EXPRESSION OF HUMAN APOLIPOPROTEINS AI-MILANO IN YEAST

Abstract: An expression vector capable of expressing, in a transformed yeast, apolipoprotein AI and mutant apolipoprotein AI-M (Milano) is described. Yeast strains, namely of S.cerevisiae, transformed by said vectors, produce the protein which may be conveniently purified and used for the treatment of atherosclerosis and cardiovascular diseases.

Description: EXPRESSION OF HUMAN APOLIPOPROTEINS Al AND AI-MILANO YEAST The present invention refers to the expression apolipoprotein Al and apolipoprotein AI-Milano (Apo- and Apo-AI-M) in yeast strains and to pharmaceutic preparations containing as active principle sa apolipoproteins for the treatment of atherosclerosis a cardiovascular diseases. The clear correlation between elevated levels serum cholesterol and the development of coronary hea disease (CHD) has been repeatedly confirmed, based epidemiological and longitudinal studies. The definitio however, of complex mechanisms of cholesterol transpo in plasma, has allowed to recognize a selective functi of circulating lipoproteins in determining the risk CHD. There are, in fact, four major circulati lipoproteins: chylomicrons (CM), very low density (VLDL low density (LDL) and high density (HDL) lipoprotei While CM constitute a short-lived product of intesti fat absorption, VLDL and, particularly, LDL responsible for the cholesterol transport into tissu among these, also into the arterial walls. In contra HDL are directly involved in the removal of choleste from peripheral tissues, carrying it back either to liver or to other lipoproteins, by a mechanism known "reverse cholesterol transport" (RCT). The "protective" role of HDL has been confirmed i number of studies (Miller et al. , Lancet, i.: 965-9 1977; Whayne et al. , Atherosclerosis 39: 411-419, 198 In these, the elevated levels of LDL, less so of VL SUBSTITUTE SHEET seem to be clearly associated with an increase cardiovascular risk. Recent interest in the study of the protectiv mechanism/s of HDL has been focussed into apolipoprotei Al (Apo Al), the major component of HDL. Plasma levels o Apo Al also bear, in fact, a negative correlation wit the risk of CHD and with the presence of coronary lesion (Maciejko et al., N.Eng. J. Med. , 309: 385-389, 1983 Sedlis et al., Circulation, 73: 978-984, 1986). In vitro studies , indicate that complexes of Apo A and lecithin can promote the efflux of free cholestero from cultured arterial smooth muscle cells (Stein et al. Biochem. Biophys. Acta, 380: 106-118, 1975). By thi mechanism HDL can also reduce the proliferation of thes cells (Yoshida et al. , Exp. Mol. Pathol. , 41: 258-266 1984). More recently, the infusion of Apo Al or of HDL i experimental animals has been shown to exert significan biochemical " changes, as well as to reduce the extent a severity of atherosclerotic lesions. After an initia report by Maciejko and Mao (Arteriosclerosis , 2: 407 1982), Badimon et al. , in two successive studie (Cardiovascular Disease, 1983, Abs. 81; Arteriosclerosis 7: 522a, 1987), clearly showed that, by infusing eithe Apo Al or HDL (d=l.063-1.325 g/ml) , one cou significantly reduce the extent of atherosclerot lesions in cholesterol-fed rabbits (-45%), while reduci the cholesterol ester content in the lesions by 58.4%. could also be shown (Esper et al. , Arteriosclerosis , 523a, 1987) that infusions of HDL can markedly change t plasma lipoprotein composition of Watanabe rabbits wi inherited hypercholesterolemia, which develop ear SUBSTITUTE SHEET arterial lesions. In these, HDL infusions can more tha double the ratio between the protective HDL and th atherogenic LDL. The potential of HDL to improve arterial disease i animal models has been further stimulated by th observation that Apo Al can exert a significan fibrinolytic activity (Saku et al. , Thromb. Res. , 39 1-8, 1985). Ronneberger (Xth Int. Congr. Pharmacol. Sidney, 1987, p 990) clearly demonstrated that extractiv Apo Al can significantly increase fibrinolysis in beagl dogs and in Cynomolgous monkeys. A similar activity ca be noted in vitro on human plasma. This Author coul confirm a reduction of