  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
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  • General Health & Medical Sciences (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
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  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)
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Abstract

L'invention se rapporte à une protéine, qui se caractérise par le fait qu'elle catalyse les processus de repliement fonctionnel des chaînes protéiniques ou peptidiques et par le fait qu'elle comporte les séquences d'acides aminés suivantes: 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; ou à un fragment ou une séquence homologue d'une telle protéine, qui conserve le pouvoir de catalyser lesdits processus de repliement fonctionnel des chaînes protéiniques ou peptidiques.
PCT/IT1994/000084 1993-06-11 1994-06-10 Proteine qui catalyse le repliement des chaines peptidiques et ses utilisations WO1994029441A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU69817/94A AU6981794A (en) 1993-06-11 1994-06-10 Protein which catalyses peptide refolding and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITRM930384A IT1261917B (it) 1993-06-11 1993-06-11 Proteine che catalizzano il processo di strutturazione funzionale di molecole proteiche, procedimento per la loro produzione e loro usi.
ITRM93A000384 1993-06-11

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WO1994029441A1 true WO1994029441A1 (fr) 1994-12-22

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AU (1) AU6981794A (fr)
IT (1) IT1261917B (fr)
WO (1) WO1994029441A1 (fr)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ANNAMARIA GUAGLIARDI ET AL.: "Isolation of a thermostable enzyme catalyzing disulfide bond formation from the archaebacterium Sulfolobus solfataricus", FEBS LETTERS., vol. 303, no. 1, 25 May 1992 (1992-05-25), AMSTERDAM NL, pages 27 - 30 *

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AU6981794A (en) 1995-01-03
ITRM930384A0 (it) 1993-06-11
IT1261917B (it) 1996-06-04

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