WO1993015198A1 - Polypeptides derives de l'apolipoproteine aiv humaine, leur preparation et leur utilisation - Google Patents
Polypeptides derives de l'apolipoproteine aiv humaine, leur preparation et leur utilisation Download PDFInfo
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- WO1993015198A1 WO1993015198A1 PCT/FR1993/000073 FR9300073W WO9315198A1 WO 1993015198 A1 WO1993015198 A1 WO 1993015198A1 FR 9300073 W FR9300073 W FR 9300073W WO 9315198 A1 WO9315198 A1 WO 9315198A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/775—Apolipopeptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- 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 present invention relates to polypeptides derived from human apolipoprotein AIV (apoAI), the nucleotide sequences encoding these polypeptides, their preparation and their use.
- apoAI apolipoprotein AIV
- Apolipoprotein AIV is a protein made up of 376 amino acids, with a molecular weight of 46,000 daltons. The peptide and nucleotide sequences of apoAIV, as well as the position of the helices are shown on sequences SEQ ID No. 1 and 2. ApoAIV is a major component of the chylomicrons secreted in the lymph, but it has the particularity of be predominantly in a form not associated with lipoproteins in the plasma (RB Weinberg et al., 1983, J. Lipid. Research, 24: 52-59). Furthermore, plasma apoAIV is polymorphic, although the nature of this polymorphism is still unknown (G. Utermann et al., 1982, J.
- the present invention results from the choice of the applicant to use apolipoprotein AIV as a target molecule for the study of factors influencing the reverse transport of cholesterol.
- the present invention is based more precisely on the use of apoAIV for the preparation of new products, making it possible, by new therapies, to combat hypercholesterolaemia and the effects which are associated with it, such as atherosclerosis.
- the present invention provides polypeptides derived from apoAIV, and thus makes it possible to pharmacologically exploit the properties of this protein.
- the present invention therefore relates to polypeptides derived from human apolipoprotein AIV. They may in particular be polypeptides having an increased plasma stability compared to the native protein (lower sensitivity to physiological mechanisms of elimination or degradation), and therefore longer service life. It may also be polypeptides having an activity stimulating the efflux of cellular cholesterol, and therefore promoting the reverse transport of cholesterol, leading to discharge the cells having accumulated cholesterol in the context of the formation of a plaque. atheroma. They can also be polypeptides having only one or more of the properties of apoAIV.
- polypeptides capable of binding cellular receptors, but lacking the activity stimulating the efflux of cellular cholesterol.
- they may also be polypeptides lacking the activities of apoAIV.
- Such polypeptides can indeed have therapeutic utility (generation of antibodies, structure-function studies, etc.).
- polypeptides of the invention are capable of binding the HDL receptor.
- the polypeptides are capable of binding the HDL receptor and of stimulating an eff lux of cholesterol.
- polypeptides according to the invention can be of several types. In relation to human apoAIV, these may be mutation or substitution derivatives, deletion derivatives or addition derivatives. It is understood that the present invention also covers the polypeptides comprising several types of modifications, such as for example mutation and deletion derivatives, deletion and addition derivatives, etc.
- the subject of the invention is non-natural polypeptides derived from human apolipoprotein AIV comprising, with respect to the sequence of human apolipoprotein ATV SEQ ID No. 2, at least one of the following modifications: (a) a point mutation relating to a functional residue, and / or,
- polypeptides of the invention comprise at least one modification according to (a) and (b).
- polypeptides of the invention comprise at least one modification according to (a) and (c).
- polypeptides of the invention comprise a modification according to (a), (b) or (c) associated with a modification according to (d).
- Mutation derivatives generally include a point mutation (deletion of one amino acid, or modification of one amino acid, or replacement of one amino acid with another), or double (modification of a pair of amino acids) .
- the mutations chosen take account of the position of the mutated residues in their respective helices, and tend a priori to modify the functional groups without disturbing too much the structure of the helix concerned, and therefore of the polypeptide.
- the term “functional residue” is understood to mean the residues capable of being involved in the activity of apoAIV, either at the level of the interaction with the receptor, or at the level of the transmission of a signal (such as for example stimulating the efflux of cholesterol).
- Preferred residues are generally charged residues, which have a potential effect in the interaction of the polypeptide with its receptor. Such residues can be replaced by others, apolar, or chemically modified by removing or adding a charge. Other preferred residues are glycosylated residues and residues involved in the formation of disulfide (cysteine) bridges.
- the polypeptides of the invention have, with respect to human apoAIV as represented in the sequence SEQ ID No. 2, at least one mutation on one of the following residues: aspartic acid in position 5, 44, 106, 153 ; glutamine in position 37, 194; asparagine in position 39; lysine at position 73, 84, 103, 169, 178; glutamic acid in position 76, 81, 87, 99, 131, 164, 187, 230; alanine at position 22; proline at position 139, 161; and serine in position 154.
- aspartic acid can be replaced by a residue S, K, A, F or G; glutamine with a T, K or F residue; asparagine with a residue A or D; lysine by a residue G, E, T, D, A, Y, H or F; glutamic acid with a residue S, R, F, K, A, G, N, or Q; alanine by R or E residue wine; proline with an R or G residue; and serine by an E or R residue (the letters correspond to an amino acid according to the recognized code).
- the polypeptides of the invention can be deletion derivatives.
- the deletion (s) may relate to one end of the protein (C-terminal or N-terminal).
- certain polypeptides of the invention are fragments of apoAIV, obtained by deletion of important parts of the protein.
- the polypeptides of the invention have, relative to apoAIV human terminal deletions of at least 10 amino acids.
- the deletion (s) may also relate to internal regions of rapoAIV, and in particular to whole helices or to pairs of helices.
- polypeptides comprising all or part of rapoATV and an additional element such as a marker, a targeting agent, a stabilizer or another active element.
- an additional element such as a marker, a targeting agent, a stabilizer or another active element.
- a purification marker there may be mentioned by way of example the decapeptide tag of sequence MRGS (H) 6 described by Hochuli et al (Bio / technol. (1988) 1321).
- this decapeptide can be fused into the N-terminal of rapoATV or of a derivative as defined above, thus allowing very rapid purification in one step, without affecting the production capacities of said polypeptide.
