CN103068369A - Method for producing a lipid particle, the lipid particle itself and its use - Google Patents

Abstract

Herein is reported a method for producing a lipid particle comprising the following steps i) providing a first solution comprising denatured apolipoprotein, ii) adding the first solution to a second solution comprising at least two lipids and a detergent but no apolipoprotein, and iii) removing the detergent from the solution obtained in step ii) and thereby producing a lipid particle.

Description

Generation four connects the method for albumen-apolipoprotein A-1 granule, thus obtained lipid granule and application thereof

The present invention relates to lipoprotein and lipid granule field.This paper has reported the method that produces the lipid granule that comprises apolipoprotein, phosphatidylcholine and lipid, wherein forms lipid granule when having synthetic detergent ampholytic detergent (Zwittergent).

Background of invention

Plasma lipoprotein is the albumen-lipid complex of solubility, and it carries out lipid transportation and metabolism in blood.Based on their density, size, chemical composition and function, the lipoprotein of several main species is distinguished.Among them, high density lipoprotein (HDL) granule that is also referred to as the high density lipid granule is comprised of from several subclass that 180kD changes to 360kDa mean molecule quantity.Their average lipid and protein content are respectively 50 % by weight.Phosphatidylcholine (PC) accounts for 38% of TL, secondly is cholesteryl ester and other polarity and non-polar lipid in a small amount, comprises free cholesterol.Main protein ingredient is apolipoprotein A-1 (Apo A-I), about 60% total protein weight among the representative HDL.

Cholesterol in human body, the especially cholesterol in the body fluids such as blood of circulation do not exist with the molecular forms that separates, but exist with complex (lipoprotein) form with some albumen.The main fraction of cholesterol and low density lipoprotein, LDL (LDL) or compound with high density lipoprotein (HDL).The LDL granule comprises apolipoprotein B as main protein compound, and the HDL granule comprises apolipoprotein A-1 as main protein compound.

The cholesterol that is absorbed by the HDL granule is by enzyme Lecithin-cholesterol acyltransferase. (LCAT) esterification.Cholesteryl ester has the hydrophobicity of increase and towards the diffusion of the core of HDL granule.HDL-cholesterol-ester granule can be delivered to liver and remove from circulation.

HDL granule and its main polypeptide apolipoprotein A-1 participate in reverse cholesterol transportation (RCT).Wherein apolipoprotein A-1 increases the outflow (for example from the cell of blood vessel wall flow out) of cholesterol from cell, the activation of the combination of lipid and lecithin cholesterol Acetylase, and flow through blood plasma by liver thus and remove cholesterol.This is the Active transport process, relates to epicyte protein ATP-combination-boxlike-transport protein-A-I (ABCA-I).

Based on the therapeutic agent of apolipoprotein A-1 and apolipoprotein, for example the HDL granule of reconstruct is identified in early days in the late period seventies and the eighties in last century.For the apolipoprotein A-1-Milano that comprises lipid granule, can show clinical evidence (meaning that significant speckle reduces in the arteriosclerotic).Apolipoprotein A-1-Milano is a kind of dimeric forms of wild type apolipoprotein A-1, designs according to the naturally occurring mutant of apolipoprotein A-1 molecule.By with amino acid residue 173 (arginine) thus changing into cysteine allow to form disulfide bond and can form dimer.

In WO2009/131704, reported the nanostructured that contains the organic and/or inorganic materials core that comprises of suitable chelating cholesterol and other molecules.In WO2009/097587, reported the method for generation of the interface bilayer of Nano grade, be included in about 1 hour that obtains mixture and from intermediate mixture, eliminate detergent.In WO2006/125304, reported the pharmaceutical composition for the treatment of or prevention coronary artery disease.In US2002/10142953, reported the compositions of the coding apolipoprotein that relates to lipid metabolism and cardiovascular disease.In WO2005/084642, reported apoprotein-common chelating agen (apoprotein-cochelate) compositions.In WO2007/137400, reported method and the chemical compound for the treatment of valvular stenosis.The pharmaceutical preparation, method and the dosage regimen that are used for the treatment of with prevention of acute coronary syndromes in WO2005/041866, have been reported.

In WO2009/097587, reported interface bilayer, application and the production method of Nano grade.In WO2005/065708, reported the hydrophobin preparations in immunogenic composition of the toleration with raising.In WO2006/069371, reported prevention, inhibition and/or reversed atherosclerotic plasma adiponectin materialization method.At US4, reported compositions, application and the method for generation reverse micelle (reverse micelle) in 608,347, described reverse micelle is used for the clarification biofluid to obtain the distortionless mensuration of clarification post analysis thing.

Summary of the invention

This paper has reported a kind of method for generation of lipid granule, and wherein said lipid granule forms when having synthetic detergent.Have been found that lipid granule can form when having synthetic detergent such as ampholytic detergent.Use synthetic detergent, for example avoided using the composition of animal origin.

An aspect of this paper report is the method that produces lipid granule, and wherein said lipid granule forms when having synthetic detergent.

In one embodiment, synthetic detergent is ampholytic detergent.In another embodiment, ampholytic detergent is ampholytic detergent 3-8 or ampholytic detergent 3-10.

In one embodiment, described method comprises the steps:

I) provide the first solution that comprises the degeneration polypeptide,

Ii) the first solution is added the second solution that comprises at least a lipid and synthetic detergent, described the second solution does not comprise described polypeptide, namely without described polypeptide, and

Iii) from step I i) remove detergent in the solution that obtains, and produce lipid granule thus.

In another embodiment, described method comprises the steps:

I) provide the solution that comprises natural polypeptides,

Ii) lipid and synthetic detergent are added i) solution, and

Iii) from step I i) remove detergent and produce thus lipid granule in the solution that obtains.

In one embodiment, polypeptide is the polypeptide that restructuring produces.

In one embodiment, polypeptide is apolipoprotein.In another embodiment, apolipoprotein is the apolipoprotein of purification.

In one embodiment, apolipoprotein has the aminoacid sequence of being selected from SEQ ID NO:01,02,04-52,66 and 67 aminoacid sequence or comprise at least one and contain aminoacid sequence SEQ ID NO:01,02,04-52,66 and 67 at least 80% continuous fragment.

In one embodiment, apolipoprotein has aminoacid sequence or is selected from SEQ ID NO:01,02,04-52, at least 80% continuous fragment of 66 or 67 aminoacid sequence.

In one embodiment, apolipoprotein is apolipoprotein A-1.In one embodiment, apolipoprotein A-1 is human apolipoprotein A-Ⅰ.In another embodiment, apolipoprotein is four company's albumen-apolipoprotein A-1s, and it has aminoacid sequence SEQ ID NO:01 or SEQ ID NO:02 or SEQ ID NO:66 or SEQ ID NO:67.

In one embodiment, apolipoprotein has the aminoacid sequence with the SEQ ID NO:06 of the sudden change that is selected from R151C and R197C.

In one embodiment, at least a lipid is selected from phospholipid, fatty acid and steroid lipid.

In one embodiment, at least a lipid is at least two kinds of lipids that randomly are independently from each other phospholipid, fatty acid and steroid lipid.In another embodiment, at least a lipid namely is selected from the group that comprises a kind of lipid, two kinds of lipids, three kinds of lipids and four kinds of lipids from 1-4 kind lipid.

In one embodiment, the second solution comprises phospholipid, lipid and detergent.

In one embodiment, the second solution is comprised of phospholipid, lipid, detergent and buffer salt.

In one embodiment, lipid is two kinds of different phospholipid.In another embodiment, lipid is two kinds of different phosphatidylcholines.In another embodiment, the difference of the first phosphatidylcholine and the second phosphatidylcholine is one or two fatty acid residue or fatty acid residue derivant, and it is esterified to be the glycerol backbone of phosphatidylcholine.In one embodiment, the first phosphatidylcholine is POPC, and the second phosphatidylcholine is DPPC.

In an embodiment of the method that this paper reports, the first solution does not contain lipid granule substantially.

In one embodiment, described method is at step I i) afterwards with step I ii) comprise the steps before iia): incubation is at step I i) in the solution that obtains.In one embodiment, incubation and/or removal are being carried out from 4 ℃-45 ℃ temperature.

In one embodiment, use synthetic detergent (ampholytic detergent) incubation polypeptide about 0.5 hour to about 60 hours.In one embodiment, use synthetic detergent (ampholytic detergent) incubation polypeptide about 0.5 hour to about 20 hours.In one embodiment, use synthetic detergent (ampholytic detergent) incubation polypeptide about 2 hours to about 60 hours.In one embodiment, use synthetic detergent (ampholytic detergent) incubation polypeptide about 12 hours to about 20 hours.In one embodiment, use about 16 hours of synthetic detergent (ampholytic detergent) incubation polypeptide.

In one embodiment, detergent is the detergent with high CMC.In another embodiment, detergent is to have at least detergent of the CMC of 5mM.

In one embodiment, remove by diafiltration or dialysis or absorption.

In one embodiment, absorption is selected from affinity chromatography or hydrophobic chromatography.In one embodiment, removal is undertaken by dialysis.

In one embodiment, the first solution has the first volume, the second solution has the second volume, apolipoprotein in the first solution exists with the concentration that limits, and each exists the lipid in the second solution and detergent with the concentration that limits, wherein at step I i) in, the concentration of apolipoprotein, the concentration of lipid and the concentration of detergent are changed/reduce, and allow to form lipid granule.

In one embodiment, described method comprises the steps:

Iv) the purification lipid granule also produces lipid granule thus.

In one embodiment, the second method comprises the steps ii):

Ii) at least a lipid and synthetic detergent are added i) solution in and regulate concentration and the concentration rate of lipid, detergent and apolipoprotein.

An aspect of this paper report is the lipid granule that obtains by the method such as this paper report.

An aspect of this paper report is pharmaceutical composition, and described pharmaceutical composition comprises the lipid granule that contains apolipoprotein that the method with this paper report obtains, and with the application for the preparation of the atherosclerotic medicine for the treatment of of the lipid granule of this paper report.

Detailed Description Of The Invention

Definition

Term " apolipoprotein " refers to the albumen that comprises respectively in lipid or hdl particle.

