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WO2001081366A2 - Purification de polypeptides inhibiteurs de l'enzyme de conversion de l'angiotensine contenant le vpp du lait - Google Patents

Purification de polypeptides inhibiteurs de l'enzyme de conversion de l'angiotensine contenant le vpp du lait Download PDF

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Publication number
WO2001081366A2
WO2001081366A2 PCT/US2001/012672 US0112672W WO0181366A2 WO 2001081366 A2 WO2001081366 A2 WO 2001081366A2 US 0112672 W US0112672 W US 0112672W WO 0181366 A2 WO0181366 A2 WO 0181366A2
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WIPO (PCT)
Prior art keywords
vpp
milk
protein
polypeptide
purification
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Ceased
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PCT/US2001/012672
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WO2001081366A3 (fr
Inventor
Joseph M. Kobzeff
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Monsanto Technology LLC
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Monsanto Technology LLC
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Priority to AU2001255469A priority Critical patent/AU2001255469A1/en
Publication of WO2001081366A2 publication Critical patent/WO2001081366A2/fr
Publication of WO2001081366A3 publication Critical patent/WO2001081366A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention generally relates to a method for producing peptides containing the tripeptide VPP. More particularly, the method encompasses collecting milk from a transgenic non-human mammal engineered to express and secrete a recombinant BSS polypeptide into its milk. This polypeptide contains significant amounts of the tripeptide VPP. The method further comprises hydrolyzing the proteinaceous matter from milk to purify the polypeptide. The VPP may then be further purified from this polypeptide.
  • Hypertension is generally known as the "silent killer" because its only readily detectable symptom is an abnormally increased blood pressure.
  • Abnormally increased blood pressure is clinically defined as a systolic blood pressure greater than 140 mmHg or a diastolic blood pressure greater than 90 mmHg.
  • Hypertension is the primary risk factor for coronary, cerebral, and renal vascular diseases, which collectively cause over half of all deaths in the United States. And no single or specific cause is known for the hypertension referred to as primary (essential) hypertension.
  • renin-angiotensin system plays an important role in salt-water homeostasis and the maintenance of vascular tone.
  • stimulation or inhibition of this system respectively, raises or lowers blood pressure, and may be involved in the etiology of hypertension. Hall, J.E., and Guyton, A.C. (1990), In Hypertension : Pathophysiology Diagnosis and Management, (Raven Press, Ltd., New York), pp .1105-1129.
  • Angiotensin converting enzyme (ACE) is an important mediator in the renin- angiotensin system.
  • ACE acts on angiotensin I, which is formed by decomposition of angiotensin secreted by the liver, by an enzyme, renin, produced in the kidney, and converts it to angiotensin II.
  • Angiotensin II then increases blood pressure by contracting the smooth muscles of the blood vessel walls and promoting secretion of aldosterone by action on the adrenal cortex.
  • ACE decomposes and inactivates a protein called bradykinin. Bradykinin dilates the blood vessels and lowers blood pressure. Therefore, a common method of decreasing blood pressure in an individual is to inhibit the activity of ACE.
  • Several compounds have been studied for their ability to reduce blood pressure by inhibiting the function of ACE in the renin-angiotensin mechanism.
  • ACE inhibiting substances are known and commonly used for the purpose of decreasing blood pressure in patients.
  • ACE inhibiting pharmaceuticals is the synthetic chemical product known as captopril (D-2 -methyl-3 -mercaptopropanoyl-L-proline) , which is an oral hypotensive agent.
  • captopril D-2 -methyl-3 -mercaptopropanoyl-L-proline
  • anti-hypertensive agents possess significant side effects including elevation of blood lipids and glucose. Also, special care must be taken to monitor the safety of synthetic chemical products for anti-hypertensive use.
  • VPP Val-Pro-Pro
  • IPP Ile-Pro-Pro
  • VPP nutritional or dietary supplements that contain VPP without the unpredictability and cost associated with current production methods.
  • VPP may then be used for pharmaceutical compositions, dietary supplements, food ingredients, and foods for the specified health uses of reducing and inhibiting hypertension and diseases related to hypertension at a low cost with no appreciable side effects.