- polypeptide P ( ⁇ N13, R93G): polypeptide having a deletion of the 13 N-terminal amino acids ( ⁇ N13) and a glycine instead of 'an arginine in position 93 (R93G).
- - P polypeptides ( ⁇ N13): polypeptide having a deletion of the 13 N-terminal amino acids, and its tag P version (tag ⁇ N13).
- polypeptide P ( ⁇ C194): polypeptide having a deletion of the last 194 amino acids.
- polypeptide P ( ⁇ N182): polypeptide having a deletion of the first 182 amino acids.
- - P polypeptides ( ⁇ hl-2): polypeptide having a deletion of helices 1 and 2 as shown in SEQ -D n ° 2 (residues D14 to V62), and its tag version.
- - P polypeptides ( ⁇ h9-10) polypeptide having a deletion of helices 9 and 10 as presented in SEQ ID No. 2 (residues P206 to A249), and its tag version.
- polypeptides ( ⁇ hll-12): polypeptide having a deletion of helices 11 and 12 as presented in SEQ ID No. 2 (residues P250 to E289), and its tag version.
- - polypeptide P ( ⁇ hll-12, L87M): polypeptide having a deletion of helices 11 and 12 as shown in SEQ ID No. 2 ( ⁇ hll-12), and a methionine instead of a leucine in position 87 (L87M ).
- - P polypeptides ( ⁇ hl3-14): polypeptide having a deletion of the helices
- polypeptides ( ⁇ h5-6): polypeptide having a deletion of helices 5 and 6 as shown in SEQ ID No. 2 (residues P118 to R161), and its tag version.
- D44F polypeptide having a phenylalanine instead of an aspartic acid in position 44.
- polypeptide P (D44A) polypeptide having an alanine instead of an aspartic acid in position 44.
- - polypeptide P (D5S) polypeptide having a serine instead of an aspartic acid in position 5.
- polypeptide P (D5K): polypeptide having a lysine instead of an aspartic acid in position 5.
- K178Y polypeptide having a tyrosine instead of a lysine in position 178.
- K178A polypeptide having an alanine instead of a lysine in position 178.
- E230K polypeptide having a lysine instead of a glutamic acid in position 230.
- Atherosclerosis is a disease which is characterized by the formation of lipid or fibro-lipid plaques in the intima of the aorta, the coronary arteries and the carotid essentially. These plaques, more or less calcified depending on the progress of the process, can be combined with lesions, and are created by the accumulation in the arteries of fatty deposits consisting essentially of cholesterol esters. These plaques then cause a thickening of the arterial wall, with enlargement of the smooth muscle, appearance of foam cells and accumulation of fibrous tissue.
- the atheromatous plaque is very clearly in relief on the wall, which gives it a stenosing character responsible for vascular occlusions by atheroma, thrombosis or embolism, which occur in the most affected patients.
- an atheroma plaque therefore results from the combination of different factors, (i) an excess of plasma cholesterol, from which arterial deposits originate, and (ii) a defect in regulation between cholesterol influx and efflux at the level of peripheral tissues, in favor of an intracellular accumulation.
- polypeptides according to the invention can in particular make it possible to slow down the formation of atheroma plaques, to induce the regression of atheroma plaques, and to reduce the risk of incidence of coronary accidents.
- polypeptides of the invention can be obtained in various ways, and in particular by chemical and / or genetic means.
- the polypeptides of the invention can be obtained either entirely by chemical synthesis, or by chemical or enzymatic modifications of the native protein. In the latter case, different chemical or enzymatic agents can be used, making it possible to modify residues (mutation derivatives), to mark residues (addition derivatives) or to fragment the protein (deletion derivatives).
- sulfo-NHS-acetate which allows the acylation of primary amines and especially of lysine
- tetranitromethane which introduces a nitrate group on tyrosines
- N-bromo-succinimide which oxidizes tryptophan residues.
- chemical and enzymatic agents making it possible to cut peptide bonds there may be mentioned more particularly cyanogen bromide, iodosobenzoate, trypsin, chymotrypsin, thermolysin or even proendopeptidase.
- these treatments can be applied both to the polypeptide produced from a chemical synthesis and to the native polypeptide.
- these treatments can also be applied to polypeptides obtained by genetic means, and therefore already comprising certain modifications.
- the polypeptides of the invention are obtained by modification at the level of the coding nucleotide sequences, and expression of said sequences in a cellular host.
- the present invention also relates to the nucleotide sequences coding for the polypeptides described above. They can be DNA sequences, RNA sequences, synthetic, semi-synthetic, hybrid sequences, etc. These sequences can be used:
- the nucleotide sequences of the invention can be obtained from a sequence coding for apoAIV, by different techniques such as in particular by genetic cutting, mutagenesis, or any other treatment altering the structure of nucleic acids. The techniques best known to those skilled in the art are mentioned below in general cloning techniques. The detail of the synthesis of the nucleotide sequences of the invention is given in part B / of the examples.
- the human apoAIV sequence used in the invention was obtained from a genomic clone containing the Kpnl-HindlII fragment of the human apoAIV gene sequence, which contains the end of intron 2, the all of exon 3 as well as the 3 'untranslated sequence.
- This clone therefore did not contain, in particular, the first two exons of the human apoAIV gene (Elshourbagy et al. JBC (1987) 262: 7973).
- the complete sequence was obtained by: - chemical synthesis of the 5 'end of the rapoAIV sequence, so that this synthetic fragment contains the codons corresponding to the part of the mature protein encoded by the first two exons of the human gene, and also the first nucleotides of the 5 ′ region of exon 3 extending to the BstEII site; then, - The binding of this synthetic fragment to a DNA fragment containing the remainder of the apolipoprotein AIV gene sequence located after the BstEII site, used to make the junction to the nearest nucleotide.
- the present invention also relates to a process for the preparation of the polypeptides of the invention described above. This process consists of carrying out the following steps:
- a nucleotide sequence as defined above coding for a polypeptide of the invention is introduced into a cell, - in a second step, the cell thus obtained is cultured under conditions of expression of said nucleotide sequence and,
- the nucleotide sequence can be introduced into the cell by different techniques.