Term " apolipoprotein A-1 " refers to have the amphipathic helix polypeptide of albumen-lipid and protein-protein interaction character.Apolipoprotein A-1 is synthetic by liver and small intestinal as the preapoprotein of 267 amino acid residues former (prepro-apoliprotein), described preapoprotein is former secreted as apolipoprotein former (pro-apolipoprotein), and it is cut into the mature polypeptide with 243 amino acid residues.Apolipoprotein A-1 is comprised of the 6-8 that is separated by blank area different aminoacid repetitive sequence, described aminoacid repetitive sequence is comprised of 22 amino acid residues respectively, described blank area is proline normally, and in some cases, the fragment that is comprised of several residues forms.Exemplary human apolipoprotein A-Ⅰ aminoacid sequence is at GenPept database login NM-000039 or database login X00566; Report among the GenBank NP-000030.1 (gi4557321).There is naturally occurring variant in human apolipoprotein A-Ⅰ (SEQ ID NO:06), such as P27H, and P27R, P28R, R34L, G50R, L84R, D113E, A-A119D, D127N, disappearance K131, K131M, W132R, E133K, R151C (amino acid residue 151 is changed to Cys by Arg, apolipoprotein A-1-Paris), E160K, E163G, P167R, L168R, E171V, P189R, (amino acid residue 173 is changed into Cys by Arg to R197C, apolipoprotein A-1-Milano) and E222K.Also comprise and have the variant that conserved amino acid is modified.

In one embodiment, four connect the fragment that albumen-apolipoprotein A-1 comprises the cleavage site of immunoglobulin A protease (IgA protease).Comprise following sequence from the known recognition site of IgA protease, wherein the position of " ↓ " expression cutting key:

Wherein select more continually and cut first three.

Term " apolipoprotein analogies " finger print is intended the synthetic polypeptide of the function of each apolipoprotein.For example, " Apolipoprotein a-i mimics " is the synthetic polypeptide that shows about Cholesterol removal (being that the reverse cholesterol flows out) biological function suitable with natural apolipoprotein A-1.In one embodiment, Apolipoprotein a-i mimics comprises at least one amphipathic alpha-helix, and it has at the positively charged amino acid residue of hydrophobic-hydrophilic interface collection bunch with at the electronegative amino acid residue of the center of hydrophilic face collection bunch.In order to simulate the function of apolipoprotein A-1, the apolipoprotein analogies comprise the repetition polypeptide from 15-29 amino acid residue, in one embodiment, comprise 22 amino acid residues (PVLDEFREKLNEELEALKQKLK (SEQ ID NO:04); PVLDLFRELLNELLEALKQKLK (SEQ ID NO:05)) repetition polypeptide.

Term " at least one " refers to one, two, three, four, five, six, seven, eight, nine or more.Term " at least two " refers to two, three, and four, five, six, seven, eight, nine, ten or more.

Term " cardiovascular disease " refers generally to disease or the disease about heart or blood vessel, such as atherosclerosis, coronary heart disease, cerebrovascular disease, arotic disease (aortoiliac disease), ischemic heart disease or peripheral blood vessel.These diseases may not can be found before the malignant event as the disease result occurs, described malignant event such as myocardial infarction, apoplexy, angina pectoris, Transient ischemic attacks, congestive heart failure, aortic aneurysm, these malignant events in most cases can cause the experimenter dead.

Term " cholate " refers to 3 α, 7 α, 12 α-trihydroxy-5 β-gallbladder-24-acid or its salt, especially its sodium salt.Can be by forming lipid granule at their transition temperatures separately with the lipid incubation apolipoprotein of detergent dissolution.

Term " critical micelle concentration " and abbreviation " CMC " thereof can be used alternately, refer to such surfactant or detergent concentration, surpass described concentration, the spontaneous reunion of individual detergent molecule (monomer) is micelle (micelle, round bar, layer structure etc.).

Term " conserved amino acid modification " refers to can not to affect or change the amino acid sequence modifications according to the characteristic of lipid granule of the present invention or apolipoprotein.Modification can be introduced by standard technique known in the art, such as the sudden change of site-directed mutation and PCR-mediation.Conserved amino acid is modified and to be comprised that amino acid residue is wherein had the modification that the amino acid residue of similar side chain substitutes.This area has defined the amino acid residue family with similar side chain.These families comprise have basic side chain aminoacid (for example, lysine, arginine, histidine), (for example has the aminoacid of acid side-chain, aspartic acid, glutamic acid), (for example has the aminoacid of uncharged polar side chain, glycine, agedoite, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), (for example has the aminoacid of non-polar sidechain, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), (for example has the aminoacid of β-side chain side chain, threonine, valine, isoleucine) and have aminoacid (for example, the tyrosine of aromatic series side chain, phenylalanine, tryptophan, histidine).Therefore " variant " albumen, refer to such molecule at this paper, its aminoacid sequence and " parent " Argine Monohydrochloride sequence different are at the most 10 interpolations, disappearance and/or displacements, in one embodiment, from about 2 to about 5 interpolation, disappearance and/or displacement.Can pass through based on Riechmann, L., etc., Nature (nature) 332 (1988) 323-327, and Queen, C., Deng, amino acid sequence modifications is carried out in the mutation of the described molecule modeling of Proc.Natl.Acad.Sci.USA 86 (1989) 10029-10033..

Term " detergent " refers to surface-active chemicals." detergent " normally has the amphipathic molecule of nonpolar hydrophobic part and polarity hydrophilic segment.Term " amphoteric detergent (zwitterionic detergent) " refers to that total electrical charge is zero and comprises simultaneously at least one positively charged part and the chemically active surface chemical compound of at least one electronegative part.In one embodiment, detergent is selected from the detergent based on sugar, based on the detergent of polyoxyalkylene, and based on the detergent of bile salts, synthetic detergent or its combination.Term " based on the detergent of sugar " refers to be selected from the detergent of n-octyl-β-D-pyranglucoside, n-nonyl-β-D-pyranglucoside, dodecyl-β-D-pyrans maltoside or 5-cyclohexyl amyl group-β-D-pyrans maltoside and derivant thereof.Term " based on the detergent of bile salts " refers to be selected from sodium cholate, cholic acid potassium, cholic acid lithium, 3-[(3-chloro-acid amide base propyl group) dimethylammonio]-Ji-propane sulfonic acid ester (CHAPS), 3-[(3 chloro-acid amide base propyl group) dimethylammonio]-2-hydroxypropanesulfonic acid ester (CHAPSO), and the detergent of its derivant.Term " based on the detergent of polyoxyalkylene " refers to be selected from Tween20, Triton X-100, the detergent of Pluronic F68 and its derivant.Term " synthetic detergent " refers to be selected from the detergent of ampholytic detergent 3-6, ampholytic detergent 3-8, ampholytic detergent 3-10, ampholytic detergent 3-12 and its derivant.

Term " high density lipoprotein granule " or its abbreviation " HDL granule " can be used alternately, refer to comprise apolipoprotein A-1 as the lipid-albumen composition of main proteinate.

Term " immunoassay " refers to the standard solid-phase immunoassay of carrying out with monoclonal antibody, be included in the antibody (capture antibody) that adsorbs/be fixed on the solid phase, antigen and and the antibody (tracer antibody) for another epi-position of antigen puted together of enzyme between form complex.Therefore, form multiple structure: solid phase-capture antibody-antigen-tracer antibody.In the reaction by multiple structure catalysis, activity and the antigen concentration in the incubation medium of the enzyme of antibody-put together are proportional.Standard sandwich method is also referred to as two antigen bridging immunoassays, because capture antibody and tracer antibody are incorporated into the different epi-positions of antigen.The algoscopy of other type is radioimmunoassay, fluorescence immunoassay and enzymoimmunoassay.It is well known by persons skilled in the art carrying out the method for described algoscopy and practical application and program.Immunoassay can be carried out with Advances in Homogeneous Immunoassay or heterogeneous immunoassay method form.

The congruent fingers such as term " increasing lipid flows out " and its grammer promote lipid to flow out from level and/or the speed of the lipid outflow of cell or speckle increase, strengthen the lipid outflow, the assistance lipid flows out, the rise lipid flows out, and improves lipid and flows out, and/or strengthen the lipid outflow.In one embodiment, lipid flows out the outflow that comprises phospholipid, triglyceride, cholesterol and/or cholesteryl ester.

Term " DMPC " refers to the phospholipid DMPC.

Term " DPPC " refers to phosphatidase 11, and 2-two-palmityl-sn-glycerol-3-phosphatidylcholine is also referred to as 1,2-, two palmityls-phosphatidylcholine.

Term " polymer " refers to the complex by two above monomer compositions.Polymer forms by noncovalent interaction between monomer.Each monomer comprises the domain of multimerization.In one embodiment, polymer comprises 2 or 3 monomers.In another embodiment, the multimerization domain interacts by the noncovalent interaction between the individual multimerization domain in being included in each monomer.Term " multimerization domain " refer to covalently or non-covalently the to associate aminoacid sequence of two above monomer molecules.The multimerization domain can from the multimerization domain interaction of different, similar or same acid sequence.In one embodiment, the multimerization domain is four to connect albumen trimerizing structural detail or derivatives thereofs, and it has the aminoacid sequence that the consensus amino acid sequences with SEQ ID NO:53 has at least 68% homogeneity.In one embodiment, at the cysteine residues of the position 50 of SEQ ID NO:53 by different radical amino acid replacements, in another embodiment, by serine residue or threonine residues or methionine residues displacement.The polypeptide that comprises the multimerization domain can associate with one or more other polypeptide that also comprise the multimerization domain.Can come initial polymer to form by mixed polypeptide under the condition that is fit to simply.In another embodiment, the multimerization domain has the aminoacid sequence of SEQ ID NO:53, wherein at the N of aminoacid sequence end or C end disappearance or 1-10 residue of interpolation.In another embodiment, the multimerization domain has the aminoacid sequence of SEQ ID NO:53, wherein at 6 or 9 amino acid residues of the N-of aminoacid sequence end disappearance.In another embodiment, the multimerization domain has the aminoacid sequence of SEQ ID NO:53, has wherein lacked N terminal amino acid residue L or N terminal amino acid residue C and L.In one embodiment, the multimerization domain is four company's albumen trimerizing structural details and the aminoacid sequence with SEQ ID NO:54.In one embodiment, polymer is same aggressiveness.

Polymer can be with aggressiveness or different aggressiveness, can make up and is attached in the described polymer because comprise the different apolipoproteins of multimerization domain.In one embodiment, polymer is the same aggressiveness of trimer.

According to an embodiment, the multimerization domain connects albumen available from four.In one embodiment, the multimerization domain comprises four company's albumen trimerizing structural details of the aminoacid sequence with SEQ ID NO:54.Four connect albumen trimerizing structural detail trimerization is caused by curling helical structure, and described curling helical structure and two other four curling helical structures that connect albumen trimerizing structural detail interact to form trimer.Four connect albumen trimerizing structural details can connect albumen available from people four, connects albumen available from rabbit four, connects albumen available from Mus four, or available from the C-type agglutinin of shark cartilage.In one embodiment, four connect the consensus sequence that albumen trimerizing structural details comprise with SEQ ID NO53 has at least 68%, or at least 75%, or at least 81%, or at least 87%, or the sequence of at least 92% homogeneity.