  • a method for producing a recombinant BSSL polypeptide comprising collecting milk from the transgenic non-human mammal wherein the milk contains the recombinant
  • the method further provides for hydrolyzing the milk containing the recombinant BSSL polypeptide to produce a polypeptide containing the VPP tripeptide .
  • Another aspect provides a pharmaceutical composition comprising VPP produced by the method of the invention and a pharmaceutically acceptable carrier, diluent or excipient.
  • a nutritional composition comprising VPP produced by the method of the invention and a nutritionally acceptable carrier, diluent or excipient.
  • Still another aspect of the invention is provided a food composition
  • a food composition comprising VPP produced by the method of the invention and a nutritionally acceptable carrier, diluent or excipient.
  • A represents alanine
  • R represents arginine
  • N represents asparagine
  • D represents aspartic acid
  • C represents cysteine
  • Q represents glutamine
  • E represents glutamic acid
  • G represents glycine
  • H histidine
  • I represents isoleucine
  • L represents leucine
  • K represents lysine
  • M represents methionine
  • F represents phenylalanine
  • P represents proline
  • S represents serine
  • T represents threonine
  • W represents tryptophan
  • Y represents tyrosine
  • V represents valine.
  • ACE angiotensin converting enzyme
  • treatment relate to any treatment of hypertensive disease and include:
  • substantially pure when referring to proteins and polypeptides, denotes those polypeptides that are separated from proteins or other contaminants with which they are naturally associated.
  • a protein or polypeptide is considered substantially pure when that protein makes up greater than about 50% of the total protein content of the composition containing that protein, and typically, greater than about 60% of the total protein content. More typically, a substantially pure protein will make up from about 75 to about 90% of the total protein. Preferably, the protein will make up greater than about 90%, and more preferably, greater than about 95% of the total protein in the composition.
  • recombinant BSSL shall mean a non- native polypeptide derived by recombinant means or a native polypeptide with an altered amino acid sequence.
  • non-human mammals are to be understood to include non-human primates, murine species, bovine species, canine species, etc.
  • Preferred non-human mammals include bovine, porcine and ovine. More preferably, the non-human mammal is the bovine spe.cies .
  • transgenic shall be understood to mean an organism harboring in its germ and somatic cells a transgene that has been introduced using recombinant technology.
  • transgene shall mean a gene inserted, using recombinant technology, into the germ and somatic cells in a manner that ensures its function, replication, and transmission as a normal gene.
  • moiety shall mean an identified sequence of polypeptide residues.
  • operably association or “operably linked” are used interchangeably and shall mean a unit of coordinated and regulated gene activity by means of which the control and synthesis of a protein is determined. It consists of a DNA coding a structural gene together with one or more regulatory regions .
  • Applicant has discovered a method for the industry scale production of polypeptides containing VPP.
  • the method comprises collecting milk from a transgenic non-human mammal engineered to secrete a recombinant polypeptide into its milk that contains a significant amount of VPP.
  • the proteinaceous matter is then hydrolyzed from the milk and VPP is further purified from the polypeptide.
  • the method of the present invention can be beneficially used to produce the anti-hypertensive tripeptide VPP.
  • This tripeptide has significant anti- hypertensive activities and therefore is useful in the diagnosis, treatment and prophylaxis of hypertension and related conditions such as left ventricular systolic dysfunction, myocardial infarction, diabetes mellitus and progressive renal impairment/failure. Furthermore, the peptide is not expected to have the negative side effects associated with the anti-hypertensive pharmaceutical products .
  • BSSL polypeptide Bile Salt-Stimulated Lipase
  • BSSL functions as a nonspecific lipase because it hydrolyzes not only triacylglyceral, but also diacyl and monoacylglyceral, as well as cholesteryl esters (Hernell et al . , (1993) J. Pediatr. Gastroenterol . Nutr .16 :426-31) .
  • BSSL is a natural constituent of milk in a limited number of species, e.g., humans, gorillas, cats and dogs, and can accumulate to approximately 1% of total milk protein in these species (Hernell et al . , (1989) in Textbook of gastroenterology and nutrition, pp 209-217; and Hamosh et al., (1986) Fed. Proc . 45:1452).
