CA2125877A1 - Polypeptides derived from human aiv apolipoprotein, preparation and use thereof - Google Patents

Abstract

The present invention relates to polypeptides derived from human apolipoprotein AIV (apoAIV), the nucleotide sequences encoding these polypeptides, their preparation and their use.

Description

WO 93/15198 ~ 7 PCT / FR93 / 00073 POLYPEPTIDES DERIVED FROM HUMAN AIV APOLIPOPROTEIN, THEIR PREPARATION AND

The present invention relates to polypeptides derived from apolipoprotein.
Human AIV (apoAI), the nucleotide sequences coding for these polypeptides, 5 their preparation and their use.

Apolipoprotein AIV (apoAIV) is a protein made up of 376 acids.
amines, having a molecular weight of 46,000 daltons. The apoAIV peptide and nucleotide sequences, as well as the position of the helices are shown on Ies sequences SEQ ID No. 1 and 2. ApoAIV is a major component of lo chylomicrons secreted in the Iymphe, but it has the particularity of being mostly in non-associated form with lipoproteins in plasma (RB
Weinberg et al., 1983, J. Lipid. Research, 24: 52-59). Furthermore, apoAIV
plasma is polymorphic, although the nature of this polymorphism is still unknown (G. Utermann et al., 1982, J. Biol. Chem. 257: 501-507). The role The physiological nature of apoAIV also remains relatively unknown. We know she can activate lecithin cholesterol acyltransferase (LCAT) in vitro (Steinmetz et al., 198S, J. Biol. C} lem., 260: 2258-2264) and that it can, like apolipoprotein AI, interf ~ rer with fixadon of HDL particles on endo ~ elial cells bovine aoitics (Savion et al., 1987, Eur. J. Biochem., 257: 41714178). These 20 two activities indicate that apoAIV most likely occurs as a mediator of reverse cholesterol transport.

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 choles ansport ~ erol. The present invention is based more precisely on

2 ~ the use of apoAIV for the preparation of new products, allowing, by new ~ therapies, to fight against hypercholesterolemia and the effects therein associated, such as atherosclerosis.
More specifically, the present invention provides polypeptides derived from apoAIV, and thus makes it possible to pharmacologically exploit the properties of this 30 p ~ otein. The present invention therefore relates to polypeptides derived from human apolipoprotein AIV. They may in particular be polypeptides having a increased plasma stability compared to native protein (lower sensitivity physiological mechanisms of elimination QU of degradation), and therefore ... . . . ~. . . . . . ... ~.

WO 93/15198 pcr / FR93 / ooo73 2125 ~ 77 2 longer service life. They may also be polypeptides having an activity stimulating the efflux of cellular cholesterol, and therefore favoring the - reverse transport of cholesterol, leading to discharge of the cells which have accumulated cholesterol against the background of the formation of an atheroma plaque. They may also be polypeptides having only one or more of the properties from apoAIV. In particular, they may, for example, be polypeptides capable of bind cell receptors, but lacking efflux-stimulating activity ~ cell cholesterol. In addition, it can also be polypeptides.
devoid of apoAlV activities. Such polypeptides can indeed have 10 therapeutic uses (generation of antibodies, structure-function studies, etc.).
. Preferably, the polypeptides of the invention are capable of binding the HDL receiver.
Even more preferably, the polypeptides are capable of binding the HDL receptor and stimulate an efflux of cholesterol.
The polypeptides according to the invention can be of several types. he can with respect to human apoAIV, these are mutation or substitution derivatives, deledon derivatives or addition derivatives. It is understood that the present invendon also covers polypepddes with several types of modifications, such as that for example mutation and deletion derivatives, deletion derivatives and 20 addition, etc.
More preferably, the invention relates to non-polypeptides natural derivatives of human apolipoprotein ~ IV including, compared to the sequence of human apolipoprotein AIV SEQ ID No. 2, at least one of following changes:
(A) a point mutation involving a functional residue, and / or, (b) a deletion of a terminal end of at least 10 amino acids, and / or, (c) a deletion of a propeller or a pair of propellers, and / or, (d) an additional heterologous part having a different biological activity or complementary to that of apoAIV, or a property of marker, of targeting30 or of stabilizer.
In a particular embodiment, the polypeptides of the invention comprise at least minus a modification according to (a) and (b).
In another particular embodiment, the polypeptides of the invention comprise at least one modification according to (a) and (c) ~

Wo 93/15198 pcr / FRs3 / ooo73 212 ~ 77 In another particular embodiment, the polypeptides of the invention comprise a modification according to (a), (b) or (c) associated with a modification according to (d).
Mutation drifts generally include a point mutation ~ deletion of an amino acid, or modification of an amino acid, or replacement of one amino acid by another), or double ~ modification of a pair of amino acids).
The mutations chosen take into account the position of the mutated residues in their respective propellers, and tend a priori to modify the functional groupings without disrupting too much the structure of the helix concerned, and therefore of the polypeptide. Within the meaning of the present invention, by functional residue is meant r ~ residuals capable of being involved in apoAIV activity, i.e. at the level of interaction with the receptor, or at the level of the transmission of a signal (such as for example stimulating the efflux of cholesterol). The preferred residues are generally the charged residues, which have a potential effect in the interaction of the polypeptide with its receiver. Such residues can be ~ replaced by others, apolar, or chemically modified by removing or adding a filler. Residues glycosylates and residues involved in the formation of disulfide bridges (cysteine) are other preferred residues.
Preferably, the polypeptides of the invention have, with respect to human apoAIV as shown; in 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 posit; on 39; Iysine in position 73.84, 103,169.17 ~; glutamic acid in position 76, B1, 87, 99, 131, 164, 187, 230;
alanine at position 22; proline at position 139, 161; and serine in position 154. More preferably, aspartic acid can be replaced by a residue S, K, A, F or &; glutamine by a T, K or F residue; asparagine by a residue A
or D; lysine by a residue G, E, T, D, A, Y, H or F; glutamic acid by a residue S, R, F, K, A, G, N, or Q; alanine with a residue R or E; proline by a residue R or G; and the serine by a residue E or R (the letters correspond to an acid amine according to recognized code).
The polypeptides of the invention can be deletion derivatives. In in this case, the deletion (s) may relate to one end of the protein (C-terminal or N-terminal). In this regard, certain polypeptides of the invention are fragments of apoAIV, obtained by deletion of important parts of the protein.
Preferably, the polypeptides of the invention have, relative to apoAIV

WO 93/15198 PC ~ r / FR93 / 00073 212 ~ 877 4 human terminal deletions of at least 10 amino acids. The deletion (s) may also relate to internal regions of apoAIV, and in particular to whole propellers or on pairs of propellers.

Another type of polypeptide according to the invention is represented by S addition derivatives. In particular, the invention relates to polypepddes comprising all or part of apoAIV and an additional element such as a marker, a targeting agent, stabilizer or other active ingredient. As a purification marker, by way of example, the decapeptide t ~ g of sequenoe MRGS (H) 6 described by Hochuli et al (Bio / technol. (1988) 1321). As illustrated in the examples, this lo decapeptide can be fused to the N-terminal of apoAIV or a derivative such as defined above, thus allowing very rapid purification in one step, without affect the production capacities of said polypeptide.
A ti ~ ed` specific examples of polypeptides according to the invention, one can preferentially cite the following polypeptides:
1 ~ - polypepdde P (~ N13, R93G): polypeptide having a deletion of 13 N-terminal amino acids (/ ~ N13) and a glycine instead of an arginine in position 93 (R93G).
- P (N13) polypeptides: polypeptide having a deletion of 13 acids N-terminal amines, and its P tag version (N13 tag).
- polypeptide P (R93G): polypeptide having a glycine instead of a arginine in position 93.
- P polypeptides (~ C44 ~: polypeptide having a deletion of 44 acids C-terminal amines, and its tag version.
- polypeptide P (/ ~ C194): polypeptide possan ~ a deletion of 194 2s of ~ last amino acids.
- polypeptide P (~ N182): polypeptide having a deletion of 182 first amino acids.
- P polypeptides (~ h1-2): polypeptide having a deletion of the helices 1 and 2 as shown in SEQ ID No. 2 (residues D14 to V62) ~ and its tag version.
- P polypeptides (l \ h7-8): polypeptide having a deletion of the helices 7 and 8 as presented in SEQ ID No. 2 (residues P162 to A205), and its version tag.

7 ~

- P polypeptides (h9-10): polypeptide having a deletion of the helices 9 and 10 as presented in SEQ ID No. 2 (residues P206 to A249), and its version tag, - P polypeptides (~ h11-12): polypeptide having a deletion of the helices s 11 and 12 as presented in SEQ ID No. 2 (residues P250 to E289), and its version tag.
- P polypeptide (hll-12, L ~ 7M): polypeptide having a deletion of propellers 11 and 12 as shown in SEQ ID No. 2 (~ hll-12), and a methionine instead of a leucine at position 87 (L87M).
- P polypeptides (ah13-14): polypeptide having a deletion of the helices 13 and 14 as presented in SEQ ID No. 2 (residues P290 to N333), and its tag version.
- P polypeptides (~ h5-6): polypeptide having a deletion of the helices 5 and 6 as shown in SEQ ID No. 2 (residues P118 to R161), and its version tag.
- polypeptide P ~): polypeptide having a phenylalanine instead of an aspartic acid in position 44.
- polypeptide P (D44A): polypeptide having an alanine instead of a aspartic acid in position 44.
- P polypeptide (DSS): polypeptide having a serine instead of an acid aspartic in position 5.
- polypeptide P (D5K): polypeptide having a lysine instead of an acid aspartic in position 5.
- pclypeptide P (K178Y): polypeptide having a tyrosine instead of a ~
Iysine in position 178.
- P polypeptide (K178A): polypeptide having an alanine instead of a Iysine in position 178.
- polypeptide P (E230K): polypeptide having a lysine instead of a glutamic acid in position 230.
Such polypeptides according to the invention are of pharmaceutical interest important, especially in the treatment and / or prevention of atherosclerosis.Atherosclerosis is a disease that is characterized by the formation of plaques lipid or fibr ~ lipid in the intima of the aorta, coronary arteries and carotid essentially. These plates, more or less calcified according to the progress of the processes, can be ~ entangled with lesions, and are created by the accumulation in WO 93/15198 PCI ~ FR93 / 00073 212S $ 77 6 the arteries of fatty deposits consisting essentially of cholesterol esters. These plaques then cause a thickening of the arterial wall, with enlargement 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 5 a stenosing character responsible for vascular occlusions by atheroma, thrombosis or emboUe, which occur in the most affected patients.
The formation of an atheroma plaque therefore results from the conjugation of different factors, (i) an excess of plasma cholesterol, from which the arterial deposits, and (ii) a defect in regulation between influx and efflux of cholesterol at o level of peripheral tissues, in favor of an intracellular accumulation. Taking into account their properties, the polypeptides according to the invention can in particular allow slowing the formation of atheroma plaques, inducing regression of atheroma plaques, and decrease the risk of accident incidence coronaries.
The polypeptides of the invention can be obtained in different ways, and in particular by chemical and / or genetic means.
In the case of chemical preparation, the polypeptides of the invention can be obtained either entirely by chemical synthesis, or by chemical or enzymatic modifications of the nadve protein. In the latter case, 20 different chemical or enzymatic agents can be used, making it possible to modify residues (mutation derivatives), mark residues (derivatives addition) or fragment the protein (deletion derivatives). Among the chemical agents which can modify or mark amino acids, more may be mentioned particularly sulfo-NHS-acetate, which allows the acylation of primary amines and 2s especially lysine; tetranitromethane which introduces a nitrate group on the tyrosines, or even N-bromo-succinimide which oxidizes t ~ yptophan residues.
Among the chemical and enzymatic agents used to cut bonds peptide, more particularly cyanogen bromide, iodosobenzoate, trypsin, chymotrypsin, thermolysin or even 30 proendopeptidase.
As stated above, these treatments can be applied as well on the polypepdde product of a chemical synthesis than on the native polypeptide. Of more, these treatments can also be applied to the polypeptides obtained by see genetic, and therefore, already with certain modifications.