- the introduction can be carried out by transformation, conjugation, or electroporation.
- the choice of one or the other of these methods is established in particular according to the chosen host.
- eukaryotic hosts which can be used in the process of the invention, mention may be made of animal cells, yeasts, or fungi.
- yeasts mention may be made of yeasts of the genus Saccharomyces. Kluyveromyces. Pichia pastoris. Schwanniomy these. or Hansenula.
- animal cells mention may be made of COS, CHO, C127 cells, etc.
- the fungi likely to be used in the present invention there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp.
- prokaryotic hosts which can be used in the context of the present invention, mention may be made of the following bacteria E.coli. Bacillus. or Streptomyces.
- the method is implemented using a prokaryotic cell as the host cell.
- the method of the invention is implemented using the E. coli bacterium as the host cell.
- the nucleotide sequence used is a genomic sequence, a cDNA sequence, a hybrid sequence sequence, etc.
- this nucleotide sequence generally comprises a region for initiating transcription and translation joined to the 5 ′ terminal end of the coding sequence, so as to direct and regulate the transcription and translation of said sequence.
- the choice of these regions may vary depending on the host used. In particular, in bacteria such as E. coli. we can use the promoter of the tryptophan operon (Ptrp), or the left and right promoters of bacteriophage lambda (PL, PR) OR the promoter of gene 10 of bacteriophage T7.
- the nucleotide sequence preferably forms part of a vector, which can be autonomously replicating or integrative. More particularly, autonomously replicating vectors can be prepared using autonomously replicating sequences in the chosen host. For example, in yeast, it may be replication origins derived from plasmids: pKDl (EP 241 435) or else chromosomal sequences (ARS) and in bacteria, it may be origins of replication derived from plasmids (pBR322, pET3, etc.). As regards integrative vectors, these can be prepared for example by using sequences homologous to certain regions of the host genome, allowing, by homologous recombination, the integration of the vector. In this regard, the use of rDNA allows multiple integration of exogenous DNA, and therefore its presence in greater number of copies per cell.
- the nucleotide sequence comprises, upstream of the coding sequence, or, where appropriate, between the region for starting transcription and translation and the coding sequence, a "leader” sequence directing the polypeptide originating in the secretion pathways of the host used.
- This “leader” sequence may be the “leader” sequence of apoAIV, but it may also be a heterologous sequence (originating from a gene coding for another protein) or even artificial. The choice of one of these sequences is notably guided by the host used.
- polypeptides of the present invention can then be isolated from the culture medium by any technique known to those skilled in the art. More particularly, part C / of the examples describes a process allowing the polypeptides of the invention to be purified under native conditions, that is to say without any denaturation step.
- compositions comprising one or more polypeptides according to the invention or one or more nucleotide sequences according to the invention.
- compositions are intended for the treatment or prevention of conditions linked to hypercholesterolaemia.
- the subject of the invention is pharmaceutical compositions comprising one or more polypeptides according to the invention, or one or more nucleotide sequences according to the invention, intended for slowing down the formation of atheroma plaques, and or for regression of atheroma plaques and / or a reduction in the risk of coronary accidents.
- the present invention also relates to the use of the polypeptides described above for the production of molecules of non-peptide or non-exclusively peptide structure having the same type of activity.
- the invention relates to the use of a polypeptide of the invention as described above for the preparation of non-peptide, or not exclusively peptide, molecules pharmacologically active on plasma cholesterol levels, by determination structural elements of this polypeptide which are important for its activity, and reproduction of these elements by non-peptide or non-exclusively peptide structures.
- the invention also relates to pharmaceutical compositions comprising one or more molecules thus prepared.
- Figure 1 Structure and construction of the plasmid pXL1697.
- Figure 2 Structure of plasmids pXL1872 and pXL1867.
- Figure 3 Structure of plasmids pXL1696 and pXL2051.
- the buffer used for the ligations has the following composition: 50 mM Tris-HCl, 10 mM MgCl 2 , 15 mM DTT, 1 mM ATP, pH 7.5.
- the oligonucleotide phosphorylation buffer has the following composition: 5 mM Tris-HCl, 1 mM MgCl, 0.6 mM DTT, pH 7.5. Mutagenesis directed in vitro by oligodeoxynucleotides is carried out according to the method developed by Taylor et al. (Nucleic Acids Res. ⁇ (1985) 8749-8764) using the kit distributed by Amersham.
- the nucleotide sequence of the apolipoprotein AIV gene described in the present invention differs from the previously published cDNA sequences (see in particular the list of cDNA sequences published by CY. Yang, ZW. Gu, I. Chong, W. Xiong, M Rosseneue, H. Yang, B. Lee, AM Gotto and L. Chan, 1989, BBA, 1002: 231-237).
- the sequence coding for the signal peptide of the protein is absent, and, upstream of the first codon of the mature protein, a start codon for ATG translation has been placed.
- this nucleotide sequence of the apolipoprotein AIV was obtained in an original way by the assembly of four oligonucleotides which have been synthesized chemically and whose size is between 86 and 107 mer (SEQ ID n ° 3-6).
- SEQ ID n ° 3-6 the sequence of the oligonucleotides that cohesive ends Xbal and EcoRI have been defined in order to allow a two-step assembly of the complete sequence of the apolipoprotein AIV.
- the four oligodeoxynucleotides which were used to assemble the apolipoprotein ATV gene were synthesized by the phosphoramidite method (LJ. Bride and MH Caruthers (1983) Tetrahedron Lett. 24: 245) using the Bioresearch synthesizer. (Model 8600) according to the manufacturers' advice.
- the oligonucleotides were purified on acrylamide gel 15. Their sequence is given on sequences SEQ ID No. 3-6.
- oligonucleotides were phosphorylated by treatment with T4 DNA kinase. Oligonucleotides A and C were paired with oligonucleotides B and D respectively under stoichiometric conditions. The hybridization of the oligonucleotides was carried out in Eppendorf tubes immersed in a beaker containing approximately 100 ml of water brought to 80 ° C. which is left to return to laboratory temperature.
- the A-B and C-D fragments were ligated in the presence of T4 DNA ligase with the replicative form of the phage M13mpl0 previously digested with the enzymes Xbal and EcoRI.