Term " noncovalent interaction " refers to non-covalent adhesion such as ionic interaction power (for example salt bridge), nonionic interaction force (for example, hydrogen bond), or hydrophobic interaction power (for example, Van der Waals force or π-accumulative facies mutual effect).

Term " phase transition temperature " refers to induce the lipid physical state, and (wherein hydrocarbon chain random orientation and mobile) changes needed temperature from orderly gel phase (wherein hydrocarbon chain fully extends and close packing) to unordered liquid crystalline phase.Can carry out the formation of lipid granule in the phase transition temperature of used phospholipid/mixture of phospholipids or the temperature more than the phase transition temperature.In following table 1, list the phase transition temperature of several phosphatidylcholines and its mixture.

Table 1: the phase transition temperature of pure phosphatidylcholine and phosphatidylcholine mixture

The phospholipid mol ratio	Phase transition temperature
		POPC
	4℃(-3℃)

The phospholipid mol ratio	Phase transition temperature
		DPPC	41℃
DPPC∶POPC3∶1	34℃
		DPPC∶POPC1∶1	27℃
DPPC∶POPC1∶3	18℃

Term " phosphatidylcholine " refers to by a glycerol part, two carboxylic moiety and the molecule that phosphocholine partly forms, wherein the glycerol part is passed through respectively ester bond with other parts, i.e. two carboxylic acid ester bonds and a phosphoric acid ester bond covalent bond, wherein 1-hydroxyl or the 3-hydroxy combining of phosphoric acid ester bond and glycerol part.Term " carboxylic moiety " refers to comprise the organic moiety of at least one acyl group (R-C (O) O).Phosphatidylcholine can be any kind or source.In one embodiment, phosphatidylcholine is selected from PC, S-PC, dipalmitoyl phosphatidyl choline, DMPC, distearoyl phosphatidylcholine, DLPC, dipalmitoyl phosphatidyl choline, 1-myristoyl-2-palmitoylphosphatidyl choline, 1-palmityl-2-myristoyl phosphatidylcholine, 1-palmityl-2-DSPC, 1-stearoyl-2-palmitoylphosphatidyl choline, DOPC, POPC, 1-oleoyl-2-palmitoylphosphatidyl choline, and its analog and derivant.

All phospholipid used herein can be from any source, i.e. (if suitable), from Semen sojae atricolor, breast, egg or even the internal of animal except the people, they can be from natural origin, or semi-synthetic source or even fully synthetic source.

The polymer that " polypeptide " is comprised of the aminoacid that connects by peptide bond is no matter it is natural generation or synthetic the generation.The polypeptide that is less than about 20 amino acid residues can be called " peptide " and will be called " albumen " by the molecule that two above polypeptide form or comprise 100 polypeptide more than the amino acid residue.Polypeptide can also comprise non-aminoacid ingredient, such as carbohydrate group, and metal ion, or carboxylate.Non-aminoacid ingredient can be added by the cell of express polypeptide, and can change with cell type.Polypeptide is limited by their amino acid backbone structure or its nucleic acid of encoding in this article.Usually do not specify the interpolation such as carbohydrate, but it can exist.

Term " POPC " refers to phosphatidase 11-palmityl-2-oleoyl-sn-glycerol-3-phosphatidylcholine, is also referred to as 1-palmityl-2-oleoyl-phosphatidylcholine.

Term " fast " refers to the process finished in 10 hours at the most.Rapid dilution is wherein in the process that the first solution is added in 10 hours at the most in the second solution.In one embodiment, this process is finished in 5 hours at the most, in another embodiment, finishes in 2 hours at the most.

Term " does not substantially contain " solution that refers to comprise albumen and one or more lipids and comprises and be less than 5% (w/w) lipid granule, is less than 2.5% lipid granule, is less than 1% lipid granule or is less than 0.5% lipid granule.

Term " variant " also comprises variant or the apolipoprotein analogies of the apolipoprotein that this paper reports, wherein in variant, the aminoacid sequence of each apolipoprotein or apolipoprotein analogies comprises one or more amino acid replacements, interpolation or disappearance.Modification can increase or reduce apolipoprotein for the affinity of apolipoprotein receptor or apolipoprotein invertase, or can compare with each carrier protein the stability that increases the apolipoprotein variant, or can compare with corresponding apolipoprotein and increase the dissolubility of apolipoprotein variant in aqueous solution, or can compare with each apolipoprotein increase the apolipoprotein variant in host cell/produced by the restructuring of host cell.

This paper report

Have been found that lipid granule can form by using the synthetic detergent as sole cleaning agent.

Use has at least, and the synthetic detergent of the CMC of 5mM is favourable.

The polypeptide incubation step (repairing such as 2-60 hour) that surpasses 1 hour in the situation of synthetic detergent can be had.

Can be in the concentration of 0.5x CMC, namely half of micelle formation needed concentration detects the formation of lipid granule.Therefore, form lipid granule and only need a small amount of detergent.This risk with adverse events when using this lipid granule in advantage such as the body reduces, because it is less to form the detergent concentration of described lipid granule needs.

In addition, with by rapid dilution in the solution that comprises detergent and at least a lipid, directly from comprising Denatured protein but do not comprise detergent and do not comprise the method combination that produces the raising of lipid granule the solution of lipid, it is more favourable using synthetic detergent.

Produce the method for lipid granule

What this paper reported is the method for producing lipid granule, and wherein lipid granule forms when having synthetic detergent.Have been found that lipid granule can form when only having synthetic detergent, wherein synthetic detergent has at least CMC of 5mM.Use synthetic detergent, composition and the permission for example having avoided using animal origin form lipid granule under the detergent of low concentration.

An aspect of this paper report is the method for producing the lipid granule that comprises polypeptide and lipid, and wherein said lipid granule forms when having synthetic detergent.

In one embodiment, described synthetic detergent has at least CMC of 10mM.In another embodiment, described synthetic detergent has at least CMC of 35mM.

In one embodiment, described synthetic detergent allows to form lipid granule at the synthesis of detergent agent concentration of 0.5x CMC.

In one embodiment, polypeptide and synthetic detergent incubation are about 2 hours to about 60 hours.

In one embodiment, described synthetic detergent is ampholytic detergent.In another embodiment, described ampholytic detergent is ampholytic detergent 3-8 or ampholytic detergent 3-10.

In one embodiment, described method comprises the steps:

From the solution that comprises polypeptide, at least a lipid and synthetic detergent, remove detergent and form thus lipid granule.

In one embodiment, described method comprises the steps:

I) incubation comprises the solution about 2 hours to about 60 hours of polypeptide, at least a lipid and synthetic detergent, and

Ii) from solution, remove detergent and also form thus lipid granule.

In one embodiment, described method comprises the steps:

I) provide the first solution that comprises the degeneration apolipoprotein,

Ii) the first solution is added comprise at least a lipid and synthetic detergent but in the second solution without apolipoprotein, and

Iii) from step I i) remove detergent and form thus lipid granule in the solution that obtains.

In one embodiment, described method comprises the steps:

I) provide the solution that comprises natural polypeptides,

Ii) at least a lipid and synthetic detergent are added i) solution in, and

Iii) from step I i) the solution removal detergent that obtains and form thus lipid granule.

In another embodiment, described method comprises the steps:

I) provide the solution that comprises natural apolipoprotein,

Ii) at least a lipid and synthetic detergent are added i) solution in, and

Iii) from step I i) the solution removal detergent that obtains and form thus lipid granule.

In one embodiment, described the second solution comprises at least two kinds of different lipids, and described lipid is independently from each other phospholipid, fatty acid and steroid lipid.In another embodiment, described at least two kinds of different lipids are two kinds of different phosphatidylcholines.In another embodiment, described the first phosphatidylcholine is POPC, and the second phosphatidylcholine is DPPC.

Reported that many polypeptide that produce from natural existence or restructuring (as for example from apolipoprotein A-1 or defat matter (delipidated) apolipoprotein A-1 of people HDL granule) produce the distinct methods of lipid granule.Wherein, for example with the apolipoprotein A-1 incubation phospholipid (such as palmityl-2-oleoyl-sn-glycerol-3-phosphatidylcholine) of purification and the aqueous mixture of detergent (such as sodium cholate), wherein use apolipoprotein A-1 with non-denatured form.After forming lipid granule by dialysis or diafiltration, remove detergent.

Have been found that for forming lipid granule, can use to have at least synthetic detergent of the CMC of 5mM.Use such synthetic detergent, need the detergent of low concentration to form lipid granule on the one hand, on the other hand, obtain the more product of homogeneous, namely have the still less product of side-product.Use has the synthetic detergent of lower CMC, as for example having the ampholytic detergent 3-12 of 2.8mM CMC, needs the synthetic detergent of higher concentration to form lipid granule, says nothing of and has also formed more inhomogenous product (seeing Figure 19).

Synthetic detergent is such detergent, it is neither naturally occurring, neither separate from natural origin the detergent of the synthetic generation that neither exist at nature.Therefore, synthetic detergent is designed by the people fully.Example with synthetic detergent of the above CMC of 5mM is ampholytic detergent 3-8 (n-octyl-N, N-dimethyl-3-ammonium-1-propane sulfonic acid ester; CMC=330mM-390mM), ampholytic detergent 3-10 (positive decyl-N, N-dimethyl-3-ammonium-1-propane sulfonic acid ester; CMC=25mM-40mM), Fos-choline-10 (CMC=11mM), CHAPS (3-[(3-chloro amido propyl group) dimethylammonio]-1-propane sulfonic acid ester; CMC=8mM), CHAPSO (3-[(3-chloro amido propyl group) dimethylammonio]-2-hydroxyl-1-propane sulfonic acid ester; CMC=8mM), n-octyl-β-D-Fructus Hordei Germinatus pyranoside (CMC=19mM).

The method of this paper report allows in one step the fully apolipoprotein A-1 of refolding and lipid degeneration.By using the method for this paper report, (i) can obtain to have the lipid granule of the product quality of raising, (ii) can save the time that the pretreatment apolipoprotein A-1 consumes, and (iii) large-scale processing of bio-pharmaceuticals production becomes possibility for the first time.

Forming the main points that process exploitation must consider for lipid granule is i) need biological activity and ii) relate to the specification requirement of the manufacturability of lipid granule.For example, comprise the lipid granule of apolipoprotein for formation, these requirements refer in the opposite direction.