  • BSSL The cDNA sequence of human milk BSSL has been characterized (Baba et al . , (1991) Biochem. 30:500-510) ( SEQ ID No: 1) .
  • BSSL is a single chain glycoprotein.
  • the deduced sequence of the mature protein contains 722 amino acids and is highly glycosylated (SEQ ID No : 2) .
  • the carboxy-terminal region contains 16 proline-rich repeating units of 11 amino acids each.
  • each repeating unit contains the tripeptide sequence VPP.
  • each BSSL polypeptide contains 16 repeats of the tripeptide sequence VPP.
  • BSSL accordingly, because it contains significant amounts of VPP and accumulates to 1% of total milk protein, is particularly advantageous for use in the present invention.
  • the present invention employs the use of recombinant technology to produce a recombinant BSSL polynucleotide.
  • BSSL cDNA has been isolated from a number of sources including humans (SEQ ID NO: 1) .
  • BSSL from any organism may be -employed to the extent that the amino acid sequence contains a significant amount of the sequence VPP.
  • the BSSL employed is from humans.
  • a recombinant polynucleotide is one in which polynucleotide sequences of different organisms have been joined together to form a single unit.
  • a cloning vector is a self-replicating DNA molecule that serves to transfer a DNA segment into a host cell.
  • the three most common types of cloning vectors are bacterial plasmids, phages, and other viruses.
  • An expression vector is a cloning vector designed so that a coding sequence inserted at a particular site will be transcribed and translated into a protein.
  • Both cloning and expression vectors contain nucleotide sequences that allow the vectors to replicate in one or more suitable host cells.
  • this sequence is generally one that enables the vector to replicate independently of the host cell chromosomes, and also includes either origins of replication or autonomously replicating sequences.
  • Various bacterial and viral origins of replication are well known to those skilled in the art and include, but are not limited to the pBR322 plas id origin, the 2m plasmid origin, and the SV40, polyoma, adenovirus, VSV and BPV viral origins .
  • the polynucleotide sequence of the present invention may be used to produce proteins by the use of recombinant expression vectors containing the sequence.
  • Suitable expression vectors include chromosomal, non-chromosomal and synthetic DNA sequences, for example, SV 40 derivatives; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA; and viral DNA such as vaccinia, adenovirus, fowl pox virus, retroviruses, and pseudorabies virus.
  • any other vector that is replicable and viable in the host may be used.
  • the nucleotide sequence of interest may be inserted into the vector by a variety of methods .
  • the sequence is inserted into an appropriate restriction endonuclease site(s) using procedures commonly known to those skilled in the art and detailed in, for example, Sambrook et al . , Molecular Cloning, A Laboratory Manual , 2nd ed. , Cold Spring Harbor Laboratory Press, (1989) and Ausubel et al . , Short Protocols in Molecular Biology, 3rd ed. , John Wiley & Sons (1995) .
  • the sequence of interest is operably linked to a suitable expression control sequence or promoter recognized by the host cell to direct mRNA synthesis.
  • Promoters are untranslated sequences located generally 100 to 1000 base pairs (bp) upstream from the start codon of a structural gene that regulate the transcription and translation of nucleic acid sequences under their control . Promoters are generally classified as either inducible or constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in the environment, e.g., the presence or absence of a nutrient or a change in temperature. Constitutive promoters, in contrast, maintain a relatively constant level of transcription. In addition, useful promoters can also confer appropriate cellular and temporal specificity. Such promoters include those that are developmentally-regulated or organelle-, tissue- or cell-specific.
  • a nucleic acid sequence is operably linked when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operatively linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide;
  • a promoter is operably linked to a coding sequence if it affects the transcription of the sequence;
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • operably linked sequences are contiguous and, in the case of a secretory leader, contiguous and in reading frame. Linking is achieved by blunt end ligation or ligation at restriction enzyme sites.