- the replicative form of the recombinant bacteriophage obtained called pXL1695 was used to transfect TG1 bacteria made competent by the CaC ⁇ method.
- This replicon pXL1695 was purified, either in its form of single-stranded DNA and its sequence was verified, or in its form of replicative double-stranded DNA for the continuation of the constructions.
- pXL1695 M13mplOABCD
- pXL1694 a plasmid carrying a KnpI-HindIII fragment of approximately 3 kb containing all of the third exon of the apoAIV gene
- Xbal and BstEII on the one hand
- EcoRI and BstEII on the other hand, respectively.
- pXL1695 a 167 base pair fragment was purified on acrylamide gel.
- the replicative form of the vector M13mpl8amIV was digested with Xbal and EcoRI and a fragment of approximately 7 kb was purified on agarose gel.
- the three fragments were pooled and ligated in the presence of T4 DNA ligase.
- pXL1696 After transfection of TG1, clones containing the replicative form called pXL1696 were obtained. The complete coding sequence of the Xbal-EcoRI fragment thus cloned in pXL1 696 was checked. This fragment was then re-extracted from pXL1696 and ligated in the presence of an EcoRI-Hind ⁇ l fragment, of approximately 3 kb originating from pXL1694 and bearing in particular the end of exon 3 of the human apoAIV gene, and in the presence also of the vector M13mpl9, cut by the enzymes Xbal and HindIII.
- the recombinant vector thus obtained, pXL1697 carries the entire coding sequence of apoAIV (1132 bp) as well as a genomic fragment of approximately 2 kb corresponding to intron 3 of the apoAIV gene (FIG. 1). .
- 5'-GCCCCTTTGGAGAGCTGAGGATCCCCTGGTGCACTGGCCCCA-3 * made it possible to introduce a BamHI site on pXL1697 immediately after the stop codon (TGA) of the apoAIV gene.
- the vector obtained was designated pXL1866.
- the vector pXL1866 described above was cut by the enzymes Xbal and bamHI, and a 1.15 kb fragment containing the entire coding sequence of apoAIV was purified on agarose gel and religated in M13mpl8amIV to give the vector pXL1872 ( Figure 2).
- the vector pXL1866 was cut with NdeI and BamHI and a 1.2 kb fragment was purified and then inserted into pET3-a, itself cut with BamHI and NdeI.
- the resulting expression vector, pXL1867 therefore contains the coding sequence of apolipoprotein AIV without any residual fragment of intron 3 originating from the genomic sequence of apolipoprotein AIV (FIG. 2).
- the 570 bp Xbal-EcoRI fragment containing the 5 ′ part of the apoAIV coding sequence was excised from the vector pXL1872, purified on agarose gel and religated in M13mpl0 to give the vector pXL1696 ( figure 3).
- the 577 bp EcoRI-BamHI fragment containing the 3 'part of the apoAIV coding sequence was excised from the vector pXL1872, purified on agarose gel and religated in M13mpl9 to give the vector pXL2051 ( figure 3).
- This mutation has the effect of changing an arginine codon to glycine in the protein. From the clone pXL1799 and another clone not carrying the additional mutation (C-> G), an NdeI-BamHI fragment of 1097 bp was purified and ligated with the vector pET3-a (AMS Biotechnology), itself cut by the enzymes NdeI and BamHI.
- the vector pXL1775 which makes it possible to express at a very high level a polypeptide possessing a deletion of the 13 N-terminal amino acids ( ⁇ N13) and a glycine instead of an arginine in position 93 (R93G ); and the vector pXL1869, which makes it possible to express at a very high level a polypeptide having a deletion of the 13 N-terminal amino acids ( ⁇ N13).
- the vector pXL2168 was constructed, in which the sequence coding for the polypeptide P ( ⁇ N13) was fused in 5 ′ to a sequence coding for the decapeptide tag MRGS (H) 6.
- the following 2 oligodeoxynucleotides were synthesized, using a Biosearch 8600 DNA synthesizer: Oligo A: 5'-TAATGCGTGGATCGCACCATCACCATCACCA-3 'Oligo B: 5'-TATGGTGATGGTGATGGTGCGGATCCACigotides Byn and and the hybridization product was ligated with the vector pXL1869, previously digested with the enzyme NdeI.
- the vector thus obtained, designated pXL2168 codes for the polypeptide P ( ⁇ N13) fused in N-terminal with the decapeptide MRGS (H) 6.
- mutagenesis directed on the single-stranded form of the plasmid pXL1872 described above was carried out using the Amersham kit according to the manufacturer's recommendations. This mutagenesis made it possible to introduce a BamHI site as well as a TGA codon into the coding sequence of apoAIV at the respective positions 1000 and 997, causing a deletion of the 44 C-terminal amino acids.
- the vector pXL2182 was constructed from the vector pXL1766.
- the vector pXL2182 thus obtained makes it possible to express at high level the polypeptide P ( ⁇ C44) fused in N-terminal with the decapeptide MRGS (H) 6.
- mutagenesis directed on the single-stranded form of the plasmid pXL1872 described above was carried out using the Amersham kit according to the manufacturer's recommendations. This mutagenesis made it possible to introduce a BamHI site as well as a TGA codon into the coding sequence of apoAIV at the respective positions 553 and 550, causing a deletion of 194 amino acids on the C-terminal side.
- the recombinant vector pXL1774 thus obtained makes it possible to express at a very high level a polypeptide having a deletion of the last 194 amino acids ( ⁇ C194).
- a mutagenesis directed on the single-stranded form of the plasmid pXL1872 described above was carried out using the Amersham kit according to the manufacturer's recommendations .
- This mutagenesis made it possible to introduce an NdeI site as well as an ATG codon in the coding sequence of apoAIV at the respective positions 544 and 547, causing a deletion of 182 amino acids on the N-terminal side.
- mutagenesis directed on the single-stranded form of the plasmid pXL1872 described above was carried out using the Amersham kit according to the manufacturer's recommendations. This mutagenesis made it possible to delete the part of the coding sequence of apoAIV lying between positions 40 and 186, causing a deletion of 49 amino acids, from residue D14 to residue V62.