From technological standpoint, saturated phospholipid (for example, two palmityls-sn-glycerol-3-phosphocholine, the DPPC of the carboxylic moiety with 16 following chains of carbon atom will be selected to comprise; Two myristoyls-sn-glycerol-3-phosphocholine, DMPC etc.).In contrast, can think unsaturated phospholipid (for example, palmityl-2-oleoyl-sn-glycerol-3-phosphocholine, the POPC that comprises the carboxylic moiety with at least 16 carbon atom chains from biological data; Stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine is more effective SOPC), and nontoxic to liver.

The selection decision of phospholipid combination comprises the effect regulating liver-QI safety of the lipid granule of apolipoprotein.In the body of the lipid granule that comprises DMPC that uses rabbit to carry out, in the research, have been found that the rabbit of processing with 30mg/kg shows serious side effect, but survive, then dead with the rabbit that 100mg/kg processes.The result clearly illustrates for the mobilization of cholesterol and therefore needs lipid (Figure 23) for the effect of molecule.

External functional test has confirmed that the lipid granule that comprises single phosphatidylcholine such as DPPC or POPC has activated LCAT.

Show that also Cholesterol Efflux is higher when lipid granule comprises the combination of different phospholipid.

Table 2: be used for the phospholipid combination of rabbit research preparation in the body, difference is that its lipid forms

These results are also by showing that data are confirmed in the body of mobilizing for the cholesterol of all combinations.Yet, for only comprising single phosphatidylcholine DPPC, or the lipid granule of the combination of DPPC and sphingomyelins (SM), can determine the increase (Fig. 1) of liver enzyme.

Therefore, the lipid granule that obtains by the method such as this paper report also is an aspect.

From technological standpoint, and form relatively with pure POPC, more convenient with pure DPPC formation lipid granule.Reduce the risk that precipitation forms by the combination of using different phospholipid.In addition, compare with the pure POPC with phase transition temperature of 4 ℃, 41 ℃ the phase transition temperature of pure DPPC is also so that the preparation lipid granule is more easy.And the product of acquisition is homogeneous more.This lipid granule analysis that also can be undertaken by SEC-MALLS confirms that described SEC-MALLS is the analytical tool (albumen-conjugate analysis) that allows to measure albumen-lipid composition.In Fig. 2, shown the chromatogram of the sample of resolving with size exclusion chromatography (UV280 detection).Can observe by the appearance at peak a plurality of separation or that partly dissociate the inhomogeneity of sample.

When using pure POPC to prepare lipid granule, the POPC molecule number of each apolipoprotein monomer is 40-85 in one embodiment in lipid granule, in one embodiment, is 50-80, in one embodiment, is 54-75.

When using pure DPPC to prepare lipid granule, the DPPC molecule number of each apolipoprotein monomer in one embodiment, is 50-150 in lipid granule, in one embodiment, be 65-135, in one embodiment, be 76-123, and in one embodiment, be 86-102.

When being that the mixture of 1: 3 POPC and DPPC is during for the preparation of lipid granule with mol ratio, the phospholipid molecule number of each apolipoprotein monomer in lipid granule, in one embodiment, about 50-about 120, in one embodiment, be that about 65-is about 105, and in one embodiment, be about 72-about 96.

When being that the mixture of 1: 1 POPC and DPPC is during for the preparation of lipid granule with mol ratio, the lipid molecular number of each apolipoprotein monomer in one embodiment in lipid granule, 50-120, in one embodiment, 60-100, in one embodiment, be 71 to 92, and be 71-85 in one embodiment.

When with mol ratio being the mixture of 3: 1 POPC and DPPC during for the preparation of lipid granule, the lipid molecular number of each apolipoprotein monomer in lipid granule in one embodiment, is 50-105.

When with mol ratio being the mixture of 3: 1 POPC and DPPC during for the preparation of lipid granule, the lipid molecular number of each apolipoprotein monomer in lipid granule in one embodiment, is 60-95.

When with mol ratio being the mixture of 3: 1 POPC and DPPC during for the preparation of lipid granule, the lipid molecular number of each apolipoprotein monomer in lipid granule in one embodiment, is 60-90.

When with mol ratio being the mixture of 3: 1 POPC and DPPC during for the preparation of lipid granule, the lipid molecular number of each apolipoprotein monomer is 60-88 in one embodiment in lipid granule.

When with mol ratio being the mixture of 3: 1 POPC and DPPC during for the preparation of lipid granule, the lipid molecular number of each apolipoprotein monomer in lipid granule in one embodiment, is 62-80.

When with mol ratio being the mixture of 3: 1 POPC and DPPC during for the preparation of lipid granule, the lipid molecular number of each apolipoprotein monomer in lipid granule in one embodiment, is 66-86.

When with mol ratio being the mixture of 3: 1 POPC and DPPC during for the preparation of lipid granule, the lipid molecular number of each apolipoprotein monomer in lipid granule in one embodiment, is 64-70.

When with mol ratio being the mixture of 3: 1 POPC and DPPC during for the preparation of lipid granule, the lipid molecular number of each apolipoprotein monomer in lipid granule in one embodiment, is about 66.

Comprise the lipid granule of apolipoprotein and POPC for preparation, in one embodiment, use 1: 40-1: 100 apolipoprotein and the mol ratio of POPC, in one embodiment, use 1: 40-1: 80 mol ratio, in one embodiment, use about 1: 60 mol ratio.

Comprise the lipid granule of apolipoprotein and DPPC for preparation, in one embodiment, use 1: 70-1: 100 apolipoprotein and the mol ratio of DPPC, in one embodiment, use 1: 80-1: 90 mol ratio, in one embodiment, use about 1: 80 mol ratio.

The lipid granule that comprises apolipoprotein, POPC and DPPC for preparation, in one embodiment, use 1: 60-1: the mol ratio of 100 apolipoprotein and (POPC and DPPC) (mol ratio of POPC and DPPC 1: 3), in one embodiment, use 1: 70-1: 90 mol ratio, and in one embodiment, use about 1: 80 mol ratio.

The lipid granule that comprises apolipoprotein, DPPC and POPC for preparation, in one embodiment, use 1: 60-1: the mol ratio of 100 apolipoprotein and (POPC and DPPC) (mol ratio of POPC and DPPC 1: 1), in one embodiment, use 1: 60-1: 80 mol ratio, and in one embodiment, use about 1: 70 mol ratio.

The lipid granule that comprises apolipoprotein, DPPC and POPC for preparation, in one embodiment, use 1: 60-1: the mol ratio of 100 apolipoprotein and (POPC and DPPC) (mol ratio of POPC and DPPC 3: 1), in one embodiment, use 1: 50-1: 70 mol ratio, and in one embodiment, use about 1: 60 mol ratio.

In one embodiment, if prepare lipid granule with the mixture of lipid, mixture has 4 ℃-45 ℃ phase transition temperature, in one embodiment, have 10 ℃-38 ℃ phase transition temperature, and in one embodiment, have 15 ℃-35 ℃ phase transition temperature.

In one embodiment, apolipoprotein and phospholipid in solution with about 1: about 1: 100 apolipoprotein of 50-: the mol ratio of phospholipid exists.In one embodiment, mol ratio is about 1: 50-1: 80.

Comprise the lipid granule of apolipoprotein for formation, known distinct methods is such as the subsequently dialysis of lyophilizing, freeze thawing, detergent dissolution, microjet, supersound process and homogenization.

Lipid granule comprises, in one embodiment, and average 1-10 apolipoprotein molecule/lipid granule, in one embodiment, average 1-8 apolipoprotein molecule/lipid granule, in one embodiment, average 1-4 apolipoprotein molecule/lipid granule.

In one embodiment, lipid granule comprises average number and is at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 apolipoprotein molecule/lipid granules.In one embodiment, average is 1.

In one embodiment, except apolipoprotein, lipid granule comprises one or more other polypeptide.

Without restriction, lipid granule can serve as enzymatic cofactor and/or carrier and be used for absorbing lipid, especially cholesterol.

Except synthetic detergent, in the lipid granule such as this paper report, can also there be one or more detergents.Described detergent can be any detergent, i.e. the medicinal detergent of non-toxic concentration or other detergent are such as nonionic or ionic detergent.Nonionic detergent can be the alkylene oxide derivative that comprises the organic compound of one or more hydroxyls.In one embodiment, nonionic detergent is selected from ethoxylation and/or propylated alcohol or ester compounds or its mixture.In another embodiment, ester is selected from the ester of Sorbitol and fatty acid, as dehydrated sorbitol mono-fatty acid ester or, Arlacel-40, oiliness sucrose ester, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene sterin ether, polyoxyethylene-poly-the third oxyalkyl ether, block polymer and cethyl ether (cethyl ether), polyoxyethylene castor oil or castor oil hydrogenated derivant and polyglycine fatty acid ester.In one embodiment, non-ionic detergent is selected from

Or

Ionic detergent can be the bile duct agent.In one embodiment, described ionic detergent is selected from cholic acid or deoxycholic acid, or their salt and derivant, or is selected from free fatty, such as oleic acid, linoleic acid and other.

In one embodiment, ionic detergent is selected from cation lipid such as C ₁₀-C ₂₄Alkylamine or alkanolamine and cation cholesteryl ester.In one embodiment, detergent is the detergent with high CMC.In another embodiment, detergent is to have at least detergent of 5mM CMC.

In one embodiment, lipid granule comprises and is less than 0.75 % by weight detergent.

In one embodiment, lipid granule comprises and is less than 0.30 % by weight detergent.

In one embodiment, lipid granule comprises and is less than 0.1 % by weight detergent.

In one embodiment, lipid granule comprises and is less than 0.05 % by weight detergent.

In one embodiment, in the method according to the invention, the first solution does not contain lipid granule substantially.

In one embodiment, described method is at step I i) afterwards at step I ii) before, comprise the steps iia) incubation is at step I i) in the solution that obtains.In one embodiment, incubation carried out about 12 hours to about 20 hours.In another embodiment, incubation carried out about 16 hours.

In one embodiment, incubation and/or removal are carried out 4 ℃-45 ℃ temperature.

In one embodiment, remove by diafiltration or dialysis.

In one embodiment, the first solution has the first volume, the second solution has the second volume, polypeptide in the first solution, have the concentration of restriction such as apolipoprotein, the lipid in the second solution and detergent have respectively the concentration of restriction, wherein at step I i) in, the concentration of the concentration of apolipoprotein, the concentration of lipid and detergent is changed/reduces, and allows to form lipid granule.

In one embodiment, described method comprises the steps:

Iv) the purification lipid granule also produces lipid granule thus.

Can form different lipid granules with the method such as this paper report.