WO 93 / lSl98 pcr / FR93 / ooo73 21 ~ 7 ~

In the case of preparation by genetic route, the polypeptides of the invention are obtained by modification of the nucleotide coding sequences, and expression of said sequences in a cellular host.
In this regard, the present invention also relates to the sequences s nucleotides for the polypeptides described above. It could be DNA sequences, RNA sequences, synthetic sequences, semi synthetic, hybrid, etc. These sequences can be used:
- for the production of the polypeptides of the invention by expression in a cell host, o - as a composition for gene therapies, - for the realization of marked probes allowing the identification of related polypeptides or the demonstration of an expression of the polypeptides of the invention, - for the preparation of new cloning or expression vectors lS containing these sequences, or - for the preparation of new eukaryotic or proca ~ yotes cells recombinant or transgenic animals containing these sequences or vectors.
The nucleotide sequences of the invention can be obtained from of a sequence coding for apoAIV, by different techniques such as in particular by ge ~ ethical cutting, mutagenesis, or any other treatment altering the structure of nucleic acids. The techniques best known to man of the trade are mentioned below in general cloning techniques. The detail of the synthesis of the nucleotide sequences of the invention is given in part B /
examples. The human apoAIV sequence used in the invention was obtained from a genomic clone containing the f ~ agment KpnI-HindIII of the human apoAIV gene sequence, which contains the end of intron 2, all of exon 3 as well as the 3 'untranslated sequence. This clone therefore did not contain, in in particular, the first two exons of the human apoAIV gene (Elshourbagy etcoll. JBC (1987) 262: 7973). The complete sequence was obtained by:
- chemical syn ~ thesis of the 5 'end of the apoAIV 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, WO 93/15198 PCI` / FR93 / 00073 212 ~ 8 ~ 7 -- the binding of this synthetic fragment to a DNA fragment containing the remaining from the apolipoprotein AIV gene sequence located after the BstEII site, used to join to the nearest nucleotide.
The details of these steps are given in part A / of the examples.
S The present invendon also relates to a process for the preparation of polypeptides of the invention described above. This process consists of carrying out the steps following:
- in a first step, a sequence is introduced into a cell nucleotide as defined above coding for a polypeptide of the invention, ~ 0 - in a second step, the cell thus obtained is cultured in conditions for expression of said nucleotide sequence and, - In a third step, the polypeptide produced is recovered.
More particularly, during the first step of the the invention, the nucleotide sequence can be introduced into the cell by different techniques. In particular, the introduction can be carried out by ~ ansf ~ mation, conjugation, or electroporation.
Regarding the transformation, different protocols have been described in anterior art. In particular, it can be achieved by treating whole cells in presence of lithium acetate and polyethylene glycol according to the technique determined by Ito et al. ~ J. Bacteriol. 153 (1983) 163-168), or in the presence of ethylene glycol and ; dimethyl sulfoxide according to the technique of Dun ~ ns et al. (Curr. Genet. 18 (1990) 7).
The transformation can also be carried out according to the technique described by Dagert at al. (Gene 6 (1979) 23-28) by treatment with a solution of CaC12 then shock thermal. An alternative protocol has also been described in the patent application 2 ~ EP 361 99 ~.
Agissa ~ t electroporation, the technique described by Karube et al. (FEBS
Letters 182 (198 ~) 90) can advantageously be used.
The choice of one or the other of these methods is established in particular by depending on the host chosen.
Among the euca ~ otes hosts usable in the process of the invention, one can cite animal cells, yeasts, or fungi. In particular, with regard to yeasts, there may be mentioned the yeasts of the genus Sac ~ bammyces, Kluvveromvces, ~ hi ~ a - ~ astoris, Schwanniomvces, or Hansenula. In the case of animal cells, we can quote the cells COS, CHO, Cl27, etc. Among the fungi likely to be .:

~ 123 & 7 ~

dw used the present invention, there may be mentioned more particularly Asper ~ illus ssp. or Trichoderma ssp.
Among the prokaryotic hosts usable within the framework of the present invention include the following bacteria ~ .5 ~, BaCi1l ~, OR Streptomvces.
s In a preferred embodiment of the invention, the method is implemented by using as a host cell a prokaryotic cell.
Even more preferably, the method of the invention is implemented using the bacteria ~, ~ QIi as the host cell.
Generally, the nucleotide sequence used is a sequence genomics, cDNA sequence, hybrid sequence sequence, etc. For a better implementation of the invention, it is however preferred to use a cDNA (cf.
example A / ~. Furthermore, this nucleotide sequence generally comprises a start area; rage of transcription and translation attached to the end S ' terminal of the coding sequence, so as to direct and regulate transcription and 1 ~ translation ~ of said sequence. The choice of these regions may vary depending on the host used. In particular, in bacteria such as E. ~ i. we can use the ~ omotn ~ of the op ~ ron tryptophan (Ptrp), or the left and right promoters of the bacteriophage lambda (PL, PR) OR the promoter of gene 10 of bacteriophage17.
Furthermore, the nucleotide sequence is preferably part of a vector, which can be autonomous or integrative replication. More particularly, autonomously replicating vectors can be prepared using sequences to autonomous replication at the chosen host. For example ~ in yeast, it can origin of replication derived from plasmids: p ~ Dl (EP 241 435) or chromosomal sequences (ARS ~ and in bacteria, they may be from replication derived from plasmids (pBR322, pET3, etc.). Regarding vectors integrradfs, these can be prepared for example by using sequences homologous to certain regions of the host genome, permissible, by recombination homologous, integration of the vector. In this regard, the use of rDNA allows a multiple integration of exogenous DNA, and therefore its presence in a larger number of copies per cell.
In a preferred mode, the nucleotide sequence comprises, upstream of the coding sequence, or, if appropriate, between the transcription start region and translation and the coding sequence, a "leader" sequence directing the ~ 12 ~ 77 ~ o Polypeptide originating in the secretion pathways of the host used. This sequence Hleader "can be the leader sequence of apoAIV, but it can also be a heterologous sequence (derived from a gene coding for another protein) or even artificial. The choice of one of these sequences is notably guided by the host s used.
The polypeptides ~. of the present invention can then be isolated from culture medium by any technique known to those skilled in the art. More in particular, part C / of the examples describes a method allowing to purify under native conditions, i.e. without any denaturation step, the polypeptides of the invention.
Another subject of the invention relates to phannaceudic compositions comprising one or more polypeptides according to the invention or one or more nucleotide sequences according to the invendon.
Preferably, such compositions are intended for the treatment or Is the prevention of conditions linked to hypercholesterolemia.
Even more preferably, the purpose of the invendon is compositions pharmaceuticals comprising one or more polypeptides according to the invention, or a or several nucleodic sequences according to the invention, intended for slowing down the formation of atheroma plaques, and / or the re ~ & ession of atheroma plaques 20 and / or to reduce the risk of coronary accidents.
In addition, the present invention also relates to the use of polypeptides described above for the production of molecules of non-peptide structure or not exclusively peptide having the same type of activity. In this regard, the invention relates to the use of a polypeptide of the invention as described 25 above for the preparation of non-pepddic molecules, or not exclusively peptides, pharmacologically active on the plasma levels of cholesterol, by determining the structural elements of this polypeptide which are important for its activity, and reproduction of these elements by non-peptide or not exclusively peptide. A subject of the invention is also pharmaceutical compositions comprising one or more senior molecules.
prepared.
The present invention will be more fully described with the aid of the examples.
; following, which should be considered as illustrative and not limiting.

WO 93/1 ~ 198 2 1 2 ~ S 7 7 PCr / FR93 / 00073 he L ~: GENDE OF FLAGURES
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 s TECHNlOUE ~ CLONING ENERALS
Conventional techniques for the purification of plasmids, the preparation of cells competent for transformation by the CaC12 method, are described in the laboratory manual: T. Maniatis et al., Molecular cloning, 1982, Cold Spring Harbor Laborato y. DNA sequences were determined by Sanger's method (Smith AJ.H., 1980, Methods in Enzymol. 65: 499-559). Protocols of cloning in M13 are described (Messing et al. 1981, Nucleic Acid Res. 9:
309-321) ~ Restric ~ ion endonucleases (New England Biolabs) were used according to the manufacturer's advice ~ The buffer used for composition ligatures `` following: 50 mM Tds-HCl, 10 mM MgCl2, 15 mM DTT, 1 mM ATP, pH 7.5. The 15 oligonucleotide phosphorylation buffer has the following composition:
Tris-HCl S mM, MgCl2 1 mM, DTT 0.6 mM, pH 7.5 ~ Site-directed mutagenesis by oligodeoxynucleotides is carried out according to the method developed by Taylor and aL (Nucleic Acids ~ es ~ ~ (1985) 8749-8764) using the kit distributed by Amersham ~
. .
20 A - ~~ YNTHESE AND ~ SEMBLAGE l) E LA_ SEOUENCE COI:) ANTE DE
APOLIPOPROTEIN AIV AND VECTOR DISTRIBUTION.
A ~ - Sv ~ èse and a ~ bla ~ ed ~ l ~! 2nte de ~ AIV.
The nucleotide sequence of the apolipoprotein AIV gene described herein invention differs from previously published cDNA sequences (see in particular 2s the list of cDNA sequences published by CY ~ Yang, ZW. Gu, I. Chong, W ~ Xiong, M ~ Rosseneue, H. Yang, B. Lee, A ~ M. Gotto and L. Chan, 1989, B ~ B ~ A., 1002:
231-237). In particular, the sequence encoding the signal signal peptide of the protein is absent, and, upstream of the first codon of the mature protein was placed a codon of start of t ~ ATG aduction ~ This therefore introduces a supernatant methionine-30 upstream of the sequence of the mature protein, and thus allows to express the gene encoding only for the mature part of the protein.