lipid deposition and arteria plaque formation in Apo Al treated animals. Plasma Apo Al is a single polypeptide chain of 24 amino acids, whose primary sequence is known (Brewer e al. , Biochem. Biophys. Res. Commun., 80: 623-630, 1978) Apo Al is synthesized as a 267 amino acid precursor i the cell. This pre-pro-apoliprotein first loses the 1 N-terminal residues intracellularly; the loss of further amino acids occurs in the plasma and/or lymph b the activity of specific proteases. The major structural requirement of the Apo A molecule is believed to be the presence of repeat unit of 11 or 22 aminoacids, presumed to exist in amphipathi helical conformation (Segrest et al., FEBS Lett. , 38 247-253, 1974). This structure allows for the mai biological activities of Apo Al, i.e. lipid binding an lecithin cholesterol acyl transferase (LCAT) activation The other functions of HDL , i.e. interaction with cell and removal of cholesterol from these, are not associate with clear features of the apolipoprotein. SUBSTITUTE SHEET The apolipoprotein AI-Milano (Apo AI-M) is the firs described molecular variant of human Apo Al (Franceschin et al. , J. Clin. Invest. , 66: 892-900, 1980). It i characterized by the substitution of Arg 173 with Cy (Weisgraber et al. , J. Biol. Chem. , 258: 2508-2513 1983). The mutant apoprotein is transmitted as a autosomal dominant trait and 8 generations of carrier have been identified (Gualandri et al. , Am. J. Hum Genet. , 37: 1083-1097, 1984). The carrier status of Apo AI-M is characterized by remarkable reduction of HDL-cholesterol. In spite o this, the affected subjects do not apparently show an increased risk of arterial disease; indeed, b examination of the genealogic tree it appears that thes subjects may be "protected" from atherosclerosis. The mechanism of the possible protective effect o Apo AI-M in the carriers seems to be linked to modification in the structure of the mutan apolipoprotein, with the loss of one -(-helix and a increased esposure of hydrophobic residues (Franceschin et al., J. Biol. Chem., 260: 1632-1635, 1985). The los of the tight structure of the multipleo(.-helices leads t an increased flexibility of the molecule, whic associates more readily with lipids, compared to norma Al. Moreover, apolipoprotein/lipid complexes are mor susceptible to denaturation, thus suggesting that lipi delivery is also improved in the case of the mutant. Another, very specific feature of the Apo AI-M, i its capacity to form dimers with itself and complexe with Apo All, in both cases because of the presence o the Cys residue. The presence of dimers and complexes i the circulation is probably responsible for the prolonge SUBSTITUTE SHEET elimination half-life of these in the carriers, recentl described in clinical studies (Gregg et al. , NATO ARW o Human Apolipoprotein Mutants: From Gene Structure t Phenotypic Expression, Limone S.G. , 1988). The modifie apolipoprotein particles may thus remain in th circulation for very long periods, thus exerting thei arterial protective activity in a better way, compared t normal Al particles. The therapeutic use of apolipoprotein Al or of it AI-M mutant is presently limited by the lack of a metho allowing the preparation of said apolipoproteins i sufficient amount and in a suitable form. The difficulty of producing apolipoprotein Al an particularly Al Milano (AI-M) from plasma fractionatio is quite considerable (Franceschini et al. , J.Biol.Chem. 260: 16321-16325, 1985). Furthermore there are a serie of risks associated with plasma fractionation products such as contamination with infectious agents an availability of starting material, that are essential t avoid. Several attempts have been made to produce human Ap Al, by way of the recombinant DNA technology. In th European patent publication No. 0267703 the preparatio of Apo Al from E.coli is described. The process describe a chimeric polypeptide where the Apo Al moiety is fuse to the N-terminal amino acid residues o beta-galactosidase or to one or more IgG-binding domain of Protein A, or to the pro sequence of human Apo Al. The above methods suffer from the fact that E.col is used as the