For example, in order to produce the lipid granule that comprises apolipoprotein, can select to comprise saturated phospholipid (for example, two palmityls-sn-glycerol-3-phosphocholine, the DPPC of the carboxylic moiety with 16 atoms and shorter chain from technological standpoint; Two myristoyl-sn-glycerol-3-phosphocholine, DMPC etc.).From angle biology, in contrast, it is contemplated that unsaturated phospholipid (palmityl-2-oleoyl-sn-glycerol-3-phosphocholine for example, the POPC of the carboxylic moiety that comprises the chain with at least 16 C atoms; Stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine, SOPC) be more effective and right and wrong-hepatotoxic.

Can use phosphatidylcholine DPPC and POPC and composition thereof to be used to form the lipid granule that comprises apolipoprotein.These exemplary phosphatidylcholines are different and have an identical esterified carboxylic moiety of phosphoglycerol main chain that is carboxylic moiety.When using DPPC, the preparation lipid granule is more easy.On the contrary, POPC is more effective in external functional examination, and particularly as the substrate that activates lecithin cholesterol Acetylase (LCAT), it is necessary that described enzyme is converted into cholesteryl ester for the cholesterol of will mobilize.Have been found that with the lipid granule that only comprises a kind of phosphatidylcholine and compare, comprise two kinds of phosphatidylcholines with different mol ratio, for example the lipid granule of the mixture of POPC and DPPC has the character (seeing, for example Fig. 4) of raising.

For example, lipid granule can only comprise POPC.When with 1: 40 until the apolipoprotein of 1: 80 mol ratio: lipid is during for generation of lipid granule, and the POPC molecule number of each apolipoprotein monomer can change between 54-75.In one embodiment, the mol ratio of apolipoprotein and POPC is 1: 40-1: 80, and in one embodiment, mol ratio is 1: 50-1: 70, in one embodiment, mol ratio is about 1: 60.

Therefore, comprise the lipid granule of apolipoprotein and POPC for generation, the mol ratio of apolipoprotein and POPC is from 1: 40 to 1: 100, and in one embodiment, mol ratio is 1: 40 to 1: 80, and in one embodiment, mol ratio is about 1: 60.

For example, lipid granule can only comprise DPPC.When with 1: 40 until the apolipoprotein of 1: 80 mol ratio: lipid is during for generation of lipid granule, and the DPPC molecule number of each apolipoprotein monomer can change between 76-123.In one embodiment, the mol ratio of apolipoprotein and DPPC is 1: 70-1: 100, and in one embodiment, mol ratio is 1: 75-1: 90, in one embodiment, mol ratio is about 1: 80.

For example, can be that 1: 3 POPC and the mixture of DPPC begin to prepare lipid granule from mol ratio.When with 1: 60 until the apolipoprotein of 1: 100 mol ratio: lipid is during for generation of lipid granule, and the phospholipid molecule number of each apolipoprotein monomer can change between 72-112.In one embodiment, apolipoprotein is 1 with the mol ratio of (POPC and DPPC): 70-1: 90, and in one embodiment, mol ratio is 1: 75-1: 85, in one embodiment, mol ratio is about 1: 80.

Therefore, comprise apolipoprotein for generation, the lipid granule of POPC and DPPC, the mol ratio of apolipoprotein and (POPC and DPPC) (POPC and DPPC mol ratio are 1: 3) in one embodiment, 1: 60-1: 100, in one embodiment, mol ratio is 1: 70-1: 90, and in one embodiment, mol ratio is about 1: 80.

For example, can be that 1: 1 POPC and the mixture of DPPC begin to prepare lipid granule from mol ratio.When with 1: 60 until the apolipoprotein of 1: 100 mol ratio: lipid is during for the preparation of lipid granule, and the phospholipid molecule number of each apolipoprotein monomer can change between 71-111.In one embodiment, apolipoprotein is 1 with the mol ratio of (POPC and DPPC): 60-1: 80, and in one embodiment, mol ratio is 1: 65-1: 75, in one embodiment, mol ratio is about 1: 70.

Therefore, comprise apolipoprotein for generation, the lipid granule of DPPC and POPC, the mol ratio of apolipoprotein and (POPC and DPPC) (POPC and DPPC mol ratio are 1: 1) in one embodiment, 1: 60-1: 100, in one embodiment, mol ratio is 1: 60-1: 80, and in one embodiment, mol ratio is about 1: 70.

For example, can be that 3: 1 POPC and the mixture of DPPC begin to prepare lipid granule from mol ratio.When with 1: 60 until the apolipoprotein of 1: 100 mol ratio: lipid is during for the preparation of lipid granule, and the phospholipid molecule number of each apolipoprotein monomer can change between 46-93.In one embodiment, apolipoprotein is 1 with the mol ratio of (POPC and DPPC): 50-1: 70, and in one embodiment, mol ratio is 1: 55-1: 65, in one embodiment, mol ratio is about 1: 60.

Therefore, comprise apolipoprotein for generation, the lipid granule of DPPC and POPC, the mol ratio of apolipoprotein and (POPC and DPPC) (wherein POPC and DPPC mol ratio are 3: 1) in one embodiment, 1: 60-1: 100, in one embodiment, mol ratio is 1: 50-1: 70, and in one embodiment, mol ratio is about 1: 60.

In one embodiment, apolipoprotein provides as the aqueous solution of apolipoprotein and can be available from Downstream processing after the apolipoprotein production in recombinant production or any other source, and can comprise the different apolipoprotein of variable concentrations purity.

Basically, the formation of lipid granule is by realizing with the lipid of detergent dissolution polypeptide at their transition temperature incubations separately.Remove detergent by dialysis and cause forming of the lipid granule that formed by lipid bilayer.

For example, lipid granule forms by four company's albumen-apolipoprotein A-1s or its polymer are realized at their transition temperature incubations separately with the lipid of detergent dissolution.Remove the formation that detergent causes lipid granule by dialysis, described lipid granule is comprised of the lipid bilayer that is centered on by the alpha-helix apolipoprotein.

Lipid granule can carry out purification by the combination of precipitation and/or chromatographic step.For example, excessive detergent namely is not that the detergent of the part of lipid granule can be removed in hydrophobic adsorption chromatography step.In one embodiment, the step of the method for purification lipid granule comprises hydrophobic adsorption chromatography step.In another embodiment, the chromatographic material for hydrophobic adsorption step is selected from Extracti Gel D (available from Pierce Biotechnology, Rockford IL, USA), CALBIOSORB ^TM(available from Calbiochem, San Diego, CA, USA), SDR30HyperD ^TMSolvent-detergent is removed chromatographic resin (available from PALL Corporation, Ann Arbor, MI, USA).Lipid granule reclaims from hydrophobic adsorbing material with the solution that does not contain detergent.This chromatographic step especially is suitable for having the detergent of low CMC.

In one embodiment, use dialysis to remove the detergent with high CMC.

Medicine and diagnosis composition:

The lipid granule that obtains by the method such as this paper report can be used for the treatment of and/or diagnose the illness or disease.

Can with connect such as four of this paper report albumen-apolipoprotein A-1s or as the lipid granule of this paper report be used for the treatment of and/or diagnose by abnormal lipid level or lipid disease or the disease in the deposition sign of body part (such as the speckle in the blood vessel).

For the protein determining to obtain-lipid complex is supported the ability of the cholesterol esterification of LCAT catalysis, by quick interpolation cholesterol alcoholic solution cholesterol is attached in the lipid granule such as this paper report.Compare with the complex of DPPC outside comprising the apolipoprotein composition that is independent of them (connecting albumen-apolipoprotein A-1s such as wild type apolipoprotein A-1 or four), the lipid granule that comprises pure POPC is better LCAT substrate (Fig. 3).

The demonstration of the starting velocity of cholesterol esterification is compared with any pure phosphatidylcholine in the lipid granule that comprises different POPC and DPPC mixture, mixture is better LCAT substrate, such as (see Table 3 and Fig. 4) that can observe from the starting velocity of cholesterol esterification.

Table 3: the starting velocity of the cholesterol esterification in the lipid granule of the different mixtures that comprises phospholipid

To be exposed to cholesterol receptor test compounds by macrophage such as the people THP1 cell that THP-1 monocytic leukemia cell is exposed to the radiolabeled cholesterol tracer acquisition of phorbol-12-myristinate-13-acetas (phorbol myristate acetate) and load.

The rate of outflow of inducing by the receptor test compounds can be calculated as radioactivity in cholesterol radioactivity in supernatant and the cell add their supernatant the radioactivity summation ratio and compare with the cell that is exposed to the medium that does not comprise receptor, and by the linear fit analysis.Can use the cell that exposes and be not exposed to the RXR-LXR agonist to carry out parallel laboratory test, the known main rise ABCA-1 of described RXR-LXR agonist and deflection are to the outflow of the transportation of ABCA-1 mediation.

In not using the pretreated cell of RXR-LXR lipid granule, relatively can observe the higher increase in Cholesterol Efflux with the outflow that connects albumen-apolipoprotein A-1 acquisition with non-lipidization four.Can in test series, observe the only on a small quantity impact (Fig. 5) of lipid mixture on flowing out.With in the RXR-LXR pretreated cell, can observe the four comparable increases of Cholesterol Efflux that connect the lipid granule of albumen-apolipoprotein A-1s that comprising non-lipid.With the comparison of arriving with not pretreated cell observation, total increase is higher.In test series, only observe the less impact (Fig. 6) of lipid mixture on flowing out.

The different lipid granule of body build-in test in rabbit.Lipid granule is used as intravenous infusion, and after using, in 96 hours, carried out serial blood sampling.Measure the value of liver enzyme, cholesterol and cholesteryl ester.For all test lipid granules that comprises initial distribution phase, plasma concentration is suitable, subsequently plasma concentration log-linear decline (Fig. 7).As observing from table 4, for all test compounds, pharmacokinetic parameter is similar.The half-life of observing was close to 1.5 days.

Table 4: the pharmacokinetic parameter of mensuration

As can from Fig. 8, observing, in blood plasma, mobilize and esterified cholesterol.Even after four concentration that connect albumen-apolipoprotein A-1 had begun to reduce, plasma cholesterol ester level continued to increase really.Reduce to about 0.5mg/ml (about 50% normal wild type apolipoprotein A-1) when blood plasma four connects albumen-apolipoprotein A-1 level, still can detect the cholesteryl ester level of increase.

Comprise four lipid granules that connect albumen-apolipoprotein A-1s and do not induce liver enzyme in rabbit and the mice, as can from Fig. 1 and 9, observing.In addition, can determine not have haemolysis (Figure 10) in the plasma sample that two hours obtain after intravenous is used.

Therefore, aspect of the present invention is pharmaceutical composition and diagnosis composition, and it comprises such as the lipid granule that contains apolipoprotein of this paper report or comprises four company's albumen-apolipoprotein A-1s of reporting such as this paper.

As in following table 5, showing, with the apolipoprotein of non-lipid and other lipid granule relatively, have character in the body of raising such as the lipid granule of this paper report.