- Ndel-BamHI fragment of 990 bp was purified and ligated with the vector pET3-a, itself cut by the enzymes Ndel and BamHI.
- the recombinant vector pXL1981 thus obtained makes it possible to express at a very high level a polypeptide having a deletion of helices 1 and 2 ( ⁇ hl-2).
- the vector pXL2183 was constructed from the vector pXL1981.
- the vector pXL2183 thus obtained makes it possible to express at high level the polypeptide P ( ⁇ hl-2) fused in N-terminal with the decapeptide MRGS (H) 6.
- mutagenesis directed on the single-stranded form of the plasmid pXL1872 described above was carried out using the Amersham kit according to the manufacturer's recommendations. This mutagenesis made it possible to delete the part of the coding sequence of apoAIV lying between positions 484 and 615 causing a deletion of 44 amino acids, from residue P162 to residue A205.
- the vector pXL2184 was constructed from the vector pXL1943.
- the vector pXL2184 thus obtained makes it possible to express at high level the polypeptide P ( ⁇ h7-8) fused in N-terminal with the decapeptide MRGS (H) 6.
- a mutagenesis directed on the single-stranded form of the plasmid pXL1872 described above was carried out using the Amersham kit according to the manufacturer's recommendations This mutagenesis made it possible to delete the part of the coding sequence of apoAIV lying between positions 616 and 747, causing a deletion of 44 amino acids, from residue P206 to residue A249.
- Ndel-BamHI fragment of 1005 bp was purified and ligated with the vector pET3-a, itself cut by the enzymes Ndel and BamHI.
- the recombinant vector pXL1982 thus obtained makes it possible to express at a very high level a polypeptide having a deletion of helices 9 and 10 ( ⁇ h9-10).
- the vector pXL2215 was constructed from the vector pXL1982.
- the vector pXL2215 thus obtained makes it possible to express at high level the polypeptide P ( ⁇ h9-10) fused in N-terminal to the decapeptide MRGS (H) 6.
- mutagenesis directed on the single-stranded form of the plasmid pXL1872 described above was carried out using the Amersham kit according to the manufacturer's recommendations. This mutagenesis made it possible to delete the part of the coding sequence of apoAIV lying between positions 748 and 867, causing a deletion of 40 amino acids, from residue P250 to residue E289. Furthermore, during the sequence verification of the derivatives obtained, certain clones carried an additional point mutation on the sequence. This mutation (L-> M) has the effect of changing the codon leucine 87 to methionine in the protein.
- NdeI-BamHI fragment of 1017 bp was purified and ligated with the vector pET3-a, itself cut , by the enzymes NdeI and BamHI.
- Two recombinant vectors were thus obtained, including the vector pXL1986 which makes it possible to express at very high level a polypeptide having a deletion of helices 11 and 12 ( ⁇ hll-12) and a methionine instead of a leucine in position 87 (L87M ).
- the vector pXL2186 was constructed from the vector obtained above, coding for the polypeptide P ( ⁇ hll-12).
- the vector pXL2186 thus obtained makes it possible to express at high level the polypeptide P ( ⁇ hll-12) fused in N-terminal with the decapeptide MRGS (H) 6.
- mutagenesis directed on the single-stranded form of the plasmid pXL1872 described above was carried out using the Amersham kit according to the manufacturer's recommendations. This mutagenesis made it possible to delete the part of the coding sequence of apoAIV lying between positions 868 and 999, causing a deletion of 44 amino acids, from residue P290 to residue N333.
- Ndel-BamHI fragment of 1005 bp was purified and ligated with the vector pET3-a, itself cut by the enzymes Ndel and BamHI.
- the recombinant vector pXL1987 thus obtained makes it possible to express at a very high level a polypeptide having a deletion of helices 13 and 14 ( ⁇ hl3-14).
- the vector pXL2217 was constructed from the vector pXL1987.
- the vector pXL2217 thus obtained makes it possible to express at high level the polypeptide P ( ⁇ hl3-14) fused in N-terminal with the decapeptide MRGS (H) 6.
- B2 Mutants produced from the plasmid pXL1696
- mutagenesis directed on the single-stranded form of the plasmid pXL1696 was carried out using the Amersham kit according to the manufacturer's recommendations. This mutagenesis made it possible to delete the part of the coding sequence of apoAIV lying between positions 352 and 483, causing a deletion of 44 amino acids, from residue P118 to residue R161.
- the vector pXL2185 was constructed from the vector pXL2071.
- the vector pXL2185 thus obtained makes it possible to express at high level the polypeptide P ( ⁇ h5-6) fused in N-terminal with the decapeptide MRGS (H) 6.
- a mutagenesis directed on the single-stranded form of the plasmid pXL1696 was carried out using the Amersham kit according to the manufacturer's recommendations. This mutagenesis made it possible to introduce an AGC codon into the coding sequence of apoAIV, causing the replacement of aspartic acid by a serine at position 5.
- Ndel-EcoRI fragment of 564 bp was purified, then ligated with the EcoRI-BamHI fragment isolated from the plasmid pXL2051 and the vector pET3-a opened by the enzymes Ndel and BamHI.
- the recombinant vector pXL2073 thus obtained makes it possible to express at a very high level a polypeptide having a serine in position 5. 3. Generation of the vector pXL2069 and preparation of the polypeptide P (D5K).
- mutagenesis directed on the single-stranded form of the plasmid pXL1696 was carried out using the Amersham kit according to the manufacturer's recommendations. This mutagenesis made it possible to introduce an AAA codon into the coding sequence of apoAIV, causing the replacement of aspartic acid by a lysine at position 5.
- Ndel-EcoRI fragment of 564 bp was purified, then ligated with the EcoRI-BamHI fragment isolated from the plasmid pXL2051 and the vector pET3-a opened by the enzymes Ndel and BamHI.
- the recombinant vector pXL2069 thus obtained makes it possible to express at a very high level a polypeptide having a lysine in position 5.
- Ndel-EcoRI fragment of 564 bp was purified, then ligated with the EcoRI-BamHI fragment isolated from the plasmid pXL2051 and the vector pET3-a opened by the enzymes Ndel and BamHI.