WO 93/15198 PCI` / FR93 / 00073 ~ 12r ~ ~ 7 On the other hand, this nucleotide sequence of the apolipoprotein AIV has been obtained in an original way by the assembly of four oligonucleotides which have were synthesized chemically and whose size is between 86 and 107 sea (SEQ ID no 3-6). In addition, it can be noted on the sequence of the oligonucleotides5 that cohesive ends XbaI and EcoRl have been defined in order to pe ~ nettre a two-step assembly of the complete sequence of the apolipoprotein AIV.
1. Synthesis of oligonucleotides The four oligodeoxynucleotides that were used to assemble the gene for apolipoprotein AIV, were synthesized by the phosphoramidite method (LJ.
10 Bride and MH Caruthers (1983) Tetrahedron Lett. ~ 4: 245) using the Bioresearch synthesizer (Model 8600) according to the advice of the manufacturers. Oligonucleotides tides were purified on acrylamide gel 15 ~ o. Their sequence is given on the SEQ ID sequences n 3-6.
2. First assembly step The oligonucleotides were phosphorylated by treatment with T4 DNA
kinase. Oligonucleoddes A and C were paired respectively with the oligonucleotides B and D 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 80C which is allowed to return to temperature.
20 of the laboratory.
The AB and CD fragments were ligated in the presence of T4 DNA ligase with the replicative form of phage M13mplO previously digested by enzymes XbaI and EcoRI.
The replicative form of the recombinant bacteriophage obtained called pXL1695 2s (M13mplQABCD) was used to transfect TGl bacteria made competent by the CaC12 method.
This pXL1695 replicon has been purified, either in the form of single-stranded DNA and its sequence has been verified, either in the form of replicative double-stranded DNA for the continuation of constructions.
30 3. Second assembly step The replicative form of pXL1695 ~ M13mplOABCD) and a plasmid called pXL1694 carrying a KnpI-HindIII fragment of approximately 3 kb containing the entire : ``. .

third exon of the apoAIV gene (NA Elhourbagv, J Biol. C: hem .. 19 & 7, 262:
79 / 3-7981) were respectively dictated by XbaI el BslEII of a pa ~ and EcoRI and BstEII on the other hand. In the case of pXL169 ~, a fragment of 167 base pairs has been purified on acrylamide gel. The replicative form of the vector ~ I13mpl8amIV was ~ ligated by XbaI and EcoRI and a fragment of approximately 7 kb was purified on gel.
agarose.
The three fragments were pooled and ligated in the presence of T4 DNA
ligase.
After transfection of TG1, clones containing the replicative form called pXL1696 have been obtained. The complete coding sequence of the fragment XbaI-EcoRI thus cloned in pXL1696 has been verified. This fra ~ gment was then re-extracted from pXL1696 and ligated in the presence of a EcoRI-HindIII fra ~ ment, approximately 3 kb from pXL1694 and bearing in particular the end of exon 3 of human apoAIV gene, and also in the presence of the cut M13mpl9 vector by the enzymes XbaI and HindIII.
The recombinant vector thus obtained, pXL1697, carries all of the coding sequence of apoAlV (1132 bp) as well as a genomic fragment of approximately 2 kb corresponding to intron 3 of the apoAIV gene (Figure 1).
Site-directed mutagenesis performed with Amersham mutagenesis ki ~
(~ PN 1 ~ 23) using the synthetic oligodeoxynucleotide SqlOB7: SEQ ID n 7, allowed to introduce on pXL1697 a BamHI site immediately after the stop codon (TGA) of the apoAIV gene. The vector obtained was designated pXL1866.
A2 - Preparation of the vectors carrying the sequence of the oAIV.
1. Preparation of the vector pXL1872.
The vector pXL1866 described above was cut by the enzymes XbaI and bamHI, and a 1.1 fragment; kb containing the entire coding sequence of. apoAIV was purified on agarose gel and religated in M13mp18amIV for give the vector pXL1 ~ 72 (fi ~ gure 2).
2. Preparation of the vector pXL1867.
The vector pXL1866 was cut with NdeI and BamHI and a fragment of 1.2 kb was purified and then inserted into pET3-a, itself cut with BamHI and NdeI. The ; expression vector. ~ ltant, pXL1867, therefore contains the coding sequence of ~:, : ~ UILLE ~ E REU ~ PLA ~ EU ~ ENT

~ 12 ~ g7'-i apolipoprolein AI ~ 'without any residual ~ intron 3 from the genomic sequence of apolipoproline AI ~ T (Figure 2).

3. Preparation of the vector pXL1696.
The 570 bp XbaI-EcoRI fragment containing the 5` part of the sequence coding of apoAIV (residues 1 to 189) was excised from the vector pXL1872, purified on agarose gel and religated in M13mplO to give the vector pXL1696 (figure 3). ...

4. Preparation of the vector pXL20 ~ 1.
The EcoRI-BamHI fragment of S77 bp containing the 3 ′ part of the sequence o co ~ ante of apoAIV (res ~ s lS8 to 377) was excised from the vector p ~ 1872, punfied on agarose gel and religated in M13rnpl9 to give the vector pXL, 2051 (figure 3).
B - GENERATION OF SEO ~ NUCLEOTIDIOUCES ENCODING FOR
~ S P.QLYPEPI'IDES DE I ~ T ~ EN ~ E ~ PRC ~ DUCT10 ~ ~ OE: S
- T; ~ T ~ T ~ r ~ T ~ C ~
1 ~ r ~ JL, ~ r ~ r 1 lL, 'L ~.
Bl Mutants produced from pXII 872 vector 1. Generation of the vectors pXL1775, pXL186 ~ and pXL2168,. And preparation of the pol ~ peptides P (~ N13, R93G), P ('\ N13) and P (tag ~ N13 ~.
With the help of the phosphonrlated oligodeoxynucleotide SEQ ID No. 8, a muta_enèse directed on the single stranded form of the vector pXL1872 described below above was made by using the Amersham kit according to the recommendations of the maker. This mutagenesis made it possible to introduce an NdeI site as well as a codonATG in the coding sequence of apoAIV a1LY positions 40 and 43 respectively, causing a deletion of the first 13 N-terminal amino acids. Otherwise, 25 during the sequence verification of the derivatives obtained, certain clones including the clone pXL1799 carried an additional point mutation in position 280 on the sequence. This mutation (C-> G) has the effect of changing an arginine codon to glycine in protein.
From clone pXL1799 and another clone not carrying the mutation (C-> G ~ additional, an NdeI-BamHI fragment of 1097 bp was purified and ~: EUILLE DE RE ~ JIPLACE ~ IENT

- ~ 125g ~ 7 ligated with the vector pET3-a (AMS Biotechnolo ~) ~ itself cut by NdeI and BamHI enzymes.
Two recombinant vectors have been obtained: The vector pXL1775, which allows to express at a very high level a polypeptide having a deletion of

5 13 N-terminal amino acids (l ~ N13) and a glycine instead of an arginine in position 93 (R93G ~; and the vector pXL1869, which allows expression at a very high level a polypeptide having a deletion of the 13 amino acids N-terrninal ~ '\ N13).
From the vector pXL1869, the vector pXL2168 was constructed, in which the sequence co ~ ant for the polypeptide P (~ N13) has been fused in S` to a 10 sequence coding for the ta decapeptide "MRGS (H) 6 ~ For this, the 2 Oligodeoxynucleotides following OM have been synthesized, using a synthesizer Biosearch 8600 DNA:
Oligo A: SEQ ID # 9 Oligo B: SEQ ID # 10 s oligodeoxynucleotides A and B were hybridized, and the prc ~ uit of hybridization was ligated with the v ~ ctor pXL1869, previously digested with llen ~ me NdeI. The vector thus obtained, designated pXL2168, code for the N-terminal fused P (~ N13) polypeptide with the MRGS decapeptide (~ I) 6.
-2. Generation of the vectors pXL1766 and pXL2182, and preparation of the polypeptide 20 P (~ C44) and its ~ rersion tag.
Using the phosphorylated synthetic oligodeoxynucleotide SEQ ID No. 11, a mutagenesis directed on the simple brisn form of the plasmi ~ e pXL1872 described below above was performed using the Arnersham kit according to the manufacturer's recommendations. This mutagenesis has pe ~ SiS to introduce a BamHI site and a codon 25. TGA in the coding sequence ~ e apoAIV at the respective positions 1000 and 997, causing a deletion of the 44 C-terminal amino acids.
From a re-feeding ck.ne (pXL176 ~) whose sequence has been verified intétJra ~ ement, an NdeI-BamHI fragment of 1004 bp was purified and ligated with the vector pET3-a, itself coup ~ é by the enz, vmes NdeI and BamHI.
30 The vector recor r ~ inant pXL1766 thus obtained perrnet to express at very high level a polypeptide p / ~ sedating a deletion of the 44 C-terminal amino acids (~ C44).
Following the pr: ~ ole described in B1 ~ 1), the vector pXL2182 was constructed at from the vector pXLI `. The vector pXL2182 thus obtained makes it possible to express at RE'MPLAC'sts SHEET ~ 9IN ~

~ 1 ~ 3 ~ 77 high level the polypeptide P (~ C4 ~) fused in l ~ -tenninal to decapeptide MRGS (H) 6.
3. Generation of the vector pXLl / l4 and preparation of the polypeptide P (~ C194).
Using the oligodeoxynucleotide s ~, ~ phosphorylated thetic SEQ ID n 12, 5 a mutaaénèse directed on the simple bnn form of the plasmid pXL1872 described above above was carried out using the Amersham kit according to the recommendations of the maker. This mutagenesis made it possible to introduce a BamHI site as well as a codon TGA in the coding sequence of apoAIV at the respective positions 553 and 550, causing a deletion of 194 arnine acids on the C-terminal side.
From a mutant clone (pXL1798) whose sequence has been verified integrally, an NdeI-BamHI fragment of 55; bp was purified and ligated with the vector pET3-a, itself cut by the enzymes NdeI and BamHI.
The recombinant vector pXL1774 thus obtained makes it possible to express at very high level a polypeptide having a deletion of the last 194 amino acids C194).
- 4. Generation of the vector pXL1817 and preparation of the polypeptide P (AN182).
Using the phosphorylated synthetic oligodeoxynucleotide SEQ ID No. 13, site-directed mutagenesis s ~ r the single-stranded form of the plasmid pXL1872 described above above was carried out using the Amersham kit according to the recommendations of the maker. This mutagenesis made it possible to introduce an NdeI site as well as a codonATG into the coding sequence of apoAIV at the respective positions 544 and 547, causing a deletion of 182 amino acids on the N-terminal side.
From ~ ir of a mutant clone (pXL176 ~) whose sequence has been verified integrally, a 579 bp NdeI-BasnHI fragment was purified and ligated with the 2 ~ vector pET3-a, itself cut by the enzymes NdeI and BamHI.
The recombinant vector pXL1817 thus obtained makes it possible to express at very high level a polypeptide having a deletion of the first 182 amino acids (~ N182).