  • oligonucleotide adapters or linkers can be used as is known to those skilled in the art (Sambrook et al . , Molecular Cloning, A Laboratory Manual , 2nd ed. , Cold Spring Harbor Laboratory Press, (1989) and Ausubel et al . , Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons (1995)) .
  • Common promoters used in expression vectors include, but are not limited to, CMV promoter, LTR or SV40 promoter, the E. coli lac or trp promoters, and the phage lambda PL promoter. Other promoters known to control the expression of genes in prokaryotic or eukaryotic cells can be used and are known to those skilled in the art.
  • Expression vectors may also contain a ribosome binding site for translation initiation, and a transcription terminator. The vector may also contain sequences useful for the amplification of gene expression.
  • Expression and cloning vectors can and usually do contain a selection gene or selection marker. Typically, this gene encodes a protein necessary for the survival or growth of the host cell transformed with the vector. Examples of suitable markers include dihydrofolate reductase (DHFR) or neomycin or hygromycin B resistance for eukaryotic cells and tetracycline, ampicillin, or kanamycin resistance for E. coli .
  • DHFR dihydrofolate reductase
  • neomycin or hygromycin B resistance for eukaryotic cells and tetracycline, ampicillin, or kanamycin resistance for E. coli .
  • expression vectors can also contain marker sequences operatively linked to a nucleotide sequence for a protein that encodes an additional protein used as a marker.
  • the result is a hybrid or fusion protein comprising two linked and different proteins.
  • the marker protein can provide, for example, an immunological or enzymatic marker for the recombinant protein produced by the expression vector.
  • alkaline phosphatase (AP) green fluorescence protein (GFP) , myc, histidine tag (His) and hemagglutinin (HA) are used as markers .
  • the end of the polynucleotide can be modified by the addition of a sequence encoding an amino acid sequence useful for purification of the protein produced by affinity chromatography.
  • Various methods have been devised for the addition of such affinity purification moieties to proteins. Representative examples can be found in U.S. Patent Nos . 4,703,004, 4,782,137, 4,845,341,
  • the present invention may employ an expression cassette that at a minimum comprises, operably linked in the 5' to 3 ' direction, a promoter, a polynucleotide of the present invention, and a transcriptional termination signal sequence functional in a host cell.
  • the promoter selected will preferably be a mammary gland tissue-specific promoter that constitutively expresses at a high level the gene to which it is operatively linked.
  • the expression cassette can further comprise a targeting sequence, transit or secretion peptide coding region capable of directing transport of the protein produced.
  • the expression cassette encodes a signal sequence precisely fused to the N-terminal portion of the polynucleotide of the present invention to ensure its secretion into the milk of the mammal.
  • the expression cassette can also further comprise a nucleotide sequence encoding a selectable marker and a purification moiety.
  • the present invention also relates to proteins encoded by the isolated BSSL polynucleotide.
  • protein includes fragments, analogs and derivatives of the BSSL protein.
  • fragment means a polypeptide that retains essentially the same biological function or activity as the recombinant BSSL protein.
  • an analog includes a proprotein which can be cleaved to produce an active mature protein.
  • the protein of the present invention can be a natural protein, a recombinant protein or a synthetic protein or a polypeptide.
  • protein also includes forms of the BSSL protein to which one or more substituent groups have been added.
  • a substituent is an atom or group of atoms that is introduced into a molecule by replacement of another atom or group of atoms.
  • groups include, but are not limited to lipids, phosphate groups, sugars and carbohydrates.
  • protein includes, for example, lipoproteins, glycoproteins, phosp oproteins and phospholipoproteins .
  • the incorporation of the transgene expression cassette into the germline of the non-human mammal may be performed using any suitable technique, e.g., as described in "Manipulating the Mouse Embryo”; A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1986.
  • a few hundred molecules of the transgene may be directly injected into a fertilized egg, e.g., a fertilized one cell egg or pro-nucleus thereof, or an embryo of the mammal of choice, and the microinjected eggs may then be transferred into the oviducts of pseudopregnant mothers and allowed to develop.
  • transgenic non-human mammal engineered to express and secret BSSL into their milk may be obtained from a number of sources (See, e.g., Blackberg et al., U.S. Patent No. 5,763,739).