- the recombinant vector pXL2062 thus obtained makes it possible to express at a very high level a polypeptide having a phenylalanine in position 44.
- a mutagenesis directed on the single-stranded form of the plasmid pXL1696 was carried out using the Amersham kit according to the manufacturer's recommendations. This mutagenesis allowed to introduce a GCG codon into the coding sequence of apoAIV, causing the replacement of aspartic acid by an alanine at position 44.
- Ndel-EcoRI fragment of 564 bp was purified, then ligated with the EcoRI-BamHI fragment isolated from the plasmid pXL2051 and the vector pET3-a opened by the enzymes Ndel and BamHI.
- the recombinant vector pXL2074 thus obtained makes it possible to express at a very high level a polypeptide having an alanine in position 44.
- a mutagenesis directed on the single-stranded form of the plasmid pXL2051 was carried out using the Amersham kit according to the manufacturer's recommendations. This mutagenesis made it possible to introduce a GCG codon into the coding sequence of apoAIV, causing the replacement of lysine by an alanine at position 178.
- the recombinant vector pXL2073 thus obtained makes it possible to express at a very high level a polypeptide having an alanine in position 178.
- a mutagenesis directed on the single-stranded form of the plasmid pXL2051 was carried out using the Amersham kit according to the manufacturer's recommendations. This mutagenesis made it possible to introduce a TAT codon into the coding sequence of apoAIV, causing the replacement of lysine by a tyrosine at position 178.
- the recombinant vector pXL2070 thus obtained makes it possible to express at a very high level a polypeptide having a tyrosine in position 178.
- mutagenesis directed on the single-stranded form of the plasmid pXL2051 was carried out using the Amersham kit according to the manufacturer's recommendations. This mutagenesis made it possible to introduce an AAA codon into the coding sequence of rapoAIV, causing the replacement of glutamic acid by a lysine at position 230.
- the recombinant vector pXL2072 thus obtained makes it possible to express at a very high level a polypeptide having a lysine in position 230.
- strain BL21 DE3 containing an expression plasmid of a polypeptide of the invention is used as described in parts B1-B3.
- this polypeptide can be stabilized by adding rifampicin during the production phase.
- the only mRNAs that can then be neosynthesized are those that do not depend on E. coli RNA polymerase.
- the cells which then produce only the single mRNA of the desired polypeptide with the exception of all the others are incubated between 90 and 180 min at 37 ° C.
- the recombinant polypeptide produced can represent from 20 to 30% of the total proteins produced by the bacteria.
- the polypeptide is thus accumulated without degradation in the bacteria in soluble form.
- the production was extrapolated in a fermenter, by high density culture of E.coli in Fed-Batch mode. This production was carried out in a 2 liter fermenter (Setric) in 2 phases: a growth phase of the microorganism up to an OD600 of 30 to 40 (duration: approximately 5 hours). This phase is carried out in the fermentation medium defined by Jung et al. (Ann.Inst.Pasteur / Microbiol. 122 (1988) 129-146); then a phase of induction of production, by adding dTPTG and rifampicin, and increasing the rate of supply of carbon and nitrogenous substrates (duration: approximately 1 hour 30 minutes).
- the cells are collected and then lysed, for example by sonication.
- a Branson sonicator (model B30, Proscience, France) was used after concentrating the cells 30 times in PBS buffer (KC1 0.2 g / 1, KH2PO4 0.2 g 1, NaCl 8 g / 1, Na2HPO4 1.25 g / 1).
- the cells are broken at 4 ° C in continuous mode (2 pulses of 5 minutes).
- the concentrated cell suspension can also be pretreated in the presence of Triton X-100 at room temperature before sonication.
- the polypeptides P (R93G), P ( ⁇ C44) and P ( ⁇ N13) were purified according to the following protocol, which takes place under native conditions and does not require any affinity step for the lipids.
- the cell culture is centrifuged at 6,000 rpm for 30 minutes and the pellet is taken up at the rate of 3 ml per gram of wet cells in buffer A (Na 2 HPO 4 81 mM, NaH 2 PO 4 19 mM; EDTA, 2 mM ; PMSF, I mM; pH 7.5; ⁇ -mercaptoethanol 10 mM).
- the suspension is treated by 8 cycles of 3 minutes of ultrasound (BRANSON model set to 250W) at 4 ° C.
- the extracts are centrifuged for 30 minutes at 15,000 rpm at 4 ° C.
- the supernatant SI is stored and the pellet is washed in the same volume of buffer A and recentrifuged under the same conditions.
- the corresponding supernatant SI ' is added to the supernatant SI.
- an aqueous solution of streptomycin sulfate is added an aqueous solution of streptomycin sulfate at 10% at the rate of 10 ml of solution per gram of protein.
- the suspension is centrifuged for 30 minutes at 15,000 rpm at 4 ° C.
- the supernatant (S2) is recovered.
- the dialysate is injected onto a QFF type ion exchange column (Pharmacia) and eluted with a NaCl gradient.
- the determination of the fractions containing the polypeptides of the invention is carried out according to an Elisa test described below.
- the interesting fractions are combined and concentrated by precipitation with ammonium sulfate (final concentration: 80% saturation). After 15 minutes of incubation at 4 ° C with shaking, the suspension is centrifuged for 30 minutes at 15,000 rpm at 4 ° C. The pellet (C80) is recovered and taken up in buffer B and dialysis against 21 of buffer B.
- the dialysate is injected on an ion exchange column of Mono Q type
- buffer D ammonium sulfate, 1.7 M; Na 2 HPO 4 8.1 mM, NaH 2 PO 4 1.9 mM ; EDTA, 2 mM; pH 7.4
- the dialysate is injected onto a hydrophobic phenyl-sepharose interaction chromatography column (Pharmacia) and eluted with buffer E (8.1 mM Na 2 HPO 4 ; 1.9 mM NaHgPO; 2 mM EDTA; pH 7.4) .