Table 5: character in the body of different apolipoproteins and lipid granule

Can determine that cholesterol mobilizes the efficient in the blood by using in vivo behind the apolipoprotein the relatively corresponding discharge (respective excursion) of T-CHOL and carrier protein concentration.For qualitative assessment, calculate the merchant of the area under the baseline correction area (AUC) under the concentration-time curve of T-CHOL and the concentration-time curve at apolipoprotein.

Such as the lipid granule of this paper report, comprise especially that four of SEQ ID NO:01 connects albumen-apolipoprotein and mol ratio is 3: 1 POPC and the lipid granule of DPPC, the cholesterol that increases in the display body is mobilized.

Four connect albumen-apolipoprotein A-1

Except the lipid granule of above-mentioned summary, this paper reports that also four connect albumen-apolipoprotein A-1.

Four company's albumen-apolipoprotein A-1s are fusion rotein that people four connects albumen trimerizing structural detail and wild type human apolipoprotein A-Ⅰ.The aminoacid sequence that people four connects protein part can shorten 9 aminoacid in front, from the isoleucine residue (naturally occurring truncate site) of position 10.As the consequence of this truncate, lacked in the position O-glycosylation site of 4 threonine residues.Connect between albumen trimerizing structural detail and the human apolipoprotein A-Ⅰ four, removed five amino acid residue " SLKGS " (SEQ ID NO:03).

Comprise N-end purification label in order to improve expression and purification, can to produce, for example six histidine-label and IgA protease cut the construct in site.As the result of specificity cutting, keep two aminoacid-as the first alanine or glycine or serine or proline with as the second proline at the four N-ends that connect albumen-apolipoprotein A-1s.Four connect the aminoacid sequence that albumen-apolipoprotein A-1 can have SEQ ID NO:01.

Four connect albumen trimerizing structural detail provides four of permission formation trimerizing to connect the polymeric domain of albumen-apolipoprotein A-1, and described polymer is by consisting of at each individual four noncovalent interaction that connect between albumen-apolipoprotein A-1 monomer.

By using alternative purification process, can save purification-label and IgA protease cutting site, forming aminoacid sequence is that four of SEQ ID NO:02 connects albumen-apolipoprotein A-1.

In one embodiment, apolipoprotein can be variant or the Apolipoprotein a-i mimics that comprises conservative amino acid replacement.

Apolipoprotein A-1 can be by enzymatic, by NMR spectroscopy or by determining with monoclonal or Anti-TNF-α-apolipoprotein-A-I antibody.Therefore, other aspects of this paper report are polyclone and monoclonal antibody, and four of its specific binding such as this paper report connects albumen-apolipoprotein A-1.Can obtain described antibody with method known to those skilled in the art.In addition, can carry out labelling for the antibody of immunoassay with method known to those skilled in the art.

In one embodiment, apolipoprotein can be the variant that comprises conservative amino acid replacement, or Apolipoprotein a-i mimics.In one embodiment, four connect the aminoacid sequence that albumen-apolipoprotein A-1 has SEQ ID NO:02 or SEQ ID NO:66 or SEQ ID NO:67, and wherein X is selected from SEQ ID NO:68-SEQ ID NO:105.

Therefore, in one embodiment, four company's albumen-apolipoprotein A-1s have following amino acid sequence IVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVDEPPQSPWDRVKDLAT VYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQEFW DNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQE GARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKEN GGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKL NTQ (SEQ ID NO:02).

In one embodiment, four connect albumen-apolipoprotein A-1 has following aminoacid sequence (A, G, S, T) PIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVDEPPQSPWDRVKDLA TVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQEF WDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQ EGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKE NGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKK LNTQ (SEQ ID NO:66).

In one embodiment, four connect albumen-apolipoprotein A-1 has following aminoacid sequence (M) HHHHHHXIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVDEPPQSPWD RVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLG PVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEP LRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAAR LEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSAL EEYTKKLNTQ (SEQ ID NO:67), wherein X can be any of following aminoacid sequence: A, G, S, P, AP, GP, SP, PP, GSAP (SEQ ID NO:68), GSGP (SEQ ID NO:69), GSSP (SEQ ID NO:70), GSPP (SEQ ID NO:71), GGGS (SEQ ID NO:72), GGGGS (SEQ ID NO:73), GGGSGGGS (SEQ ID NO:74), GGGGSGGGGS (SEQ ID NO:75), GGGSGGGSGGGS (SEQ ID NO:76), GGGGSGGGGSGGGGS (SEQ ID NO:77), GGGSAP (SEQ ID NO:78), GGGSGP (SEQ ID NO:79), GGGSSP (SEQ ID NO:80), GGGSPP (SEQ ID NO:81), GGGGSAP (SEQ ID NO:82), GGGGSGP (SEQ ID NO:83), GGGGSSP (SEQ ID NO:84), GGGGSPP (SEQ ID NO:85), GGGSGGGSAP (SEQ ID NO:86), GGGSGGGSGP (SEQ ID NO:87), GGGSGGGSSP (SEQ ID NO:88), GGGSGGGSPP (SEQ ID NO:89), GGGSGGGSGGGSAP (SEQ ID NO:90), GGGSGGGSGGGSGP (SEQ ID NO:91), GGGSGGGSGGGSSP (SEQ ID NO:92), GGGSGGGSGGGSPP (SEQ ID NO:93), GGGGSAP (SEQ ID NO:94), GGGGSGP (SEQ ID NO:95), GGGGSSP (SEQ ID NO:96), GGGGSPP (SEQ ID NO:97), GGGGSGGGGSAP (SEQ ID NO:98), GGGGSGGGGSGP (SEQ ID NO:99), GGGGSGGGGSSP (SEQ ID NO:100), GGGGSGGGGSPP (SEQ ID NO:101), GGGGSGGGGSGGGGSAP (SEQ ID NO:102), GGGGSGGGGSGGGGSGP (SEQ ID NO:103), GGGGSGGGGSGGGGSSP (SEQ ID NO:104), and GGGGSGGGGSGGGGSPP (SEQ ID NO:105).

If in coli strain, produce heterologous polypeptide, usually can not effectively cut with protease and remove amino-end methionine residues, the aminoterminal methionine residues partly is present in the polypeptide of generation thus.

Following embodiment is provided, and sequence table and Tu Lai assist and understand the present invention, the true scope that proposes in appended claims.Be appreciated that and without departing from the premise in the spirit of the present invention, described method modified.

Sequence table is described

Accompanying drawing is described

The result that Fig. 1 studies with the interior rabbit of body that five kinds of different lipid granules of its lipids composition carry out.Top: cholesterol is mobilized, and therefore can be for the standby batch demonstration effect of the ownership.Bottom: for the increase of noticing the liver enzyme by the lipid granule that uses DPPC to produce as single phospholipid.

Fig. 2 analyzes according to the SEC-MALLS of the lipid granule of POPC of the present invention and apolipoprotein; Mol ratio is 1: 20-1: 160.

Fig. 3 DPPC and POPC are on the impact of LCAT activity.

The starting velocity of the cholesterol esterification of Fig. 4 in the lipid granule that comprises POPC and/or DPPC.

The outflow of Fig. 5 foam cell that cholesterol is derived to THP-1 in the cell that excites with the RXR-LXR agonist.

Fig. 6 cholesterol after using the RXR-LXR agonist to activate the ABCA-I approach flows out to the foam cell that THP-1 derives.

The time dependence plasma concentration of the different apolipoprotein compositionss of Fig. 7.

Fig. 8 cholesterol is mobilized and time and the concentration process of esterification in blood plasma.

Fig. 9 injects the comparison that the liver enzyme of the different components that comprises apolipoprotein of the present invention behind the 100mg/kg. in mice discharges in single dose intravenous.

Spontaneous haemolysis in Figure 10 body in the rabbit research-blood plasma.

Figure 11 uses 250mM Tris-HCl, 140mM NaCl, the analysis SEC of the lipid granule that pH7.5. carries out.

Figure 12 uses 50mM K ₂HPO ₄, the analysis SEC of the lipid granule that 250mM arginine monohydrochloride, 7.5% trehalose carry out at pH7.5.

Figure 131: 20-1: the POPC of 320 mol ratios and four connects the non-degeneration PAGE (swimming lane 1: non-degeneration label of the lipid granule of albumen-apolipoprotein A-1; Swimming lane 2: mol ratio 1: 320; Swimming lane 3: mol ratio 1: 160; Swimming lane 4: mol ratio 1: 80; Swimming lane 5: mol ratio 1: 80 (f/t); Swimming lane 6: mol ratio 1: 40; Swimming lane 7: mol ratio 1: 20; Swimming lane 8: apolipoprotein (forming six aggressiveness)).

Figure 141: 20-1: the SEC-MALLS that the POPC of 160 mol ratios and four connects the lipid granule of albumen-apolipoprotein A-1 analyzes.

The even stack of the SEC chromatogram (UV280 signal) of the lipid granule of albumen-apolipoprotein A-1 of Figure 15 POPC and four.

Figure 16 mol ratio is the SEC-MALLS analysis that 1: 40 POPC and four connects the lipid granule of albumen-apolipoprotein A-1.

Figure 17 is with 1: 20-1: the DPPC that 100 mol ratios obtain and four connects the non-degeneration PAGE (1: the molecular weight marker thing of the lipid granule of albumen-apolipoprotein A-1; 2: do not contain four of lipid and connect albumen-apolipoprotein A-1; 3:1: 20; 4:1: 40; 5:1: 60; 6:1: 80; 7:1: 100).

The POPC that Figure 18 obtains in the mol ratio of 1: 60 (curve of going up most)-1: 100 (minimum curve): DPPC=3: 1 and four SEC-MALLS of lipid granule that connect the mixture of albumen-apolipoprotein A-1s analyze (UV280 signal)

Figure 19 uses cholate, ampholytic detergent 3-8, the non-degeneration PAGE SDS of the lipid granule of four company's albumen-apolipoprotein A-1s that 3-10 and 3-12 carry out.Swimming lane 1 on each gel: pure apolipoprotein; The sample of the swimming lane 2:0.1x CMC cholate lipid on each gel as a reference.

Figure 20 uses 3x CMC ampholytic detergent 3-8 and POPC (apolipoprotein: the four even SEC-MALLS protein conjugate analyses of the lipid granule of albumen-apolipoprotein A-1 of phospholipid mol ratio=1: 60) carrying out.

Figure 21 uses 2x CMC ampholytic detergent 3-10 and POPC (apolipoprotein: the four even SEC-MALLS protein conjugate analyses of the lipid granule of albumen-apolipoprotein A-1 of phospholipid mol ratio=1: 60) carrying out.