5. Generation of the vectors pXL1981 and p ~ L2183, and preparation of the polypeptides 30 P (~ hl-2) and P (tagAh1-2).
Using oligodeo. ~ Synthetic phospho cynucleotide ~ ylé SEQ ID n 14, a mutagenesis directed on the single-stranded form of the plasmid pXL1872 described above above was done using the Amersham kit according to the recommendations of the - maker. (: this mutagenesis has made it possible to delete the part of the coding sequence of '' RE9JIPLACE SHEET ~ E ~ I

~ 12 ~ ~ 7 7 apoAIV compnse between positions 40 and 186, causing a deletion of 49 amino acids, from residue D14 to residue V62.
From a mutant clone whose sequence has been fully verified, a 990 bp NdeI-BamHI fragment was purified and ligated with the vector pET3-a, Even cut by the enzymes NdeI and BamHI
The recombinant vector pXL1981 thus obtained perrnet d`e ~: primer at very high level a polypeptide having a delhion of helices 1 and 2 (hl-2).
Erl according to the protocol described in B1.1), the vector p ~ 2183 was constructed at from the vector pXL1981. The vector pXL2183 thus obtained perrnet to express to lo high level the polypeptide P (hl-2) fused in N-terminal to the decapeptide MRGS (H) 6.

6. Generation of the vectors pXL1943 and p ~ 2184, and preparation of the polypeptides P (h7-8) and P (tag ~ h7-8). .
Using the phosphorylated synthetic oligodeoxynucleotide SEQ ID n 1 ~, a mutagenesis directed on the simple brL ~ form of the plasrnide pXL1872 described above above was carried out using the Amersham kit according to the recommendations of the ~ abricant. This mutagenesis made it possible to cite the parh of the coding sequence of - the apoAIV between positions 484 and 61; causing a deletion of 44 amino acids, from residue P162 to residue A205.
~ 0 From a mutant clone whose sequence has been fully vetified, a NdeI-BamHI fragment of 1005 bp was purified and ligated with the vector pET3-a, itself cut by the enzymes NdeI and BamHI.
The recombinant vector pXL1943 thus obtained makes it possible to express at very high level a polypeptide having a deletion of helices 7 and 8 ll ~ h7 8).
Following the protocol described in B1.1 ~, the vector pXL2184 was constructed at from Yector pXL1943. The vector pXL2184 thus obtained makes it possible to express at high level the polypeptide P (~ h7-8) fused in N-terminal to the decapeptide MRGS (H) 6.

7. Generation of the vectors pXL1982 and pXL2215, and preparation of the polypeptides P (/ ~ h9-10) and P (tag h9-10).
Using the synthetic phosphorylated oligodeoxynucleotide SEQ ID No. 16, a mutagenesis directed on the single-stranded form of the plasmid pXL1872 described above above was carried out using the Amersham kit according to the recommendations of the maker. This mutagenesis made it possible to delete the part of the coding sequence from REPLACEMENT SHEET ~: E ~ EN`.

= ~ ~

~ 12 ~ 7 apoAIV between positions 616 and 74 /, causing a deletion of 44 amino acids, from residue P206 to residue A249.
From a mutant clone whose sequence has been fully verified, a NdeI-BamHI fragment of 1005 bp was purified and ligated with the vector pET3-a, s itself cut by the enzy ~ es NdeI and BamHI.
The recombinant vector pXL1982 thus obtained perrnet to express at very high level a polypeptide having a deletion of helices 9 and 10 (~ h9-10).
Following the protocol described in B1 1), the vector pXL2215 was constructed at from the vector pXL1982. The vector pXL2215 thus obtained makes it possible to express at 10 high level P (h9-10) polypeptide fused in N-terminal to decapeptide MRGS (H) 6.

8. Generation of the vector pXL1986 and preparation of the polypeptide P (~ h11-12, L87M).
Using the phosphorvlated synthetic oligodeoxynucleotide SEQ ID No. 17, a mutagenesis directed on the simple ~ rin form of the plasmid pXL1872 described above 15 above was carried out using the Amersh m kit according to the manufacturer's recommendations. ~ this mutagenesis made it possible to delete the part of the coding sequence of rapoAIV between positions 748 and 867, causing a deIetion of 40 amino acids, from residue P250 to residue E289. In addition, when checking sequence of derivatives obtained, some clones carried a point mut ~ tion ~ o additional on the sequence. This mutation ~ L-> M) has for e ~ and to change the codon leucine 87 to methionine in the protein.
From this clone and from another clone not carrying the mutation (L-> M) additional, a 1017 bp NdeI-BamHI fragment was purified and ligated with the vector pET3-a, itself cut by the enzymes NdeI and BamHI.
2 ~ Two recombinant vectors were thus obtained, including the vector pXL1986 which allows to express at a very high level a polypeptide having a deletion of helices 11 and 12 ~ hll-12) and a methionine instead of a leucine in position 8f (L87M ~.
Following the protocol described in B1.1), the vector pXL2186 was constructed at From the vector obtained above, coding for the polypeptide P (~ h11-12). The pXL2186 lever thus obtained makes it possible to express the polypeptide at a high level.
P (~ h11-12) fused in N-terminal to the MRGS decapeptide (H) ~.

9. Generation of the vectors pXL1987 and pXL2217, and preparation of the polypeptides P (~ hl3-14) and P (tag / ~ hl3-14 ~.

REPLACEMENT SHEET ~ IENT

5 ~ ' 8 ~ 7 l9 With the aid of the phosphorvlated svnthetic oligodeoxynucleolide SEQ ID n 1 ~, a mutagenesis directed on the simp] e stranded form of the plasmid pXL1872 described above above was carried out using the Amersham kit according to the recommendations of the maker. This muta_enesis made it possible to delete the part of the coding sequence of apoAIV between positions 86 ~ and 999, causing a deletion of 44 amino acids, from residue P290 to residue N333.
. From a mutant clone whose sequence has been fully verified, a ~ Raoment NdeI-BamHI of 1005 bp has been purified and li ~ ,, ~ ture with the çctor pET3-a, itself ~ cut by the enzymes NdeI and BamHl.
lo The recombinant vector pXL1987 thus obtained makes it possible to express at very high level a polypeptide having a deletion of helices 13 and 14 (~ h13-14).
Following the protocol described in 131.1), the vector pxr ~ 17 was constmil 2 from the vector pXL198 /. The vector pXL2217 thus obtained makes it possible to express at high level pol, vpeptide P (hl3-14) fused to N-terminaI with decapeptide MRGS (H) 6 ~

.
~ 2. Mutants produced by ~ ir of the plasmid pXL1696 1. Generation of the vectors pXL2071 and pXL2185, and preparation of the polypeptides P (~ h5-6) and P (tag ~ h5-6).
Using the phosphorylated synthetic oligodeoxvnucleotide SEQ ID No. 19 ~
a mutagenesis directed on the single-stranded form of the plasmid pXL1696 (cf. example A2) was carried out using the Amersham kit according to the ~ recommendations of the maker. This mutagenesis made it possible to delete the par ~ ie of the coding sequence of apoAIV between positions 352 and 483, causing a deletion of 44 2 ~ amino acids, from residue P118 to residue R161.
From a mutant clone whose sequence has been fully verified. a 432 bp NdeI-EcoRI fragment was purified, then ligated with fragment E: coRI-BamHI isolated from plasmid pXL2051 and the vector pET3-a opened by the enzymes NdeI and BamHI.
The recombinant vector pXL2 ~ 71 thus obtained makes it possible to express at very high level a polypeptide having a deletion of helices 5 and 6 (~ hS-6).
Following the protocol described in B1.1), the vector pXL2185 was constructed at from the vector pXL20 / 1. The vector pXL2185 thus obtained perrnet to express at RE SHEET ~! APLACE ~ JIENT

~ 12S8 ~ 7 haul level the polypeptide P (~ 6) fused in ~ -te ~ final with decapeptid ~
MRGS (H) 6.
2. Generation of the vector pXL2063 and preparation of the polypeplide P (D5S).
Using the synthetic phosphor oligodeoxynucleotide, vle SEQ ID No. 20, S a mutagenesis directed on the single-stranded form of the plasmid pXL1696 (cf. example A2 ~ was performed using the Amersham kit according to the recommendations of maker. This rnutagenesis made it possible to introduce an AGC codon in the sequence.
coding of apoAIV, prov ~ as the replacement of aspartic acid by a serine in position 5.
From a mutant clone whose sequence has been fully verified, a NdeI-EcoRI fragment of ~ 64 bp was purified, then li. ~ atured with the fraoment EcoRI-BamHI isolated from plasmid pXL20 ~ 1 and the vector pET3-a or ~ ert by enzymes NdeI and BamHI
The recombinant vector pXL2073 thus obtained makes it possible to express at very high 1 ~ level a polypeptide having a serine in position ~.
3 ~ Generation of the vector pXl :, 2069 and preparation of the polypeptide P (D ~ K ~.
Using the synthetic phosphor oligodeoxynucleotide, vle SEQ ID No. 21, a mutagenesis directed on the single-stranded form of the plasmid pXL16 ~ 6 (cf. example A2) was carried out using the Amersham kit according to the recommendations of maker. This mutagenesis made it possible to introduce a codon ~ A into the sequence coding for apoAIV, causing the replacement of aspartic acid with a lysine in position 5.
From a mutant clone whose sequence has been fully verified, a fra ~ ment NdeI-EcoRI of 564 bp was purified, then li ~ ature with ~ ec fragment EcoRI-2 ~ BarnHI isolated from the plasmid pXL2051 and the vector pET3-a opened by the enzymes NdeI and BamHI.
The recombinant vector pXL2069 thus obtained makes it possible to e ~; primer at very high level a polypeptide having a lysine in position 5.
4. Generation of the vector pXL2062 and preparation of the polyF ~ eptide P (D44F).
Using the phosphorylated synthetic oligodeoxynucleotide SEQ ID No. 22, a mutagenesis di ~ igée on! a single stranded form of the plasmid pXL1696 (Cf example A2) was carried out using the Amersham kit according to the recommendations of fabrican ~. This mutagenesis made it possible to introduce a codon Tl`C into the sequence .

i ~ RENIPLACE HEAD ~ AENT

- ~ 12 ~ 77 coding for aooAIV, causing the replacement of aspartic acid by a phenylalanine in position 4 ~.
From a mutant clone whose sequence has been fully verified, a 564 bp NdeI-EcoRI fragment was purified, then ligated with the EcoRI- fragment BamHI isolated from plasmid pXL2051 and the vector pET3-a opened by the enzymes NdeI and BamHI.
The recombinant vector pXL2062 thus obtained makes it possible to express at very high - level a polypeptide having a phenylalanine in position 44. ----5. Generation of the vector pXL2074 and preparation of the polypeptide P (D44A).
Using the phosphorvlated synthetic oligodeoxynucleotide SEQ ID No. 23, a muta, enesis directed on the single-stranded form of the plasmid pXL1696 ~ see example A2) was carried out using the Arnersham kit according to the recommendations of fab ~ icant. This mutagenesis made it possible to introduce a GCG codon into the sequence coding of apoAIV, causing the replacement of aspartic acid by a alanine in position 44 ~
; From a mutant clone whose sequence has been fully verified, a NdeI-EcoRI fragment of ~ 64 bp was purified, then ligated with the EcoRI fragment BamHI iso! E of the plasmid pXL20 ~ 1 and the vector pET3-a opened by the enzymes ~ ~ ~ NdeI and BamHL
; ~ ~ 20 The recombinant vector pXL2074 thus obtained perrnet to express at very high level a polypeptide having an alanine at position 44.