transformant organism. The expresse products are not the mature human Apo Al but contai substantial sequences of heterologous origin. Furthermo SUBSTITUTE SHEET the products are not secreted from cells but accumulat intracellularly in the E.coli host organism, thu enhancing the risk of enzymatic degradation of th expressed product. A more convenient system for expression of mammalia polypeptides seems to be eukaryotic cells and several at tempts have been made to express foreign genes in euka ryotes, especially in yeast. Expression of interferon i yeast is described in European patent publication No 0060057 and expression and secretion in yeast of protein heterologous to yeast is described in European paten publication Nos. 0088632, 0116201 and 0123544. The object of the present invention is to provid Apo Al and Apo AI-M which are generated in high yields i yeast with correctly folded structure and the propertie of natural human Apo Al, namely structural features .lipid binding and activation of fibrinolysis and of th lecithin: cholesterol acyl transferase (LCAT) (Mahley e al., J.Lipid. Res., 25: 1277-1294, 1984). According to the present invention the gene encoding the Apo Al and Apo AI-M, were provided wit DNA-sequences encoding yeast-recognizable secretion an processing signals fused upstream to the gene for th mature proteins. In particular a modified MF © 1 leade sequence was used in which the last residues where HisGlySerLeuAspLysArg. In this modified MF oC 1 leader the processing signa was the dibasic sequence Lys-Arg, which is then fused t the 5' terminus of the Apo Al gene. It was found that th leader sequence was cleaved off in all the constructions i.e. cleavage occured at the dibasic sequence Lys-Ar upstream to all the Apo Al and Apo AI-M polypeptides. SUBSTITUTE SHEET This invention provides a method for producing A Al and Apo AI-M in yeast. By this method a yeast stra transformed with an expression vector comprising DNA-sequence encoding the Apo Al and Apo AI-M proteins cultivated in a suitable culture medium and the Apo and Apo AI-M molecules are recovered from the cultu medium. When cultivating such transformed yeast strain high yields of the Apo Al and Apo AI-M are isolated fr the culture broth. The expression products were isolated and Apo immunoreactive material was purified and characterized microsequence analysis. It was found that th polypeptide has the same characteristics as the natur Apo Al. The expression vectors of the invention comprise replication system for stable maintenance in a yea host, DNA-sequences encoding the Apo Al or Apo AI-M a promoter and terminator sequences. The expression vector may, upstream to DNA-sequen encoding the desired product, contain a preregi ensuring direction of the expressed product into t yeast secretory pathway and secretion of the express product into the growth medium. This preregion, whi might be a naturally occurring signal or leader pepti or a synthetic sequence providing secretion, is general cleaved from the desired product during secretio leaving the mature product ready for isolation from t culture broth. A well suited leader sequence for yeast is the yea MF o ^ 1 leader sequence (Kurjan, j. and Herskowits, I Cell 30: 933-946, 1982). SUBSTITUTE SHEET The expression vector may be a plasmid capable o replication in the host microorganism or capable o integration into the host chromosome. The vector employe may code for expression of repeated copies of the desire DNA-sequence, each separated by selective cleavage sites. The expression of the desired DNA-sequence will b under control of a promoter sequence, correctl positioned to the DNA-sequence encoding the desire product to result in expression of the desired product i the host organism. Preferably a promoter from a gen indigenous to the yeast host is used, e.g. the promote of TPI (triose phosphate isomerase) gene or the MF< ~ _ 1-promoter. The DNA-sequence for the desired product will b followed by a transcription terminator sequence, preferably a terminator sequence from a gene indigenou to the yeast host, e.g. the terminator of the TPI-gene o the MFø 1-gene. The present