  • transgenic non-human mammal engineered to express and secrete BSSL into its milk is suitable for use in the present invention.
  • the mammal will be selected from the group consisting of non-human primates, murine species, bovine species, porcine species, ovine species, and canine species. More preferably, the transgenic non-human mammal will include bovine, porcine, and ovine. Even more preferably, the transgenic non-human mammal will be a bovine.
  • a milk-collecting device may then be attached to each nipple and milk may be collected by gentle massage of the mammary gland according to methods generally known in the art. It should be noted, however, that the amount of milk collected from the particular mammal is highly dependent upon the day of lactation. The milk is then stored in a suitable container and under appropriate conditions until use. One generally skilled in the art can readily determine these conditions.
  • hydrolysis is employed to purify the polypeptide according to the method of this invention. More particularly, the proteinaceous matter is measured, and mixed with a liquid such as water, preferably distilled water, or a buffer (for example, a Tris-HCl buffer or a phosphate buffer) . Before hydrolysis, the protein and liquid mixture is typically homogenized. Homogenization of the mixture improves the activity of cleaving agent used to hydrolyze the target polypeptide from the protein mixture.
  • the concentration of target protein in the mixture is generally in the range of about 1 to about 50% (w/v) , and more preferably about 10%.
  • the mixture is thoroughly homogenized, it is adjusted to a pH that is favorable for the activity of the selected cleaving agent utilized in the hydrolysis reaction.
  • an acidic cleaving agent like pepsin
  • the most favorable pH range is about 0.1 to about 4, and more preferably about 2.
  • an alkaline cleaving agent would work most effectively in a pH range of about 8 about to 10.
  • the pH adjusted mixture is raised to a boiling temperature for a period of approximately 5 to 60 minutes, preferably for about 10 to 15 minutes. This boiling step inactivates all endogenous enzymes present in the source protein.
  • the pH in course of reaction can, if necessary, be adjusted with a base like aqueous sodium hydroxide solution, or an acid, like hydrochloric acid. Any suitable acid or base, however, may be employed to adjust the pH of the reaction solution.
  • Polypeptides contained in the protein solution used in the invention can be prepared by a process to hydrolyze or digest the selected protein with a cleaving agent .
  • the target protein can be hydrolyzed via chemical or proteolytic methods.
  • a cleavage agent like a protease is added to the mixture to hydrolyze the protein material within the mixture.
  • proteases such as thermolysin, pepsin, trypsin, chymotrypsin, papain, Pronase E, Proteinase K, or Actinase E may be used to digest the protein.
  • Thermolysin is particularly preferred as the digesting enzyme.
  • the optimal concentration of the cleaving agent within the digestion mixture is dependent upon the cleaving agent selected.
  • the additional amount of the enzyme thermolysin is varied depending on its titer, the final concentration within the reaction mixture is preferably about 100 ⁇ g/ml to about 1000 ⁇ g/ml, and more preferably the final thermolysin concentration is approximately 800 to 880 ⁇ g/ml based on the protein. It is also possible to add part of the thermolysin in the course of the reaction.
  • the temperature of the reaction mixture during the reaction may be maintained between about 30° C to about 50° C, and more preferably about 37° C.
  • the reaction time varies depending on the amount of the enzyme, reaction temperature and reaction pH. But the time is typically about 1 to about 24 hours, and more preferably about 1 to about 6 hours .
  • the digestion reaction can be stopped according to a known method, for example, according to inactivation of the cleaving agent either by heating of the reaction mixture or by pH change with addition of an organic acid such as citric acid or malic acid, an inorganic acid such as hydrochloric acid or phosphoric acid or an alkali such as sodium hydroxide or potassium hydroxide, or according to separation of the enzyme by filtration using an ultrafiltration membrane or the like.
  • an organic acid such as citric acid or malic acid
  • an inorganic acid such as hydrochloric acid or phosphoric acid or an alkali such as sodium hydroxide or potassium hydroxide
  • separation of the enzyme by filtration using an ultrafiltration membrane or the like.