333: Mutants made from ~ plasmid pXL20 ~ 1 1. Generation of the vector pXL2073 and preparation of the polypeptide P (K178A).
Using the phosphorylated ~ nthetic oligodeoxynucleotide SEQ ID No. 24, 2 ~ a mutagenesis directed on the single-stranded form of the plasrnid pXL20 ~ 1 (cf. example A2j was carried out using the Arnersham kit according to the recommendations of maker. This mut ~ en ~ allowed to introduce a GCG codon in the sequence coding of apoAIV, ~ c ~ invoking the rernplacernent of lysine by an alanine in position 178.
From a mutant whose sequence has been fully verified, a Fragmènt EcoRl- ~ 3am l- ~ de ~ 72 bp was purified, then li ~ ature with fra ~ ment l ~ deI-REPLACEMENT FEI.JILLE ~ i ~ ENT
: ~.

~ 125 ~ 77 EcoRI isolated from plasmid pXL1696 and the vector pET3-a opened by the enzymes NdeIet BamHI.
The recombinant vector pS ~ L20 / 3 thus obtained ~ makes it possible to express at very high level a polypeptide having an alanine at position 178.
2. Generation of the vector pXL2070 and preparation of the polypeptide P (K178Y).
Using the phosphorylated s ~ nthetic oligodeoxynucleotide SEQ ID n 25, a mutagenesis directed on the simple bnn form of the plasmid p ~ 20 ~ 1 (Cf example A2) was carried out using the Amersham kit according to the recommendations of maker. This mutagenesis made it possible to introduce a TAT codon into the sequence ~ o coding apoAIV, causing the replacement of Iysine by a tyrosine in position 178.
From a mutant clone whose sequence has been fully verified, a EcoRl-BamHI fragment of 572 bp was purified, then ~ ga ~ with fraO ~ nent NdeI-EcoRI isolated from plasmid pXL1696 and the vector pET3-a opened by enzymesl ~ NdeI and B ~ mHI.
The recombinant vector pXL2070 thus obtained makes it possible to express at very high level a polypeptide having a tyrosine in position ~ tion 178.
3 ~ Generation of the vector pXL2072 and preparation of the polypeptide P ~ E230K ~.
Using the phosphorylated syn * letic oligodeoxynucleotide SEQ ID No. 26, a directed snutagenesis on the single br, in form of the plasmid pXL2051 (C ~ example A2) was carried out using the Amersham kit according to the recommendations of maker. This mutagenesis has pe ~ nis to introduce a codon A ~ A in the sequence coding of apoAIV, causing the replacement of lutamic acid by a lysine in position 230.
2 ~ From a mutant clone whose sequence has been fully verified. a EcoRI-BamHI fragment of ~ 72 bp was purified, then ligated with the NdeI- fragment EcoRI isolated from the plasmid pXL1696 and the vector pET3-a opened by the enzymes NdeIel BàmHI.
The recombinant vector pXL2072 thus obtained makes it possible to express at very high level a polypeptide having a lysine in position 230.
B4: Production of the polvpeptides described in parts Bl-B3.

SHEET OF RE ~ lPLA ~ :: E. ~ 3 ~ ENT

WO 93/15198 ~ 12 5 ~ 7 7 pcr / FR93 / ooo73 NdeI-EcoRI isolated from the plasmid pXL1696 and the vector pET3-a opened by the enzymes NdeI and BamHI.
The recombinant vector pXL2070 thus obtained makes it possible to express at very high level a polypeptide having a tyrosine at position 178.
s 3. Generation of the vector pXL2072 and preparation of the polypeptide P (E230K).

Using the following phosphorylated synthetic oligodeoxynucleotide:
5'-AAGAAGAACGCC ~ GAGCTCAAGGCCAG-3 ', a directed mutagenesis on the single-stranded form of the plasmid pXL2051 (cf. example A2) was produced in using the Amersham kit according to the manufacturer's recommendations. This mutagenesis lo made it possible to introduce an AAA codon into the coding sequence of apoAlV, causing the replacement of glutamic acid by a Iysine at position 230. From a mutant clone whose sequence has been fully verified, a 572 bp EcoRI-BamHI fragment was purified, then ligated with k: NdeI- fragment EcoRl isolated from the plasmid pXL1696 and the vector pET3-a opened by the enzymes NdeI15 and BamHI.
The recombinant vector pXL2072 thus obtained makes it possible to express at very high level a polypepdde having a lysine in position 230.
B4; Production ~ olvpeptid ~ writings ~ n ~ les ~ arties B1- ~ 3.
1. Production: For example, the strain BL21 DE3 (pLysS) containing a 20 expression plasmid of a polypeptide of the invention as described in the parts B1-B3.
Overnight preculture in LB medium containing ampicillin (100, ug / ml) and chloramphenicol (50 ~ g / ml ~, LB ~ Cm, at 37C, used to inoculate at 1100th the production culture ~ same environment ~. The culture is agitated at 37C to a density 2s optics (measured fi 610 nm) of 0.5. IPTG is then added to the concentration final 1 mM and induced cells for expression of RNA polymerase 17 are incubated for 90 or 180 min. Analysis of bacterial extracts by gel electrophoresis stained with coomassie blue reveals an accumulation in extracts of 90 min cultures of the polypeptide of the invention. Furthermore, this The 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 WO 93/15198 PCl '/ FR93 / 00073 E.col RNA polymerase ;. We can add for example 20 min after adding In`G of rifampicin at concentrations which can be between 50 and 200 ~ g / ml. The cells which then produce only the single mRNA of the desired polypeptide except all others are incubated between 90 and 180 min at s 3PC.
In this case, the recombinant polypeptide produced can represent from 20 to 30 ~ o of the total proteins produced by the bacteria. In addition, the polypeptide is thus accumulated without degradation in the bacteria in soluble form.
The production was extrapolated as a fermenter, by high density culture of lo E.coli in Fed-Batch mode. This production was carried out in a ferrnenteur of 2 liters (Setric) in 2 phases: a phase of ~ microorganism growth up to DO600 from 30 to 40 (duration: about 5 hours). This phase is carried out in the middle fermentation defined by Jung et al. (Ann.Inst.Pasteur / Microbiol. ~ (1988) 129-146); then a phase of induction of production, by addition of lPTG and derifampicin, and increase in the rate of supply of carbon and nitrogen substrates (duration: approximately 1 hour 30 minutes).

2. Cell lysis and recovery of the recombinant polypeptide.
After the production culture, the cells are collected and then lyzed, by example by sonication. On the laboratory scale, a sonicator was used Branson (mod ~ le B30, Proscience, France) after concentrating the cells 30 times in PBS buffer (KCl 0.2 g / l, KH2PO4 0.2 g / l, NaCl 8 g / l, Na2HPO4 1.25 g / l ~.
The cells are broken at 4C in continuous mode (2 pulses of 5 ~ unutes ~. It is also possible to pretreat the concentrated cell suspension by 2 ~ presence of Triton X-100 at ~ room temperature before sonication.
C / PURIF ~ CATION OF PQLYPEPTID ~ S FROM: THE INVENTION.
C1. The polypeptides P (R93G), P (/ ~ C44) and P (~ N13) were purified according to the following protocol, which takes place in native conditions and requires no steps affinity for 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
(81 mM Na2HPO4, 19 mM NaH2PO4; 2 mM EDTAt; PMSF, 1 mM; pH 7.5;
~ 10 mM mercaptoethanol). The suspension is ~ ited by 8 cycles of 3 minutes :
~ ::

WO 93/15198 PCr / FR93 / 00073 21 ~

of ultrasound (BRANSON model set at 250W) at 4C. The extracts are centrifuged for 30 minutes at 15,000 rpm at 4C. The supernatant Sl is preserved and the pellet is washed in the same volume of buffer A e $ recentrifuged under the same conditions.
The supernatant comprising S1 'is added to the supernatant S1.
After a determination of the proteins of the fraction (Sl + Sl ') is added a aqueous solution of streptomycin sulfate at 10 ~ o at a rate of 10 ml of solution per ~ protein core. After 15 minutes of incubation at 4C with gentle shaking, the suspension is centrifuged for 30 minutes at 15,000 rpm at 4C. The supernatant (S2) is recovered. To this fraction S2 is added ammonium sulfate (concentration final: 25% saturationl. After 1 ~ minutes of incubation at 4C with shaking, the suspension is centrifuged for 30 minutes at 15,000 rpm at 4C. To the supernatant S25 is added ammonium sulfate (final concentration: 50% saturation) .After 15 minutes of incubation at 4C with shaking, the suspension is centrifuged for 30 min at 15,000 rpm at 4C. The base (C50) is recovered and taken up in the buffer B (Tris-HCI, 10 mM; EDTA, 2 mM; pH8.8) and dialyzed against 21 of buffer B which is changed ~ times during 48 hours.
The dialysate is injected on a QFF type ion exchange column (Pharmacia) and eluted with a NaCl gradient. The determination of the fractions contained The polypeptides of the invention are carried out according to an Elisa test described below.
The interesting fractions are collected and concentrated by precipitation in sulfate ammonium (final concentration: 80% saturation). After 15 minutes incubation at 4C with shaking, the suspension is centrifuged for 30 minutes at 15,000 rpm at 4C. The pellet (C80) is recovered and taken up in buffer B and dialyzed center 21 of buffer B.
The dialysate is injected on an ion exchange column of the Mono Q type (Pharmacia ~ and eluted with a NaCl gradient. The interesting fractions, identified by the Elisa test described below and gel SI) S 15 ~ o, are combined and concentrated by ammonium sulphate precipitation (final concentration: 80% saturation). After 15 minutes incubation at 4C with shaking, the suspension is centrifuged and taken up in buffer D (ammonium sulfate, 1.7 M; Na2HPO4 8.1 mM, NaH2PO4 1.9 mM; EDTA, 2 mM; pH 7.4 ~ and dialyzed against 21 of buffer D.
The dialysate is injected onto a chromatography column; a wealth of interactions hyd ~ Phenyl-Sepharose ophobes (Pharmacia) and eluted with buffer E ~ Na2HP04 8.1 mM; 1.9 mM NaH2PO4; EDTA, 2 mM; pH 7.4). The interesting fractions are 35 identified by Elisa test and SDS polyacrylamide gel.