invention describes also th manipulation of the previously cloned Apo Al and Apo AI- .sequences (Sharpe CR, Sidoli A, Shelley CS, Lucero MA Shoulders CC and Baralle FE, Nucl. Acids Res., 12 ' 3917-3932, (1984) to make them suitable for secretion i yeast, using a chemically synthesized adaptor of 4 nucleotides, cloned in the unique Ncol site of Apo Al. The Apo AI-M mutation was generated by partiall cloning the mutant Apo AI-M gene from a carrier and b replacing part of it into the wild type sequence. In the drawings: - Figure 1 shows the sequence of the 46-nucleotid adapter containing the end of the MF O 1 leade sequence and the amino-terminal sequence of mature Ap SUBSTITUTE SHEET Al . - Figure 2 shows the construction of the plasm pUC/AF-AI from ρUC8. - Figure 3 shows the construction of pUC/AF-AI-M starti from the plasmid of Figure 2. - Figure 4 shows the preparation of the expressi vectors pYES/AI and pYES/AI-M, starting from pYES a from the plasmids of figures 2 and 3. Construction of the Apo Al and Apo AI-M sequences The Apo Al sequences were obtained from o previously described cDNA clone (Sharpe et al Nucl.Acids Res . , 1984), from which the sequence codi for the signal peptide and the propeptide were remov and modified with the following strategy. The Nco I - BamHI fragment from the original cD sequences (plasmid pAIA) was ligated simultaneously wi a chemically synthesized fragment Hind III - Nco containing the end of the MF <?(_ 1 leader sequence and t amino terminal sequence of mature Apo Al (fig. 1) and Hind III - Bam HI digested pUC 8 (see fig. 2). This clo is named pUC/AF-AI. The Apo AI-M sequence was obtained by cloning pa of the gene from an Apo AI-M carrier. The methodolo used was as follows: a) DNA was prepared from peripheral white blood cel following standard methods (Kunkel LM, Smith K Boyer SH, Borgaonkar DS, Watchel SS, Miller BJ, Be WR, Jones HW and Rary JM. , Proc. Natl. Acad. Sc USA, 1245-1249, 1977). b) The fragment between nucleotides 1792 and 2240 (s fig. 3) (Shoulders CC , Kronblihtt AR, Munro BS a F.E. Baralle Nucl. Acids Res. , 11: 2827-2837, 198 SUBSTITUTE SHEET was produced by the amplification procedure known a Polymerase Chain Reaction (PCR. Saiki RK, Gelfan DH, Stoffel S, Scharf S, Higuchi R and Horn GT Science 239: 487-491, 1988), using two syntheti oligonucleotides as primers in the enzymati reaction on the Apo AI-M genomic DNA, prepared fro a carrier, having the following sequences: 5'-CTGAGGCAAGAGATGAGCAA-3' ; 5' end in 1792. 5'-CTCAGGAAGCTGACCTTGAA-3' ; 5' end in 2240. c) This fragment was subjected to Xho II and Stu digestions and the resulting product was ligate with the Hind III - Xho II and Stu I - BamH fragments from pUC/AF-AI into a pUC-8 BamHI-Hindll linearized vector. The obtained clone is named pUC/AI-M. Plasmid construction Genes encoding the human Apo Al and Apo AI-M wer combined with fragments coding for the TPI promote (TPI ) (T. Alber and G. Kawasaki. J.Mol. Applied Genet I: 419-434, 1982), the MF oi.1 leader sequence (J. Kurkia and I. Herskowitz., Cell 30: 933-943, 1982) and th transcription termination sequence from TPI of S cerevisiae TPIt,. These fragments provide sequences to ensure a hig rate of transcription for the apo Al encoding gene an also provide a presequence which can effect th localization of Apo Al and Apo AI-M polypeptides into th secretory pathway and its eventual excretion into th growth medium. The expression plasmids further comprise the yeas 2μ origin o replication and a selectable marker LEU2. During in vivo maturation of o_-£actor in yeast, th SUBSTITUTE SHEET last (C-terminal) six amino acids of the MF 0(_ 1 leade peptide (Lys-Arg-Glu-Ala-Glu-Ala) are removed from th -factor precursor by the sequential action of a endopeptidase recognizing the Lys-Arg sequence and a aminodipeptidase which removes the Glu-Ala residue (Julius D, Blair L, Brake A, Sprangue G and Thorner J Cell 32: 839-852, 1983). To eliminate the need for th yeast aminodipeptidase, the sequence coding for th C-terminal Glu-Ala-Glu-Ala of the MF o_ 1 leader wa removed via in vitro mutagenesis. The Hind III-Bam HI fragments from pUC/AF-AI an