  • the digestion mixture is cooled to approximately 0° C to 30° C, and most preferably about 4° C.
  • the polypeptides can be isolated from the resulting digestion solution through solid-liquid separation, for example, centrifugation or filtration.
  • the resulting liquid can be fractionated by ultrafiltration, gel filtration or
  • the cooled mixture is transferred into centrifuge tubes and separated into solid and liquid phases by centrifugation at approximately 1500 to about 3000 rpm. Centrifugation is conducted for a period of about 10 to 20 minutes, and more preferably for about 15 minutes. The supernatant is then removed and transferred to a clean centrifuge tube and centrifuged a second time at approximately 8000 rpm for a period of about 10 to 20 minutes, and more preferably for about 10 minutes, at a temperature below about 30° C. The contents of the centrifuge tubes are then dried under nitrogen gas at a temperature of approximately 30° C to 80° C, and more preferably about 37° C. A mobile phase solvent is added to the centrifuge tubes and the dried contents are resuspended to form a mobile phase mixture. The mobile phase mixture is then centrifuged at about 1000 to about 10,000 rpm, and more preferably approximately 8000 rpm at a temperature below about 30° C for a period of about 10 to 20 minutes.
  • the resulting supernatant, comprising VPP may then be further purified by chromatographic methods such as liquid chromatography, HPLC, FPLC, or the like.
  • the VPP, or other desired peptides are then isolated according to their respective retention times.
  • VPP is substantially purified by any means generally known in the art .
  • the VPP tripeptide has anti- hypertensive activity that effectively depress elevated blood pressure, but that, unlike current anti-hypertensive pharmaceutical compounds, do not have adverse side effects such as altering normal blood pressure, over depressing blood pressure or other side effects.
  • the VPP produced by the method of the invention may be added to a number compositions and administered to treat hypertensive disease.
  • the VPP may be added to nutritional compositions (e.g. dietary supplements), food compositions, vaccine compositions, drug and pharmaceutical compositions.

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Abstract

L'invention porte sur un procédé de production du tripeptide hypotenseur VPP, et sur des préparations pharmaceutiques nutritionnelles et alimentaires utilisant le VPP comme principe actif.
PCT/US2001/012672 2000-04-21 2001-04-19 Purification de polypeptides inhibiteurs de l'enzyme de conversion de l'angiotensine contenant le vpp du lait Ceased WO2001081366A2 (fr)

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AU2001255469A AU2001255469A1 (en) 2000-04-21 2001-04-19 Purification of ace inhibiting polypeptides containing vpp from milk

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006114441A1 (fr) * 2005-04-28 2006-11-02 Dsm Ip Assets B.V. Hydrolysats proteiques abaissant la pression arterielle
WO2006067163A3 (fr) * 2004-12-22 2007-05-10 Dsm Ip Assets Bv Peptides abaissant la pression sanguine obtenus en une seule operation enzymatique

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2782142B2 (ja) * 1992-07-23 1998-07-30 カルピス株式会社 アンジオテンシン変換酵素阻害剤及びその製造法
IS4130A (is) * 1993-03-01 1994-09-02 Ab Astra Ný fjölpeptíð
WO1996017054A1 (fr) * 1994-12-01 1996-06-06 Oklahoma Medical Research Foundation Technique et compositions permettant de reduire l'absorption de cholesterol

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006067163A3 (fr) * 2004-12-22 2007-05-10 Dsm Ip Assets Bv Peptides abaissant la pression sanguine obtenus en une seule operation enzymatique
US7879804B2 (en) 2004-12-22 2011-02-01 Dsm Ip Assets B.V. Blood pressure lowering peptides in a single enzymatic step
WO2006114441A1 (fr) * 2005-04-28 2006-11-02 Dsm Ip Assets B.V. Hydrolysats proteiques abaissant la pression arterielle
EA012972B1 (ru) * 2005-04-28 2010-02-26 ДСМ АйПи АССЕТС Б.В. Трипептиды мар и itp или их соли, белковые гидролизаты и смеси, содержащие указанные трипептиды или их соли, для снижения кровяного давления

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