Wo 93/15198 PCl / FR93 / 00073 ~ 12 ~ 877 26 Briefly, the ELISA test developed consists of adsorbing on a plate with a polyclonal rabbit serum directed against dwarf apoAIV (diluted 1 / lOOOème), saturate this plate with gelatin, incubate the test sample and finally, react an equimolar mixture of two monoclonal antibodies directed against apoAlV (~ 03 and M05, SERLlA, Institut Pasteur de Lille), followed by a revelation immunoenzymatic using a polyclonal anti-mouse semm coupled to peroxidase.
This test is easily calibrated with a range of dilutions of plasma apoAIV.
These fractions are combined and dialyzed against tarnpon F (sulfate ammonium, 40% saturation; 8.1 mM Na2HPO4; NaH2PO4 1, ~ mM; EDTA, 10 2 mM; pH 7.4) and stored at 4C.
C2. For the polypeptides P (~ hl-2) 1 P (~ h5-6), P (~ h13-14) and P (~ \ N182), the fraction S2 is directly injected onto QFF type coloMe (without precipitation ammonium sulfate fractionated) equilibrated in buffer B. The polypeptide is excluded (P (~ N182)) or eluted with a plateau at 250 mM NaCl in buffer B (P (~ 2)). The The polypeptide is then concentrated by precipitation into ammonium sulfate (final concentration 50 ~ o of saturation for P (~ h1-2), 10 ~ o for P (/ ~ N182)). The purification of P (~ h1-2) is terminated by centrifugation (30 min, 4C, 10,000 g).
The pellet is recovered, taken up in PBS buffer, desalted on a PD10 gel-filtration column.
(I? Hamlacia) and dried at - 20C. The polypeptide P (~ N182) precipitated with sulfate 20 of ammonium is injected onto an interaction chromatography column hydr ~ phenyl-sepharose phobes (Pharmacia) and eluted with buffer B. The polypepdde is identified by SDS polyacrylamide gel, and the interesting fractions are collected and dialyzed against buffer B and stored at 4C.
C3. The polypeptides P (tag ~ N13), P (tag ~ C44), P (tagl ~ 21, P (tagQh7-8), 25 P (tagl ~ h9-10), P (tag ~ h11-12), P (tag ~ h13-14) and P (tag ~ h5-6 ~ were purified according to the following protocol, which includes 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
(81 mM Na2HPO4, 19 mM NaH2PO4; EDTA, 2 mM; PMSF, 1 mM; pH 7.5;
30 ~ 10 mM mercaptoethanol). The suspension is treated by 3 cycles of 5 minutes of ul-beams (BRANSON model set at 250W) at 4C. The extracts are centrifuged during dant lh at 11,000 g at 4C. The nucleic acids present in the supernatant have been precipitated by adding 10% (w / v) of streptomycin sulfate (10 ml / g protein), incubation 30 min. at 4C, then recentrifuged under the same conditions as 35 previously.

:

WO 93/15198 2 1 2 ~ g 7 7 PCI / FR93 / 00073 The recombinant proteins present in the supernatant have been purified by affinity chromatography on a chelated metal ion. The purification was carried out on agarosei-nitrilotriacetic acid-nickel resin (NTA-Ni) according to the manufacturer's recommendations, with the following modifications: The solution 5 protein is desalted on a Tris-Acryl GF-05 oolane balanced with a buffer 100 mM phosphate 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 has then loaded onto a Ni-NTA column balanced with the same buffer

10 phosphate 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, by a phosphate / citrate buffer pH 5. The protein fractions obtained at pH 5 were neutralized with 1M sodium hydroxide additive (30 IlVml) and supplemented with 2 inhibitors of proteases: EDTA 0, 1M and PMSF 0.2M. The fractions were then analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) containing 15 ~ o acrylamide / bis-acrylamide, according to Laemmli (Nature; ~ (1970) 680), then grouped according to purity and quantity. The pooled fractions were incubated in presence of L (-) - histidine powder (final concentration 50 mM), for 1 h at 4C, 20 in order to destroy the Ni-poly-His-protein bond. The released nickel and histidine have then removed by desalting on a Tris-Acryl GF-05 column balanced with a phosphate buffer pH 7.4 containing 2mM EDTA.
The recombinant proteins thus purified were analyzed by SDS-PAGE electrophoresis and immlLnoblotting ~. Immunobloning was carried out with A mixture of anti-ApoAIV monoclonal antibodies conjugated to peroxidase, and anti-mouse goat antibody conjugated to peroxidase. Peroxidase activity bound was revealed by incubation with ~ e solution of 4-Chlor ~ l-Naphtol as substrate.
The different chromatograms were followed by UV detection at 280 nm.
The protein concentration was determined according to the Bradford technique (Anal. Biochem. ~ (1976) 248).

Wo 93/15198 PCr / FR93 / 00073 21 '~ 5 ~ 77 28 , D / BIOLOGIOU ~ CHARACTERIZATION OF POL ~ EErrII2ES DE
, THE INVENTION.
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 S their effect on the emux of cholest ~ rol. These 2 parameters account for the potential cholesterol-lowering pharmacological of the polypeptides of the invention. The protocol for determining these parameters is given below as well as the ~ results obtained.
1. Prot, measuring ocols.
0 a) Affinity for the HDI receiver, The purified polypeptides are first used to ecstituate pr ~ teoliposomes with DMPC, the s ~ cture of which appears identical, in view of the behavior in chron ~ atography of exclusion, to that of oliposomes reconstituted with native apoAIV. These proteoliposomes 15 reconstituted are then tested for their ability to attach to murine adipocytes (line Obl77) according to a protocol already described.
b) Efflux of cholesterol The efflux of cholesterol from murine adipocyte cells (lineage Obl77) caused by the DMPC-polypeptide proteoliposomes of the invention is 20 determined after ~ 5 hours of incubation.
2. Results Polypeptide Kd (~ o) Bmax (~ o) Efflux at 5h (%) *
ApoAIV 100 ** 100 ** 28 P (R93G) 115 97 27.5 P (~ h1-2) 105 94 23.5 P (~ C44) na na 32 P (/ \ N13) 96 94 24 (*) Expressed as% of the initial cholesterol of the charged cells, the base efflux obtained in the presence of DPMC alone after 5 hours is 11%.
30 (**) Carried out on three independent experiments.

.5. ~

21 ~ ~ ~, 7 i LIST OF SEQlJENCES
~]) IN ~ GENERAL ORMATION:
(i) DEPOSITOR:
(A) NAME: RHONE-POULENC RORER SA
~ B) STREET: 20, avenue Raymond ARON
(C ~ CITY: ANTONY
(E) COUNTRY: ERANCE
(F) POSTAL CODE: 92165 (ii) TITLE OF THE INVENTION: NEW POLYPEPTIDES, THEIR PREPARATION AND
THEIR USAGE.
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(A) TYPE OF SUPPORT: Tape (B) COMPATIBLE IBM PC COMPUTER
(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: PatentIn Release ~ 1.0, Version ~ 5 (EPO) (v) CURRENT DEMAND DATA:
DEPOSIT NUMBER: PCT / FR93 / 00073 ~ 2) ~ NFORMATION E ~ OUR SEO ID NO: 1:
(i) CHARACTERISTICS OF THE SEQUENCE
(A) LENGTH: 1134 base pairs (B) TYPE: nucleic acid ~ C) NUMBER ~ E BR ~ NS double (D) CONFIGURATION: linear (ii) YPE l ~ E MOLECULE: cDNA
(iii) MORTGAGE NO
(iii) ~ TI-SENS: NO
(vi) ORIGIN:
(A) ORGANISM: Nucleotide sequence of apolipoprotein AIV
~ ix) ADDONAL CHARACTERISTICS:
(A) NAME / KEY: CDS
(B) LOCATION: 1..1134 (ix) ADDITIONAL FEATURE:
(A) NAME / KEY: Loop (B) LOCATION: 40..120 RE SHEET ~ PLA ~ Er '~

WO 93/15198 PCI` / FR93 / 00073 ~ .. ' 21 2587 '~ 30 "

(ix) ADDITIONAL FEATURES:
(A) NAME / KEY: Loop (B) LOCATION: 121..186 (ix) ADDITIONAL ERIC CHARACI:
(A) NAME / KEY: Loop (B) LOCATION: 187,285 (ix) ADDlTlONEELE CHARACTERISIIQUE:
(A) NAME / KEY: Loop ~ B) LOCATION: 286..351 10 (ix) ADDITIONAL FEATURES:
(A) NAME / KEY: Loop (B) Eh ~ PLACEMENT: 352..417 (ix) AD ~ ITIONAL CHARACTERISTICS:
(A) NAME / KEY: Loop (B) LOCATION: 418 .. 483 (ix) ADDITIONAL FEATURE:
(A) NAME / KEY: Loop (B) LOCATION: 484..549 (ix) CHARACll ~ lSllQUE ADDlTlONELLE:
(A) NAME / KEY Loop (B) LOCATION: 550..61 ~
(ix) CHARACTERlSIlQUE ADDITIONFl 1 F.
(A) NAME / KEY: Boude (B) LOCATION: 616..681 25 (ix) ADDITIONAL CHARACTERISrlOUE:
(A) NAME / KEY: Loop - (B) LOCATION: 682 .. 747 (ix) ADDITIONAL FEATURES:
(A ~ NAME / KEY: Loop (B) LOCATION: 748 .. 801 (ix) CHARACTERISIIQUEADr) l'rlONELLE:
(A) NAME / KEY: Loop (B) LOCATION: 802..B66 ~ ix) ADDITIONAL FEATURE:
(A) NAME / KEY: E ~ oucle (B) LOCATION: 867 .. ~ 33 (ix) ADDlTlONEl CHARACTERISIlQUE l F ~
(A) NAME / KEY: Loop (B) LOCATION: 934..999 wo 93 / lslg8 2 ~ 2 5 ~ ~ ~ PCr ~ FR93 / 00073 (LX) ADDlTlONELLE CHARACI'ERlSrIQUE:
(A) NAME / KEY: Loop (B) LOCATION: 1000..1131 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