The four SEC-MALLS protein that connect the lipid granule of albumen-apolipoprotein A-1 that Figure 22 uses POPC to carry out are puted together analysis.Above: from the four natural lipid granules that company's albumen-apolipoprotein A-1 forms; Below: connect the lipid granule that albumen-apolipoprotein A-1 forms from four of degeneration.

Figure 23 connects the result (a) of rabbit research in the body that albumen-apolipoprotein A-1 carries out with four of DMPC (1: 100) (DMPC) lipid and is used among the PBS not four of lipid and connects in the body that albumen-apolipoprotein A-1 carries out rabbit and study (b) result.

Figure 24 comprises wild type apolipoprotein A-1 (A) and connects the SE-HPLC chromatogram of the lipid granule of albumen-apolipoprotein A-1s (B) such as four of this paper report 5 ℃ and 40 ℃ storages.

Materials and methods

Size exclusion-HPLC:

The Tosoh Haas TSK3000SWXL post that is used in the ASI-100HPLC system (Dionex, Idstein, Germany) carries out chromatography.Monitor eluting peak by UV diode array detector (Dionex) at 280nm.Behind 1mg/ml, use the buffer by 200mM potassium dihydrogen phosphate and 250mM potassium chloride pH7.0 to wash until obtain stable baseline on post in the sample dissolution that will concentrate.(isocratic condition) analyzes round under the condition of flow velocity at permanent solvent composition of room temperature use 0.5ml/min in 30 minutes.With chromatogram and Chromeleon (Dionex, Idstein, Germany) manual integration.Determine the polymerization that represents with % by the AUC than the area under curve (AUC) of higher molecular weight form and monomer peak value.

Dynamic light scattering (DLS):

DLS measures granular size, typically the Noninvasive technology in the sub-micron scope.In the present invention, will have the Zetasizer Nano S device (Malvern Instruments, Worcestershire, UK) of temperature control quartz cuvette (25 ℃) for the magnitude range between the monitoring 1nm-6 μ m.Detect the light intensity of backscatter laser 173 ° angles.Intensity is at the rate fluctuation that depends on the granule diffusion velocity, and described granule diffusion velocity is controlled by granular size again.The fluctuation of light intensity that therefore can be by analyzing scattering produces granular size data (Dahneke, B.E (ed.), Measurement of Suspended particles by Quasielectric Light Scattering (granule that suspends by intending electronic light scattering measurement), Wiley Inc. (1983); Pecora, R., Dynamic Light Scattering:Application of Photon Correlation Spectroscopy (dynamic light scattering of photon correlation spectroscope is used), Plenum Press (1985)).Use the size distribution of many narrow modes calculating strength of DTS software (Malvern).Test with undiluted sample.

SEC-MALLS：

SEC-MALLS is the combination of size exclusion chromatography and three detector systems: i) UV detects, and ii) refractive index detects and iii) scattering measuring.For by size separation, use the Superose6 post 10/300GL post from GEHealthcare.Described method is carried out with the permanent solvent composition of the PBS buffer ground of pH7.4, uses the flow velocity of 0.4ml/min.Connect three kinds of detector systems serially.By complete lipid granule (albumen-lipid granule) signal of refractive index detector monitoring, and at the definite signal of being induced by protein part of the UV absorbance of 280nm mensuration.The ratio of lipid fraction is by obtaining albumen UV signal simple deduction from complete signal.Apply light scattering and allow to detect the separately molecular weight of kind, and therefore can the detailed description lipid granule of complete sum.

Detergent is measured

Carry out the mensuration of remaining detergent by the reverse-phase chromatography (RP-ELSD) with the evaporative light scattering detector coupling.Use is from the Luna C184.6x150mm of Phenomenex (Aschaffenburg, Germany), 5 μ m,

As post.After centrifugal by the 10kDa film, the thing that flows through of 90 μ l is used for HPLC and separates.Under the condition of permanent solvent composition, under the condition of permanent solvent composition, carry out eluting with 74% (v/v) methanol solution that comprises 0.1% (v/v) trifluoroacetic acid.Column temperature is set in 30 ℃.Detect by evaporative light scattering detector, use 30 ℃ vapo(u)rizing temperature, 80 ℃ evaporating temperature and the air-flow of 1.0l/min.By being in the cholate situation of 0.22 μ g-7.5 μ g cholate in scope, the Criterion curve carries out the quantification of remaining detergent.

Protein determination:

Measure protein concentration by the optical density (OD) that is determined at 280nm, use the molar extinction coefficient that calculates based on aminoacid sequence.

Recombinant DNA technology:

The Application standard method operates DNA, as at Sambrook, and J., etc., Molecular cloning:A laboratory manual (molecular cloning experiment guide); Cold Spring Harbor Laboratory Press (cold spring harbor laboratory), the cold spring port, New York is described in 1989.Use molecular biology reagent according to manufacturer's description.

Embodiment 1

Preparation and description colibacillus expression plasmid

Prepare four by recombination form and connect albumen-apolipoprotein A-1 fused polypeptide.N holds the aminoacid sequence of expression fused polypeptide of C extreme direction as follows:

-amino acids methionine (M),

-have an interferon sequence fragment of the aminoacid sequence of CDLPQTHSL (SEQ ID NO:55),

-GS joint,

-have six histidine mark things of the aminoacid sequence of HHHHHH (SEQ ID NO:56)

-GS joint,

-have an IgA protease cutting site of the aminoacid sequence of VVAPPAP (SEQ ID NO:60), and

-have four of SEQ ID NO:02 aminoacid sequence to connect albumen-apolipoprotein A-1.

Aforesaid four company's albumen-apolipoprotein A-1 fused polypeptide are Precursor Peptides, and four connect albumen-apolipoprotein A-1 fused polypeptide by coming to discharge from described Precursor Peptide with the outer enzymatic cutting of IgA proteasome.

Precursor Peptide coding fusion gene assembles by be connected the nucleic acid fragment that is fit to technology with known recombination method.Nucleotide sequence by the chemosynthesis preparation confirms by dna sequencing.Be prepared as follows the expression plasmid that connects albumen-apolipoprotein A-1s for generation of four of the SEQ ID NO:01 of the fusion rotein of coding SEQ ID NO:31.

The preparation colibacillus expression plasmid

Plasmid 4980 (4980-pBRori-URA3-LACI-SAC) is the expression plasmid for the core-Succ-PEG-DSPE of expressing escherichia coli.It is by connecting from plasmid 1966 (1966-pBRori-URA3-LACI-T-repetitive sequence; In EP-B1422237, report) the long core-Succ-PEG-DSPE generation core-Succ-PEG-DSPE colibacillus expression plasmid of the long EcoRI/CelII-carrier segments of 3142bp and the coding EcoRI/CelII-fragment of 435bp comprise following element:

-the origin of replication from carrier pBR322 (corresponding to Sutcliffe, G. is etc., the described 2517-3160bp of Quant.Biol.43 (1979) 77-90 position) that is used for copying escherichia coli,

URA3 gene (the Rose of the saccharomyces cerevisiae of-coding orotidine 5 '-phosphate decarboxylase, M.et al.Gene29 (1984) 113-124), it allows to carry out plasmid by the complementation of escherichia coli pyrF mutants which had (uracil auxotrophy) and selects

-core-Succ-PEG-DSPE expression cassette comprises

-T5 hybrid promoter is (according to Bujard, H., Deng .Methods.Enzymol (Enzymology method) .155 (1987) 416-433 and Stueber, D., Deng, the described T5-PN25/03/04 hybrid promoter of Immunol.Methods (immunological method) IV (1990) 121-152), comprise according to Stueber, D. wait described synthetic ribosome binding site (seing above)

-core-Succ-PEG-DSPE gene,

The transcription terminator in-two phage-sources, λ-T0 terminator (Schwarz, E., etc., Nature272 (1978) 410-414) and fd-terminator (Beck E. and Zink, B.Gene1-3 (1981) 35-58),

-from colibacillary lacI repressor gene (Farabaugh, P.J., Nature274 (1978) 765-769).

By using unique side to connect EcoRI and CelII restricted enzyme cleavage site excises core-Succ-PEG-DSPE structural gene from carrier 4980, and the nucleic acid of EcoRII/CelII restriction site side neighbour's coding Precursor Peptide is inserted the long EcoRI/CelII-4980 carrier segments of 3142bp prepare and express the four final expression plasmids that connect albumen-apolipoprotein A-1 Precursor Peptides.

Embodiment 2

Express four and connect albumen-apolipoprotein A-1

For expressed fusion protein, use escherichia coli host/vector system, its complementation by escherichia coli auxotroph (PyrF) can be carried out the plasmid of antibiotic-free and select (EP0972838 and US6,291,245).

Carry out electroporation with expression plasmid p (IFN-His6-IgA-four connects albumen-apolipoprotein A-1) and transform e. coli k12 strain CSPZ-2 (leuB, proC, trpE, th-1, Δ pyrF).At first the Bacillus coli cells that transforms is cultivated at agar plate at 37 ℃.

Fermentation scheme 1:

About in advance fermentation, use according to Sambrook etc. (Molecular Cloning:A laboratory manual (and molecular cloning: laboratory manual). publishing house of cold spring harbor laboratory; The M9 culture medium of the 2nd edition (in December, 1989), it has replenished about 1g/l L-Leu, about 1g/l L-PROLINE and about 1mg/l thiamine-HCl.

About in advance fermentation, store 2ml in the ampoule with original inoculum and be seeded in the 300ml M9-culture medium in the 1000ml conical flask (Erlenmeyer-flask) with baffle plate.At 37 ℃, cultivate 13 hours at rotary shaker, until observe the optical density (578nm) of 1-3.

For fermentation, use according to the described batch culture bases such as Riesenberg (Riesenberg, D. is etc., J.Biotechnol.20 (1991) 17-27): 27.6g/l glucose * H ₂O, 13.3g/l KH ₂PO ₄, 4.0g/l (NH ₄) ₂HPO ₄, 1.7g/l citrate, 1.2g/l MgSO ₄* 7H ₂O, 60mg/l ferric citrate (III), 2.5mg/l CoCl ₂* 6H ₂O, 15mg/l MnCl ₂* 4H ₂O, 1.5mg/l CuCl ₂* 2H ₂O, 3mg/l H ₃BO ₃, 2.5mg/l Na ₂MoO ₄* 2H ₂O, 8mg/l Zn (CH ₃COO) ₂* 2H ₂O, 8.4mg/l Titriplex III, 1.3ml/l Synperonic 10% defoamer.Replenish 5.4mg/l thiamine-HCl and 1.2g/l L-Leu and L-PROLINE for respectively the batch culture base.Charging 1 solution comprises the 700g/l glucose, has replenished 19.7g/l MgSO ₄* 7H ₂O.The aqueous slkali that is used for pH regulator is 12.5% (w/v) NH that has replenished respectively 50g/l L-Leu and 50g/l L-PROLINE ₃Aqueous solution.All the components is dissolved in the deionized water.