pUC/AF-AI-M were cloned in the Hind III-Bam HI linearize pYES vectors, as shown in fig. 4. The resulting plasmid for yeast transformation and expression of Apo Al and Ap AI-M genes, are called pYES/AI and pYES/AI-M respectively. Transformation Plasmids prepared as above described wer transformed into S. cerevisiae strains carrying deletion into the TPI gene by selecting for growth on glucose Such strains are normally unable to grow on glucose a the sole carbon source and grow very slowly on galactos lactate medium. This defect is due to a mutation in t triose phosphate isomerase gene, obtained by deletion an replacement of a major part of this gene with the cerevisiae LEU 2 gene. Because of the growth deficienc there is a strong selection for a plasmid containg a ge coding for TPI. Expression of the Apo Al and Apo AI-M precursors in yeast The yeast strains containing plasmids encoding f the apolipoproteins were grown on YPD medium (Sherman, SUBSTITUTE SHEET et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory 1981). For each strain, two cultures of 1 litre each in a baffled flask of 2 litres were shaken at 30°C until they reached an OD at 600 nm of approx. 15 (approx. 48 h). After centrifugation the supernatant was removed for further analysis. Purification of Apo Al and Apo AI-M culture broth The culture broth was centrifuged at 15,000 rpm fo 30 min. and the supernatant dialysed against 0.15 M NaCl, 0.05 M K-phosphate buffer, 0.05% EDTA, pH 7.4 (buffer A). Triglyceride-rich particles (TGRP) were isolate from commercial phospholipid-triglyceride emulsions, b ultracentrifugal washing at 4°C, and resuspended i buffer A. The supernatant from culture broth wa incubated with TGRP at 37 β C for 60 min., with gentl agitation and incubations terminated by immersion in a ice-water bath. TGRP were reisolated b ultracentrifugation at 27,000 rpm for 35 min. at 40°C an delipidated with diethylether:ethanol 3:1. Th precipitated protein was collected by low spee centrifugation and passed through a Sephacryl S-20 column and/or an anti-apo Al-Sepharose column. Structural characterization of the recombinant apolipoprotein The structural features of the recombinant Apo A and AI-M were tested by aminoacid analysis and protei microsequencing, confirming the purity of th apolipoprotein preparations. The secondary structure wa analyzed by circular dichroism, indicating that th recombinant apolipoprotein are properly folded: th θC-helix content was calculated as 52% and 43% fo recombinant Apo Al and Apo AI-M, respectively, as a 49 SUBSTITUTE SHEET content for extractive Apo Al. Thus recombinant AI-M, a extractive AI-M (Franceschini et al. , J. Biol. Chem. 260: 16321-16325, 1985), has a lower < -helix conten compared to normal Al. Biological evaluation of recombinant Apo AI-M vs Apo Al All reported experiments were carried out b comparing Apo Al and Apo AI-M, obtained from S cerevisiae, with human Apo Al, extracted from plasma Human Apo Al was prepared from human HDL, separated b preparative ultracentrifugation. Following delipidazatio of the isolated HDL fraction, Apo Al was separated by ge filtration and ion exchange chromatography (Baker et al. Biochemistry, 12: 3866-3871, 1983). In vitro studies Activation of fibrinolysis Agents promoting the conversion of plasminogen t plasmin, thus initiating fibrinolysis, are gainin increased interest in the treatment of myocardia infarction (Laffell and Braunwald, N. Engl. J. Med. , 311 710-717, 1984). Previous data have shown that the Apo can activate fibrinolysis in vitro (Saku et al. , Thromb Res. , 39: 1-8, 1985). Experiments were carried out by the fibrin pla method (Glas-Greenwalt et al., J. Lab. Clin. Med., 104 962-976, 1984) in order to evaluate the potentiation o inhibition of urokinase activity. A bovine (plasminogen-rich) fibrinogen solution, a final concentration of 0.1% in a volume of 6 ml, w clotted with 0.2 ml of a bovine thrombin solution (20 N Units/ml). Recombinant Apo Al and Apo AI (concentrations between 10 and 500 μ/g), diluted in Tr SUBSTITUTE SHEET buffer were rapidly mixed with urokinase (f.c. 3.3 CA U/ml) and applied to the fibrin plates. These were