Met Glu Val Ser Ala Asp Gln Val Ala Thr Val Met Trp Asp Tyr Phe 10. Ser Gln Leu Ser Asn Asn Ala Lys Glu Ala Val Glu His Leu Gln Lys Ser Glu Leu Thr Gln Gln Leu Asn Ala Leu Phe Gln Asp Lys Leu Gly Glu Val Asn - Thr Tyr Ala Gly Asp Leu Gln Lys Lys Leu Val Pro Phe Ala Thr Glu Leu His Glu Arg Leu Ala Lys Asp Ser Glu Lys Leu Lys GAG GAG ATT GGG} ~ AG GAG CTG GAG GAG CTG AGG GCC CGG CTG CTG CCC 288 Glu Glu Ile Gly Lys Glu Leu Glu Glu Leu Arg Ala Arg Leu Leu Pro:

His Ala Asn Glu Val Ser Gln Lys Ile Gly Asp Asn Leu Arg Glu Leu Gln Gln Arg Leu Glu Pro Tyr Ala Asp Gln Leu Arg Thr Gln Val Asn Thr Gln Ala Glu Gln Leu Arg Arg Gln Leu Thr Pro Tyr Ala Gln Arg Met Glu Arg Val Leu Arg Glu Asn Ala Asp Ser Leu Gln Ala Ser Leu Arg Pro His Ala Asp S; lu Leu Lys Ala Lys Ile Asp Gln Asn Val Glu Glu Leu Lys Gly Arg Leu Thr Pro Tyr Ala Asp Glu Phe Lys Val Lys Ile Asp Gln Thr Val Glu Glu Leu Arg Arg Ser Leu Ala Pro Tyr Ala Gln Asp Thr Gln Glu Lys Leu Asn His Gln Leu Glu Gly Leu Thr Phe WO 93/15198 PCI` / FR93 / 00073 ~ 12 ~) 87 ~ 32 Gln Met Lys Lys Asn Ala Glu Glu Leu Lys Ala Arg Ile Ser Ala Ser GCC GAG GAG CTG. CGG CAG AGG CTG GCG CCC TTG GCC GAG GAC GTG CGT 768 Ala Glu Glu Leu Arg Gln Arg Leu Ala Pro Leu Ala Glu Asp Val Arg Gly Asn Leu Arg Gly Asn Thr Glu Gly Leu Gln Lys Ser Leu Ala Glu Leu Gly Gly His Leu Asp Gln Gln Val Glu Glu Phe Arg Arg Arg Val Glu Pro Tyr Gly Glu Asn Phe Asn Lys Ala Leu Val Gln Gln Met Glu Gln Leu Arg Gln Lys Leu Gly Pro His Ala Gly Asp Val Glu Gly His Leu Ser Phe Leu Glu Lys Asp Leu Arg Asp Lys Val Asn Ser Phe Phe AGC ACC TTC AAG GAG AAA G ~ G AGC CAG GAC AAG ACT CTC TCC CTC CCT 1056 Ser Thr Phe Lys Glu Lys Glu Ser Gln Asp Lys Thr Leu Ser Leu Pro Glu Leu Glu Gln Gln Gln Glu Gln Gln Gln Glu Gln Gln Gln Glu Gln Val Gln Met Leu Ala Pro Leu Glu Ser 370,375 (2) 1NFORMATIONPOI ~ l ~ ~ QIP NQ ~
(i ~ FEATURES OF THE SEQI) ENCE:
4d (A) LENGTH: 377 ~ amino acids, s (13) ~ YPE: a ~ from amin, (OF CONFIGURATION: linen, area (ii) MOLECULE YPE: prot, ine ~ 0 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
Met Glu Val Ser Ala Asp Gln Val Ala Thr Val Met Trp Asp Tyr Phe ~ 5 Ser Gln Leu Ser Asn Asn Ala Lys Glu Ala Val Glu His Leu Gln Lys ~ 25 30 Ser Glu Leu Thr Gln Gln Leu Asn Ala Leu Phe Gln Asp Lys Leu Gly W 0 93/151 ~ 8 21 ~ ~ 8 7 7 PCT / FR93 / 00073 Glu Val Asn Thr Tyr Ala Gly Asp Leu Gln Lys Lys Leu Val Pro Phe Ala Thr Glu Leu His Glu Arg Leu Ala Lys Asp Ser Glu Lys Leu Lys Glu Glu Ile Gly Lys Glu Leu Glu Glu Leu Arg Ala Arg Leu Leu Pro His Ala Asn Glu Val Ser Gln Lys Ile Gly Asp Asn Leu Arg Glu Leu Gln Gln Arg Leu Glu Pro Tyr Ala Asp Gln Leu Arg Thr Gln Val Asn Thr Gln Ala Glu Gln Leu Arg Arg Gln Leu Thr Pro Tyr Ala Gln Arg Met Glu Arg Val Leu Arg Glu Asn Ala Asp Ser Leu Gln Ala Ser Leu Arg Pro His Ala Asp Glu Leu Lys Ala Lys Ile Asp Gln Asn Val Glu Glu Leu Lys Gly Arg Leu Thr Pro Tyr Ala Asp Glu Phe Lys Val Lys 180 185 190: ~
Ile Asp Gln Thr Val Glu Glu Leu Arg Arg Ser Leu Ala Pro Tyr Ala 195. 200 205 Gln Asp Thr Gln Glu Lys Leu Asn His Gln Leu Glu Gly Leu Thr Phe Gln Met Lys Lys Asn Ala Glu Glu Leu Lys Ala Arg Ile Ser Ala Ser Ala Glu Glu Leu Arg Gln Arg Leu Ala Pro Leu Ala Glu Asp Val Arg Gly Asn Leu Arg Gly Asn Thr Glu Gly Leu Gln Lys Ser Leu Ala Glu Leu Gly Gly His Leu Asp Gln Gln Val Glu Glu Phe Arg Arg Arg Val Glu Pro Tyr Gly Glu Asn Phe Asn Lys Ala Leu Val Gln Gln Met Glu 2gO 295 300 Gln Leu Arg Gln Lys Leu Gly Pro His Ala Gly Asp Val Glu Gly His Leu Ser Phe Leu Glu Lys Asp Leu Arg Asp Lys Val Asn Ser Phe Phe Ser Thr Phe Lys Glu Lys Glu Ser Gln Asp Lys Thr Leu Ser Leu Pro Glu Leu Glu Gln Gln Gln Glu Gln Gln Gln Glu Gln Gln Gln Glu Gln 355 360 365:
Val Gln Met Leu Ala Pro Leu Glu Ser 370,375 wo 93 / lslg8 ~ 1 ~ 5 ~ 7 I PCr / FR93 / 00073 ~ 2) INFORMATION FOR SEO ID NO: 3:
(i) CARACTERIST. IQUES OF THE SEQUENCE:
S (A) LENGTH: 84 base pairs (B) TYPE: nucl acid, i ~ ue (C) NUMBER OF STRANDS: single ~ D) CONFIGURATION: linen, area (u) MOLECULE mE: cDNA
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
~ S
(vi) ORIGIN:
: ~ (A) ORGANIZATION: OLIGONUCLEOTIDE A

20 ~ xi) DESCRIlrrlON OF THE SEQUENCE: SEQ ID NO: 3:
: CTAGACATAT GGAGGTCAGT GCTGACCAGG TGGCCACAGT GATGTGGGAC TACTTCAGCC 60 ~ 2) INFORMATION FOR SEO ID NO: 4:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 94 base pairs :: 30 (B) TYPE: nuclear acid (C) NUMBER OF STRANDS: single (D) CON ~ IGURATION: flax, area (ii) TYPE OF MOEECULE: cDNA
(iii ~ HYPOTHETIC: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN:
(A) ORGANISM: o1igonucleotide B

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
4 ~

50 (2) INFORMATION FOR SEO ID NO: 5:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 86 base pairs (B) TYPE: nuclear acid 2125 ~

~ 2) INFORMATIOI ~ FOR L. ~ SEO IT ~! ~ O 5:
(ij CHARACTERlSTICS OF L. ~ SEQUENCE:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii ~ TYPE OF MOLECULE: A ~ Nc (iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO:
(vi) ORIGIN:
(A) ORGANIZATION: OLIGONUCLEOTIl: ~ EC
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5-:
CCACGGCCTC CTTGGCATTG T ~ GCTCAGCT GGCTGAAGTA GTCCCACATC ACTGTGGCCA 6 INFORMATTON FOR SEO ID NO: 6-(i) CHARACTERISTICS OF T_A SEQUENCE:
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(iii) HYPOTHETICAL NO
~ iii) ANTl-SENS: NO
- ~ vi) QRIGINE:
2 ~ (A) ORGANIZATION: OLIGONUCLEOTIDE D
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
AATTCGGTCA CCTGCGTAAG TGTTCACTTC TCCAAGTTTG TCCTGGA ~ GA GGGCATTGAG 60 . TTGCTGGGTG AGTTCAGATT TCTGGAGATG TT 92 30 (2) I. ~ FQRMATTO ~ FOR SEO ~ ~ Q:?:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LON & UEI ~ R: 42 base pairs (B) TYPE: nucleic acid ~ C) ~; LOMBRE DE BRINS: simple SHEET OF RE911PLA ~: E ~

(D) CONFlGUR4.TlON: linear (ii) MOLECULE IYPE: cDNA
(vi) ORIGIN:
(A) ORGANIZATION: OLIGONUCLEOTIDEsqlO87 (xi) DESC ~ I ~ ION OF THE SEQUENCE: SEQ ID NO: 7:
GCCCCTTTGG AGAGCTGAGG ATCCCCTGGT GCAC ~ GGCCC CA ~ 2 t2! INFOR1 ~ AIION FOR SEO rD I ~ O: 8:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CO ~ FIGURATION: linear:
(ii ~ TYPEDEMOLECULE cDNA
(vi ~ ORIGIN:
(A) OLIGONUCLEOTIDE ORGANIZATION
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:

~ 2) INFORMATION FOR SEO ID NO: 9:
~ it CHARACTER ~ SEQUENCE STICKS
(A) LENGTH: 31 pure bases (B) I ~ YPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: paragraph ~ re (ii) TYPE OF MOLECULE: cDNA
(vi) ORIGIN:
(A) OLIGONUCLEOTIDE A ORGANISM
(xi) DESCRIPTION OF THE SECTOR: SEQ ID NO: 9 - (2) INFORMATIO ~ FOR SEO 11 :) ~ O: 10 (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 32 base pieces (B) IYPE nucleic acid - (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE DF. ~ OLECULE: cDNA