- buffer E 8.1 mM Na 2 HPO 4 ; 1.9 mM NaHgPO; 2 mM EDTA; pH 7.4
- the ELISA test developed consists of adsorbing on a plate with a polyclonal rabbit serum directed against human apoAIV (diluted to l / 1000th), saturating this plate with gelatin, incubating the test sample and finally, react an equimolar mixture of two monoclonal antibodies directed against apoAIV (MO3 and MO5, SERLIA, Institut Pasteur de Lille), followed by an immunoenzymatic revelation using a polyclonal anti-mouse serum coupled with peroxidase.
- This test is easily calibrated with a range of dilutions of plasma apoAIV.
- buffer F ammonium sulfate, 40% saturation; Na 2 HPO 4 8.1 mM; NaH 2 PO 4 1.9 mM; EDTA, 2 mM; pH 7.4
- the fraction S2 is directly injected on a QFF type column (without fractional precipitation with ammonium sulfate) equilibrated in buffer B.
- the polypeptide is excluded ⁇ ° ( ⁇ N182)) or eluted with a plateau at 250 mM NaCl in buffer B (P ( ⁇ hl-2)).
- the polypeptide is then concentrated by precipitation in ammonium sulfate (final concentration 50% of saturation for P ( ⁇ hl-2), 10% for P ( ⁇ N182)).
- the purification of P ( ⁇ hl-2) is terminated by centrifugation (30 min., 4 ° C, 10,000 g).
- the pellet is recovered, taken up in PBS buffer, desalted on a gel of filtration PD10 QPharmacia) and dried at -20 ° C.
- the polypeptide P ( ⁇ N182) precipitated with ammonium sulphate is injected onto a chromatography column for hydrophobic phenyl-sepharose interactions (Pharmacia) and eluted with buffer B.
- the polypeptide is identified by polyacrylamide gel SDS, and the fractions of interest are collected and dialyzed against buffer B and stored at 4 ° C.
- polypeptides P (tag ⁇ N13), P (tag ⁇ C44), P (tag ⁇ hl-2), P (tag ⁇ h7-8), P (tag ⁇ h9-10), P (tag ⁇ hll-12), P (tag ⁇ hl3-14) and P (tag ⁇ h5 -6) were purified according to the following protocol, which comprises only one step.
- the cell culture is centrifuged at 6,000 rpm for 30 minutes and the pellet is taken up at the rate of 3 ml per gram of wet cells in buffer A (Na 2 HPO 4 81 mM, NaH 2 PO 4 19 mM; EDTA, 2 mM ; PMSF, 1 mM; pH 7.5; ⁇ -mercaptoethanol 10 mM).
- buffer A Na 2 HPO 4 81 mM, NaH 2 PO 4 19 mM
- EDTA 2 mM
- PMSF 1 mM
- pH 7.5 pH ⁇ -mercaptoethanol 10 mM
- the suspension is treated by 3 cycles of 5 minutes of ultrasound (BRANSON model set at 250W) at 4 ° C.
- the extracts are centrifuged for 1 hour at 11,000 g at 4 ° C.
- the nucleic acids present in the supernatant were precipitated by the addition of 10% (w / v) of streptomycin sulfate (10 ml / g protein), incubation for 30 min. at 4 ° C, then recentrifuged under the same conditions as above.
- the recombinant proteins present in the supernatant were purified by affinity chromatography on a chelated metal ion. The purification was carried out on agarose-nitrilotriacetic acid-nickel (NTA-Ni) resin according to the manufacturer's recommendations, with the following modifications:
- the protein solution is desalted on a Tris-Acryl GF-05 column balanced with a 100 mM phosphate buffer pH 8.
- the harvested protein fractions are combined, diluted in the same phosphate buffer to a final concentration of 4 mg / ml, and supplemented with Hecameg (powder), final concentration 25 mM.
- the mixture was then loaded onto a balanced Ni-NTA column, with the same phosphate buffer containing 25 mM Hecameg. No fixed protein was eluted by washing the column with the same buffer.
- the weakly bound proteins were eluted with a phosphate / citrate buffer pH 6, and the recombinant proteins, with a phosphate / citrate buffer pH 5.
- the protein fractions obtained at pH 5 were neutralized by addition of 1M sodium hydroxide (30 ⁇ l / ml ) and supplemented with 2 protease inhibitors: EDTA 0.1M and PMSF 0.2M.
- the fractions were then analyzed by electrophoresis on polyacrylamide gel (SDS-PAGE) containing 15% acrylamide / bis-acrylamide, according to Laemmli (Nature 227 (1970) 680), then grouped according to purity and quantity.
- the pooled fractions were incubated in the presence of L (-) - histidine powder (final concentration 50 mM), for 1 h at 4 ° C, in order to destroy the Ni-poly-His-protein bond.
- the liberated nickel and histidine were then removed by desalting on a Tris-Acryl GF-05 column equilibrated with a pH 7.4 phosphate buffer containing 2 mM EDTA.
- the recombinant proteins thus purified were analyzed by SDS-PAGE electrophoresis and immunoblotting.
- the immunoblotting was carried out with a mixture of anti-ApoAIV monoclonal antibodies conjugated to peroxidase, and goat anti-mouse antibodies conjugated to peroxidase. Bound peroxidase activity was revealed by incubation with a solution of 4-Chloro-1-Naphtol as a substrate.
- the biological activity of the polypeptides of the invention was evaluated mainly on the basis of 2 parameters: their affinity for the HDL receptor and their effect on the efflux of cholesterol. These 2 parameters account for the cholesterol-lowering pharmacological potential of the polypeptides of the invention.
- the protocol for determining these parameters is given below as well as the results obtained.
- the purified polypeptides are used initially to reconstitute proteoliposomes with DMPC, the structure of which appears identical, in view of the behavior in exclusion chromatography, to that of the proteoliposomes reconstituted with native apoAIV. These reconstituted proteoliposomes are then tested for their capacity to bind to murine adipocytes (line Obl77) according to a protocol already described.
- NAME RHONE-POULENC RORER S.A.