incubated for 17 h a 37°C. The lysis area measurements indicate that both proteins significantly increased th urokinase lysis areas, at the highest tested doses, b 64.0% and 67.9% respectively in the case of Apo Al (vs 59.7% and 66.2% for extractive Apo Al; Saku et al., Thromb. Res., 39: 1-8, 1985) and by 96.7% and 112.1% i the case of Apo AI-M (Table I). In this system, therefore, Apo AI-M appears to be significantly mor potent than Apo Al. Removal of cholesterol from cultured macrophages The so called "reverse cholesterol transport" (RCT) , i.e. the transport of cholesterol from tissues to plasma, is believed to be regulated by HDL, acting as th acceptor lipoprotein (Glomset J. , Adv. Int. Med., 25: 91-116, 1980). Since Apo Al is believed to be responsibl for the removal capacity of HDL, experiments were carrie out with J-774 mouse macrophages equilibrated with cholesterol. The monolayers were incubated with "Minimu Essential Medium", solvent-extracted delipidized cal serum protein, egg phosphatidylcholine (PC) an esterified cholesterol (Rothblat et al. , In vitro, 12 3 554-557, 1976). Equilibration with H cholesterol was fo 48 h, after which the radioactive medium was removed, th cells extensively washed and added with fresh medium supplemented with 5 mg/ml delipidized calf serum protein After overnight incubation, the cells were agai extensively washed and incubated with HEPES-buffere Williams Medium E in 35 mm flat dishes, with the additio of 2 ml of each acceptor particle. Each 90 min., 0.1 m aliquots of the incubation medium were taken, in the fis SUBSTITUTE SHEET 6 h and then every 3 h, for a total 24 h period a counted in order to determine the amount of labell cholesterol released by the cells (Rothblat and Phillip J. Biol. Chem. , 250: 4775-4782, 1982). Proteoliposomes containing recombinant Apo Al or A AI-M were prepared with egg PC according to the chola dialysis procedure, as described by Mats and Jonas ( Biol. Chem., 257: 4535-4540, 1982). Mixtures we generally of apolipoprotein/phospholipid/cholate (ma ratio of 1/2.5/1.5-2.5) in lOmM Tris/lmM EDTA/l NaN,/150 mM NaCl (pH 8.0). In order to size t apolipoprotein/phospholipid particles, a gel filtrati on Sepharose CL/4B (1.5 x 80 cm column) eluted with 0. M NaCl/0.02% EDTA, pH 7 was carried out. Peak fractio were concentrated on ultrafiltration cells (Amicon mod 52). By determination of the chemical composition a molecular microscopic features of the particl containing Apo Al or Apo AI-M, no significant differenc could be found. To evaluate the capacity to remove ce cholesterol, two different concentrations of t apolipoprotein component (50 and 100 μg/ml, i. corresponding to concentrations of PC/ml medium of 1 and 300 μg) were added to the cells. Since, under the experimental conditions, the unidirectional flux cholesterol is first-order with respect to cell fr cholesterol concentrations, it is possible to calculate half-time for the cholesterol efflux (tl/2) in t different conditions. A shown in Table 2, at both concentrations apolipoprotein in the liposomes, the efflux of fr cholesterol from J-774 cells was considerably faster wi Apo AI-M, compared to Apo Al. Again, no difference cou SUBSTITUTE SHEET be detected between the recombinant and extractive Apo Al (tl/2: 17.9±3.2). In vivo study of arterial atherosclerosis regression This experiment evaluated the capacity of extractive Apo Al and recombinant Apo AI-M continuous infusions for 4 weeks, to reduce the arterial lesions, established with a JQ.5% cholesterol rich diet for 2 months in rabbits. Twenty-five New Zealand male rabbits (starting weight 2.2-2.5 kg) received a 0.5% cholesterol rich diet for 2 months. At this time 4 animals were sacrified. Their aortas showed 45.3±7.6% involvement with atherosclerotic lesions. At this point, the animals were divided into t;hree groups, each of 7 animals. Group 1 was placed on a standard, cholesterol-free diet with no treatment. Group 2 was continuously infused with Apo Al, by way of a subcutaneously implanted Alzet Minipump, connected to an ear vein. Group 3 was treated in the same way with Apo AI-M. The daily total protein dose was 