RE SHEET ~ IIPL ~ CEES ~ NT

-(~ i) ORIGIN:
(A) ORGAI '; IS ~ SE: OLIGONUCLEOTIDE B
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10:
TATGGTGATG GTGATGGTGC GGATCCACGC A ~ 32 (2) INFORM. ~ TTON FOR IA SEQ ID NO: 11:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 46 base pairs ~ B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) MOLECULE mE: cDNA
(~ i) ORIGIN:
~ A) ORGANIZATION: OLIGONUCLEO'I-IDE
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:
1 ~ CTGAGG ~. ~ CA AGGTCAACTG AGGATCCAGC ACCTTCAAG ~ AGAAAG 4 G
~ 2) INFORMATIOI ~ FOR SEO ID NQ: 12:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single CONF ~ GURATION: linear ii) TYPE OF MOLECULE: cDNA
(vi) ORIGIN:
(A) ORGANIZATION: OLIGONIJCLEOTIDE
2 ~ (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12:
CTC ~ GG ~ AC GCCTTACGTG A ~ GATCCGAC GA ~ TTCCAAA GTC 4 ' (~) INFORMATIO ~ l FOR SEO ID NQ 13:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A ~ LENGTH: 39 base pairs (B) ~ PE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) MOLECULE LYPE: cDNA
(vi) ORIGIN:
3 ~ (A) ORGANIZATION: OLIGONUCLEOTIDE

REPLACEMENT SHEET ~ ENT

212 ~ 8 ~ 7 (xi) DESCRIPTION OF L. ~ SEQl LENCE: SEQ ID NO: 13:
GAGCTC ~ GG GACGCCAT. ~ GCCC ~ AC ~ Cm GAC ~ mTC 3 (~ INFORMATION FOR SEOID NO:] 4 (i) CHARACTERISTICS OF THE SEQUENCE:
~ A) LENGTH: 30 base pairs ~:
(B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) MOLECULE YPE: cDNA
(vi) ORIGIN:
(A) ORGANIZATION: OLIGONUCLEOTIDE
(xi) DESCRIPllON OF THE SEQUENCE: SEQ ID NO: 14:

~) INFORMATION FOR SEQ ID NO: 1 ~:
(i) CHARACTERISTICS OF THE SEQUE ~ CE
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGUR: ATION: lineaife (ii ~ l YPE OF MOLECULE: cDNA
(vi) ORIGIN:
(A) ORGANIZATION: OLIGONUCLEOTIDE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CAGGCCTCGC ~ GAGGCCCTA TGCTCAGGAC 30 25 (2 ~ INFORMATION FOR SEO ID NO ~
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) MOLECULE LYPE: cDNA
(vi ~ ORIGIN:
(A) ORGANIZATION: OLIGON11CLEOTIDE
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16:
35 CGCCGCAGCr TGG ~ TCCCTT G ~ CCGAGGAC. 3 RE SHEET ~ IIPL ~ C ~ NT

212 ~ '~ 77 , 9 (21 I ~ FORMATIOI ~ FOR SEO ID NO: l7:;:
~ it CHARACTERISTICS OF THE SEQUENCE::
(A) LENGTH: 30 base pairs (B) ~ YPE: nucleic acid -(C) NUMBER OF BE ~ NS: simple (D) CONFIGURATION: linear (u) TYPE OF MOLECULE: cDNA
(vi ~ ORIGIN:
(A) OLIGONUCLEOTIDE ORGANIZATION
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17:

(2) 1 INFORMATION FOR 1D SEO NO: 18:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 30 base pairs 1 ~ (B) acidenucleic YPE :::
:: ~: (C) NUMBER OF BRlNS: simple.
(D) ~ ONFIGURATiON: linear u ~ TYPE OF MOLECULE: cDNA
. (vi) ORIGIN:
. (A ~ ORGANIZATION: OLIGONUCLEOTIDE
DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18:
CGACGCCGGG TGGAGTCCTT CTTCAGCACC. ~ 30 ) TNFORMATiON FOR IP SEO NO: l2 ~ -(i) CHARACTERISTICS OF THE SEQUENCE:
2 ~ (A ~ LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA
(vi) ORIGIN:
(A) ORGANIZATION: OLIGONUCLEOTIDE
(xi) DESCRIPTI (:) SEQVENCE N: SEQ ID NO: l ~:

~ 2) INFORMAT101`1 FOR SEO ID NQ: 20:
3 ~ (i) CHARACTERISTICS OF THE SEQUENCE:

. .
REUPLACE HEAD ~ NT

- ~

: 2 1 2 ~ d 8, 7 ~ o (A) LINGER: 2 ~ base pairs (B) TYPE: nucleic acid (C) NUMBER: OF STRANDS: sirr, ple (D) CONFIGURATION: read ~, éaire (ii) TYPE OF MOLECULE: cDNA
(vi) O ~ GINE
(A) ORGANIZATION: OLIGONUCLEOTIDE
~ xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20:
GAGGTCAGTG CTAGCCAGGT GGCCACA. 27 10 (2! INFORMATION FOR I ~ A SEO ID NO: 21:
~ i) CHARACTERISTICS OF THE SEQUENCE:
tA) LENGTH: 27 basic paues tB) TYPE: nucleic acid (C) NUMBER OF STRANDS: single 1 ~ (D) CONFIGURATION: l ~, s2, ire ii) TYPE OF MOLECULE: cDNA
~ vi) ORIGIN:
(A) ORGANIZATION: OLIGONUCLEOTIDE
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21:
20 GAGGTCAGTG CTAAACAGG $ GGCCACA. 2i ~ 2) INFORMATTON FOR SEO ID NO: 22:
(i) CHARACTERISTICS OF THE SEQUENCE:
~ A) LENGTH: 27 pa ~ res of ba, ses (8) TYPE: acid, nucleic acid (C) NUMBER OF STRANDS: sim, ple (D) CONFIGURATION: linear ~ ire ii) MOLECULE I YPE: cDNA
(vi) ORIGIN:
(A) ORG ~ NISME: OLIGONUCLEOTIDE
30 . (XJ,) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22:
GCCCTCTTCC AGTTCAAACT TGGAGAA 27`
(O INFORMATION PQUR I ~ A SEO IP NO: 23:
~ i) CHARACTERISTICS OF THE SEQUENCE:
tA) LENGTH: 27 base pairs (B) TYPE: nucleic acid C) NOLE ~ BRE OF STRANDS: .simple ~: ElJILLE DE REeJlPLA ~ g '~

(D) CON ~ lGURATION: the area (ii) T'r PE OF MOLECULE: cDNA
(vi) ORIGIN:
(A) ORGANIZATION: OLIGONUCLEOTIDE
S (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23:

(2 ~ INFORMATION FOR SEO ID NO: 24:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 27 base pairs (B) ~ YPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) MOLECULE IYPE: cDNA
(vi) ORIGIN:
(A) ORGANIZATION: OLIGONUCLEOTIDE
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 24:

~ 2) I ~ PQUR EA SEO ID N TRAINING! ~;
(i) CHARACTERISTICS OF THE SEQUENCE:
2 ~ (A) LENGTH: 27 base pairs (B) l YPE: nucleic acid (C) NUMBER OF B} ~ INS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA
2 ~ (vi) ORIGIN:
(A) ORGANIZATION: OLIGONUCLEOTIDE
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25:

~ 2) Il`lFORMAT ~ ON PQUR LA SEQ TD NQ ~ 26:
(i) C: ARACTERISTICS OF THE SEQUENCE:
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RENIPLACE SHEET ~ NT

21258 ~ 7 ``

(vi) ORIGIN:
(A) ORGANIZATION: OLIGONUCLEOTIDE
(xi) I: ~ SEQUENCE ESCRIPTION: SEQ ID NO: 26:

, LILL OF REPLACEMENT ~ r

Claims (17)

1. Polypeptide derived from human apolipoprotein AIV characterized in that that it comprises, with respect to the sequence of the human apolipoprotein AIV SEQ ID
No 2, at least one of the following modifications:
(a) a point mutation involving a functional residue, and/or, (b) a deletion of a terminal end of at least 10 amino acids, and/or, (c) a deletion of a helix or pair of helices, and/or, (d) an additional heterologous part possessing a different biological activity or complementary to that of apoAIV, or a marker, targeter or stabilizer property. 2. Polypeptide according to claim 1 characterized in that it comprises at minus one modification according to (a) and (b). 3. Polypeptide according to claim 1 characterized in that it comprises at minus one modification according to (a) and (c). 4. Polypeptide according to claim 1, characterized in that it comprises a modification according to (a), (b) or (c) associated with a modification according to (d). 5. Polypeptide according to one of claims 1 to 4 characterized in that the functional residue is chosen from charged residues, site residues of glycosylation and residues involved in disulfide bridges. 6. Polypeptide according to claim 5 characterized in that the residue functional is chosen from the following residues: aspartic acid at position 5, 44, 106, 153; glutamine at position 37, 194; asparagine at position 39; lysine in position 73, 84, 103, 169, 178; glutamic acid at position 76, 81, 87, 99, 131, 164,187, 230; alanine at position 22; proline at position 139,161; and serine at position 154. 7. Polypeptide according to one of claims 1 to 6 characterized in that it is chosen from the following polypeptides described in the examples: P(.DELTA.N13,R93G), P(.DELTA.N13), P(tag.DELTA.N13), P(R93G), P(.DELTA.C44), P(tag.DELTA.C44), P(.DELTA.C194), P(. DELTA.N182), P(.DELTA.h1-2), P(tag.DELTA.h1-2), P(.DELTA.h7-8), P(tag.DELTA.h7-8), P(.DELTA.h9-10 ), P(tag.DELTA.h9-10), P(.DELTA.h11-12), P(tag.DELTA.h11-12), P(.DELTA.h11-12,L87M), P(.DELTA.h13-14), P(tag.DELTA.h13-14), P(. DELTA.h5-6), P(tag.DELTA.h5-6), P(D44F), P(D44A), P(D5S), P(D5K), P(K178Y), P(K178A), and P(E230K). 8. Nucleotide sequence coding for a polypeptide according to one any of claims 1 to 7. 9. Nucleotide sequence according to claim 8 characterized in that it is a cDNA, gDNA, RNA or synthetic sequence or semi-synthetic. 10. Nucleotide sequence according to claim 9, characterized in that it is a cDNA sequence. 11. Vector comprising a nucleotide sequence according to one of claims 8 to 10. 12. Recombinant cell containing a vector according to claim 11. 13. Process for the preparation of a polypeptide according to any one of claims 1 to 7 characterized in that a recombinant cell according to claim 12 and recovering the produced polypeptide; 14. Use of a polypeptide according to any one of claims 1 to 7 for the preparation of non-peptide molecules or not exclusively pharmacologically active peptides on plasma levels of cholesterol, by determining the structural elements of this polypeptide which are important for its activity, and reproduction of these elements by structures not peptides or not exclusively peptides. 15. Pharmaceutical composition comprising one or more polypeptides according to any one of claims 1 to 7, or one or more sequences nucleotides according to one of claims 8 to 10, or one or more molecules prepared according to claim 14. 16. Composition according to claim 15 intended for the treatment or prevention of hypercholesterolemia-related conditions. 17. Composition according to claim 16 intended for slowing down the formation of atherosclerotic plaques, and/or regression of atherosclerotic plaques and/or reducing the risk of coronary events.