- RUE 20, avenue Raymond ARON
- CAG CTC AGG CAG AAA CTG GGC CCC CAT.GCG GGG GAC GTG GAA GGC CAC 960
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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EP93904117A EP0624194A1 (fr) | 1992-01-27 | 1993-01-26 | Polypeptides derives de l'apolipoproteine aiv humaine, leur preparation et leur utilisation |
JP5512981A JPH07503367A (ja) | 1992-01-27 | 1993-01-26 | ヒト アポリポタンパク質aivから透導されたポリペプチド,その製造及びその利用 |
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FR92/00806 | 1992-01-27 | ||
FR9200806A FR2686605B1 (fr) | 1992-01-27 | 1992-01-27 | Nouveaux polypeptides, leur preparation et leur utilisation. |
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EP (1) | EP0624194A1 (fr) |
JP (1) | JPH07503367A (fr) |
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Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001000803A2 (fr) * | 1999-06-25 | 2001-01-04 | Genset | Proteine associee a l'apolipoproteine a-iv (aa4rp): sequences polypeptidiques, polynucleotidiques et marqueurs bialleliques de ces dernieres |
WO2003045993A2 (fr) * | 2001-11-23 | 2003-06-05 | Syn.X Pharma, Inc. | Marqueurs biopolymeres proteiques predictifs des diabetes de type ii |
WO2003045983A1 (fr) * | 2001-11-23 | 2003-06-05 | Syn.X Pharma, Inc. | Apolipoproteines utilisees comme marqueurs biopolymeres pour la prediction du diabete de type ii |
WO2003046000A2 (fr) * | 2001-11-21 | 2003-06-05 | Syn.X Pharma, Inc. | Marqueurs biopolymeres d'apolipoproteine predicitifs d'une resistance a l'insuline |
WO2003054014A2 (fr) * | 2001-12-13 | 2003-07-03 | Syn.X Pharma, Inc. | Marqueurs biopolymeres d'apolipoproteine indiquant la maladie d'alzheimer |
FR2893136A1 (fr) * | 2005-11-10 | 2007-05-11 | Sanofi Aventis Sa | Procedes de diagnostic de maladies hepatiques et de criblage de molecules pour le traitement de ces maladies |
WO2012100010A1 (fr) * | 2011-01-19 | 2012-07-26 | University Of Cincinnati | Apolipoprotéine aiv utilisée en tant que peptide antidiabétique |
WO2013109342A1 (fr) * | 2012-01-19 | 2013-07-25 | University Of Cincinnati | Procédé de traitement du diabète à l'aide d'apolipoprotéine a-iv non glycosylée |
JP2015524808A (ja) * | 2012-07-25 | 2015-08-27 | ユニバーシティ・オブ・シンシナティ | アポリポタンパク質aivを用いたi型糖尿病の治療法 |
US10232019B2 (en) | 2012-07-25 | 2019-03-19 | University Of Cincinnati | Method of treating hyperglycemic disorders using apolipoprotein AIV |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1068239A2 (fr) * | 1998-03-31 | 2001-01-17 | University Of Cincinnati | Peptides antioxydants |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1990012879A2 (fr) * | 1989-04-20 | 1990-11-01 | Cesare Sirtori | Expression d'apolipoproteines ai-milano humaines dans la levure |
-
1992
- 1992-01-27 FR FR9200806A patent/FR2686605B1/fr not_active Expired - Fee Related
-
1993
- 1993-01-26 EP EP93904117A patent/EP0624194A1/fr not_active Withdrawn
- 1993-01-26 WO PCT/FR1993/000073 patent/WO1993015198A1/fr not_active Application Discontinuation
- 1993-01-26 CA CA002125877A patent/CA2125877A1/fr not_active Abandoned
- 1993-01-26 JP JP5512981A patent/JPH07503367A/ja active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990012879A2 (fr) * | 1989-04-20 | 1990-11-01 | Cesare Sirtori | Expression d'apolipoproteines ai-milano humaines dans la levure |
Non-Patent Citations (2)
Title |
---|
BIOTECHNOLOGY vol. 6, no. 11, Novembre 1988, NEW YORK US pages 1321 - 1325 E. HOCHULI ET AL. 'genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent' cité dans la demande * |
JOURNAL OF BIOLOGICAL CHEMISTRY vol. 262, no. 17, 15 Juin 1987, BALTIMORE, MD US pages 7973 - 7981 NABIL A. ELSHOURBAGY ET AL. 'Structure and expression of the human apolipoprotein A-IV gene' * |
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WO2001000803A3 (fr) * | 1999-06-25 | 2001-12-27 | Genset Sa | Proteine associee a l'apolipoproteine a-iv (aa4rp): sequences polypeptidiques, polynucleotidiques et marqueurs bialleliques de ces dernieres |
WO2001000803A2 (fr) * | 1999-06-25 | 2001-01-04 | Genset | Proteine associee a l'apolipoproteine a-iv (aa4rp): sequences polypeptidiques, polynucleotidiques et marqueurs bialleliques de ces dernieres |
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US7314762B2 (en) | 2001-11-21 | 2008-01-01 | Nanogen, Inc. | Apolipoprotein biopolymer markers indicative of insulin resistance |
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US7125678B2 (en) | 2001-11-23 | 2006-10-24 | Nanogen, Inc. | Protein biopolymer markers predictive of type II diabetes |
WO2003045993A3 (fr) * | 2001-11-23 | 2003-11-20 | Syn X Pharma Inc | Marqueurs biopolymeres proteiques predictifs des diabetes de type ii |
WO2003045983A1 (fr) * | 2001-11-23 | 2003-06-05 | Syn.X Pharma, Inc. | Apolipoproteines utilisees comme marqueurs biopolymeres pour la prediction du diabete de type ii |
WO2003045993A2 (fr) * | 2001-11-23 | 2003-06-05 | Syn.X Pharma, Inc. | Marqueurs biopolymeres proteiques predictifs des diabetes de type ii |
WO2003054014A3 (fr) * | 2001-12-13 | 2003-08-21 | Syn X Pharma Inc | Marqueurs biopolymeres d'apolipoproteine indiquant la maladie d'alzheimer |
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Also Published As
Publication number | Publication date |
---|---|
FR2686605A1 (fr) | 1993-07-30 |
JPH07503367A (ja) | 1995-04-13 |
EP0624194A1 (fr) | 1994-11-17 |
FR2686605B1 (fr) | 1994-03-11 |
CA2125877A1 (fr) | 1993-08-05 |
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