7 mg/rabbit for each of the two apolipoproteins. After 4 weeks of treatment, both the extent of aortic fatty lesions and the aortic lipid content were evaluated in the three groups. The data (Table 3) show a more than 30% regression of lesions with Apo Al, and a more than 60% regression with Apo AI-M, with a corresponding highly significant reduction of aortic total cholesterol and cholesteryl esters in both groups, Apo AI-rM being significantly more effective, compared to normal Apo Al. The reported findings confirm the remarkable activity of Apo Al, in an in vivo condition (Badimon et al. Arteriosclerosis, 7: 522a, 1987) and indicate that Apo AI-M, probably because of its physico-chemical SUBSTITUTE SHEET properties, is more effective in inducing the regressio of atherosclerotic lesions, compared to normal human Al. SUBSTITUTE SHEET Table 1. Effect of the addition of Apo Al or of Apo AI-M fibrinolysis (X±SD; n = 4 per dose applied) Total protein applied (μg) 0 10 20 50 100 2 CO Lysis area (mm ) OD Apo Al 562±31 582±34 601±29 666±22* 702±39* 92 CO -j Apo AI-M 578±26 596±17 636±33 751±47** 804±44**° 113 c H —4 jγj *p < 0.05; **p < 0.01; ***p < 0.001 vs control CO Q p < 0.05; ° °p < 0.01; 000 p < 0.001 vs Apo Al X m Table 2. Comparison of half-times for cholesterol efflux from J-7 Apo Al: egg PC Apo AI-M: egg P acceptor acceptor Half-time (t 1/2 h) 18.3±4.2 12.1±2.2** (n=ll) ( n=12) * CO p < 0.01 c CD H H c H m CO X m m Table 3. Extent of aortic lesions, cholesterol and cholesteryl ester aortic content in rabbits after Apo Al and Apo AI-M infusion (n=7 per group, X±SD) % aortic lesions Cholesterol Cholesteryl esters Group 1 44±4 16.4±2.1 14.8±1.6 (control) Group 2 28±3** 7.9±1.4 6.2±0.8 CO (Apo Al) c DC- Group 3 17±2**°° 5.3±0.7**°° 4.0±0.4**°° i- CO zi (Apo AI-M) H rHπ **p < 0.01 vs group 1; oo p 0.01 vs group 2

Claims: CLAIMS 1. An expression vector capable of expressing, in transformed yeast, apolipoprotein Al and apolipoprotei AI-M (Milano) comprising a promoter sequence autologou to the yeast to be transformed, a DNA sequence coding fo Apo Al or Apo AI-M, a sequence coding for a leade peptide able to induce secretion of the expressio product in the culture medium, a transcription terminato sequence autologous to the yeast to be transformed. 2. An expression vector according to claim 1 wherei the leader sequence is the modified MF o_ 1 leade sequence encoding for the residue His Gly Ser Leu Asp Ly Arg. 3. An expression vector according to claim 1 or capable of expressing Apo Al or Apo AI-M, which i derived form plasmid pUC8. 4. An expression vector according to any one of th previous claims consisting of plasmids pYES/AI an pYES/AI-M. 5. A synthetic DNA encoding for the residue His Gly Se Leu Asp Lys Arg Asp Glu Pro Pro Gin Ser Pro Trp havin the following sequence CCATGGAAGCTTGGATAAAAGAGACGAGCCACCGCAGAGCCCATGG 6. A DNA sequence coding for apolipoprotein Al-Mila obtainable by: a) amplification of the fragment between nucleotid 1792- and 2240 of DNA coding for Apo Al using primers the synthetic oligonucleotides of formula 5'-CTGAGGCAAGAGATGAGCAA-3' ; 5'-CTCAGGAAGCTGACCTTGAA-3' ; SUBSTITUTE SHEET b) digestion of the fragment obtained in a) with the restriction enzymes Xho II and StuI; σ) ligation of the fragment obtained in b) with the fragments 1-1791 and 2241-2396. 7. A host yeast transformed with the expression vector of claims 1-5. 8. A- host yeast according to claim 7 which is a strain of Saccharomyces cerevisiae. 9. A protein obtainable from culture of transformed host yeast of claim 7 or 8. 10. Pharmaceutical compositions containing as the active principle a protein of claim 7 and a pharmaceutically acceptable carrier. SUBSTITUTE SHEET

Inventors: BARALLE FRANCISCO, FRANCESCHINI GUIDO, SIDOLI ALESSANDRO, SIRTORI CESARE, BARALLE, FRANCISCO, FRANCESCHINI, GUIDO, SIDOLI, ALESSANDRO, SIRTORI, CESARE
Applicants: BARALLE FRANCISCO, FRANCESCHINI GUIDO, SIDOLI ALESSANDRO, SIRTORI CESARE, BARALLE, FRANCISCO, FRANCESCHINI, GUIDO, SIDOLI, ALESSANDRO, SIRTORI, CESARE
Application year: 1990
Main classification: C12N15
IPC codes: A61K38, A61P3, A61P9, C07H21, C07K14, C12N1, C12N15, C12P21, C12R1