Fermentation is carried out in 10l Biostat C DCU3 fermentor (Sartorius, Melsungen, Germany).Add 300ml with the fermentation batch culture base of 6.4l sterilization and begin from pre-fermented inoculum, at 37 ℃, pH6.9 ± 0.2, the Ventilation Rate of 500mbar and 10l/min carries out batch fermentation.After the glucose consumption that begins to replenish was complete, with thermoregulation to 28 ℃, fermentation entered feed supplement-batch mode.At this moment, agitator speed by adding the constant increase of charging 1 combination (in 10 hours 550rpm-1000rpm and in 16 hours from 1000rpm-1400rpm) and Ventilation Rate (in 10 hours from 10l/min-16l/min and in 5 hours from 16l/min-20l/min), the relative value of dissolved oxygen (pO2) is remained on 50% (DO-stat, see for example Shay, L.K., Deng, J.Indus.Microbiol.Biotechnol.2 (1987) 79-85).Add aqueous slkali when cultivating when pH reaches adjusting lower limit (6.70) after about 8 hours, cause supplying other aminoacid.Induce restructuring treatment protein expression by add 1mM IPTG in optical density 70.

When fermentation ends, before collecting, use heating steps (wherein the whole culture medium in fermentor be heated to 50 ℃ reach 1 or 2 hour), four of kytoplasm and solubility expression connects albumen-apolipoprotein A-1 and is transferred to insoluble protein aggregation body, so-called occlusion body (seeing, for example EP-B1486571).Subsequently, the content of fermentor is centrifugal and the Biomass of results is stored in-20 ℃ until further processing with concurrent centrifuge (13,000rpm, 13l/h).Only in insoluble cell debris fraction, find four synthetic company's albumen-apolipoprotein A-1 precursor proteins of insoluble protein aggregation bodily form formula (so-called occlusion body (IBs)).

Only in insoluble cell debris fraction, find the synthetic fusion rotein of insoluble protein aggregation bodily form formula (so-called occlusion body (IBs)).

The sample that from fermentor, reclaims with the SDS-Polyacrylamide Gel Electrophoresis, one is the sample before inducing, other be the sample of the time point of appointment after induced protein is expressed.From each sample, with the cell (OD of same amount _Target=5) Eddy diffusion destroys in 5mL PBS buffer and by the supersound process on ice.Then, with every kind of suspension of 100 μ L centrifugal (15,000rpm, 5 minutes), and reclaim each supernatant, it is transferred in the independent bottle.This is in order to distinguish solubility and insoluble expression target protein.Add 300 μ L and each precipitation (=soluble) fraction to each supernatant (=solubility) fraction and add the SDS sample buffer (Laemmli, U.K., Nature227 (1970) 680-685) of 400 μ L.In the situation of shaking, sample is heated 15 minutes to dissolve and reduction all albumen in sample at 95 ℃.Behind cool to room temperature, every kind of sample of 5 μ L is forwarded in the 4-20%TGX Criterion Stain Free polyacrylamide gel (Bio-Rad).In addition, with 5 μ l molecular weight standards (Precision Plus Protein Standard, Bio-Rad) and the quantitative criteria with 3 kinds of amounts (0.3 μ l, 0.6 μ l and 0.9 μ l) of known products protein concentration (0.1 μ g/ μ l) be arranged on the gel.

Move electrophoresis 60 minutes at 200V, and subsequently gel is forwarded to GelDOC EZ imager (Bio-Rad) and use UV treatment with irradiation 5 minutes.Use Image Lab analysis software (Bio-Rad) to analyze gel images.Use three standards, calculate the linear regression curve, coefficient＞0.99, and use it to calculate the concentration of the target protein in primary sample.

Fermentation scheme 2:

About in advance fermentation, use according to Sambrook etc. (Molecular Cloning:A laboratory manual (and molecular cloning: laboratory manual). publishing house of cold spring harbor laboratory; The M9 culture medium of the 2nd edition (in December, 1989), it has replenished about 1g/l L-Leu, about 1g/l L-PROLINE and about 1mg/l thiamine-HCl.

About in advance fermentation, improve in the M9-culture medium from agar plate or the 300ml that is seeded in the 1000ml conical flask with baffle plate with the 1-2ml the original inoculum storage ampoule.At 37 ℃, cultivate 13 hours at rotary shaker, until observe the optical density (578nm) of 1-3.

Connect albumen-apolipoprotein A-1 for fermentation and high yield expression four, use following batch cultivation base and charging:

8.85g/l glucose, 63.5g/l yeast extract, 2.2g/l NH ₄Cl, 1.94g/l L-Leu, 2.91g/l L-PROLINE, 0.74g/l METHIONINE, 17.3g/l KH ₂PO ₄* H2 _O, 2.02g/lMgSO ₄* 7H ₂O, 25.8mg/l thiamine-HCl, 1.0ml/l Synperonic10% defoamer.Charging 1 solution comprises 333g/l yeast extract and 333g/l85%-glycerol, has replenished respectively 1.67g/l METHIONINE and 5g/l L-Leu and L-PROLINE.Charging 2 is the solution of 600g/l L-PROLINE.The alkaline solution that is used for pH regulator is 10% (w/v) KOH solution, uses 75% glucose solution as acid.All the components is dissolved in the deionized water.

Fermentation is carried out in 10l Biostat C DCU3 fermentor (Sartorius, Melsungen, Germany).Add 300ml with the fermentation batch culture base of 5.15l sterilization and begin from pre-fermented inoculum, at 25 ℃, pH6.7 ± 0.2, the Ventilation Rate of 300mbar and 10l/min carries out batch fermentation.Before the glucose consumption that begins to replenish was complete, culture reached 15 optical density (578nm), and fermentation enters charging-batch-mode, and charging this moment 1 begins with 70g/h.Monitoring glucose concentration in culture is increased to maximum 150g/h with charging 1, avoids simultaneously the glucose accumulation and keep pH to approach regulating the upper limit 6.9.Optical density 50 (578nm), charging 2 begins with the constant feed rate of 10ml/h.By parallel increase agitator speed (500rpm-1500rpm), Ventilation Rate (10l/min-20l/min) and pressure (300mbar-500mbar) keep dissolved oxygen (pO ₂) relative value more than 50%.Induce restructuring treatment protein expression by the optical density adding 1mM IPTG 90.

Seven samples that from fermentor, reclaim with the SDS-Polyacrylamide Gel Electrophoresis, one is the sample before inducing, other be the sample of the time point of appointment after induced protein is expressed.From each sample, with the cell (OD of same amount _Target=5) Eddy diffusion destroys in 5mL PBS buffer and by the supersound process on ice.Then, with every kind of suspension of 100 μ L centrifugal (15,000rpm, 5 minutes), and reclaim each supernatant, it is transferred in the independent bottle.This is in order to distinguish solubility and insoluble expression target protein.Add 300 μ L and each precipitation (=soluble) fraction to each supernatant (=solubility) fraction and add the SDS sample buffer (Laemmli, U.K., Nature227 (1970) 680-685) of 200 μ L.In the situation of shaking, sample is heated 15 minutes to dissolve and reduction all albumen in sample at 95 ℃.Behind cool to room temperature, every kind of sample of 5 μ L is forwarded in the 10%Bis-Tris polyacrylamide gel (Novagen).In addition, with 5 μ l molecular weight standards (Precision Plus Protein Standard, Bio-Rad) and the quantitative criteria with 3 kinds of amounts (0.3 μ l, 0.6 μ l and 0.9 μ l) of known products protein concentration (0.1 μ g/ μ l) be arranged on the gel.

Electrophoresis 200V operation 35 minutes, is then dyeed to gel with Coomassie brilliant blue R dyestuff, fade and it is forwarded to the water that heats and carry out digitized (GS710, Bio-Rad) on the photodensitometer.Use Quantity One1-D analysis software (Bio-Rad) to analyze gel images.Use three standards, calculate the linear regression curve, coefficient＞0.98, and use it to calculate the concentration of the target protein in primary sample.

When fermentation ends, before collecting, use heating steps (wherein the whole culture medium in fermentor be heated to 50 ℃ reach 1 or 2 hour), four of kytoplasm and solubility expression connects albumen-apolipoprotein A-1 and is transferred to insoluble protein aggregation body, so-called occlusion body (seeing, for example EP-B1486571).Behind the heating steps, only in insoluble cell debris fraction, find four synthetic company's albumen-apolipoprotein A-1 precursor proteins of IBs form.

The content of fermentor is cooled to 4-8 ℃, uses concurrent centrifuge (13,000rpm, 13l/h) centrifugal, and the Biomass of results is stored in-20 ℃ until further processing.The construct that depends on expression, the Biomass productive rate scope of total results is at 39g/l and 90g/l dry.

Embodiment 3

Preparation four connects albumen-apolipoprotein A-1

By (0.1M replenishes 1mM MgSO at buffer solution of potassium phosphate or Tris buffer solution ₄, pH6.5) bacterial cell of middle resuspending collection carries out the occlusion body preparation.After adding DNAse, destroy cell by the pressure homogenate at 900bar.The buffer solution that will comprise 1.5M NaCl and 60mM EDTA adds the cell suspension of homogenate.After with 25% (w/v) HCl pH value being adjusted to 5.0, further obtaining final occlusion body serosity after the centrifugation step.Serosity is stored in-20 ℃, in the plastic bag of the sterilization that single uses until further processing.

Occlusion body serosity (about 15kg) is dissolved in the guanidine hydrochloride solution (150l, 6.7M).After by depth-type filtration clarification Solubilizates, solution is added in the affinity chromatograph material of Zn-chelating.By Zn-chelating chromatographic material purification fused polypeptide and by the cutting of IgA protease.Subsequently, be further purified polypeptide with anion-exchange chromatography and cation-exchange chromatography step.These steps are namely carried out under Denaturing at bag urea-containing solution (7M).These steps are used for removing polypeptide fragment, endotoxin and other impurity.Comprise the diafiltration in the solution of 6.7M guanidine hydrochloride.The final solution that obtains comprises four of degeneration and connects albumen-apolipoprotein A-1.

Embodiment 4

Four connect refolding and the lipid of albumen-apolipoprotein A-1

A) conventional method

Pure crystallization POPC or DPPC (Lipoid, Switzerland) are dissolved in the aqueous buffer solution (lipid buffer), and described buffer was with 1: 1.35 mol ratio phospholipid: cholate comprises cholate.With described mixture incubation and in room temperature (POPC) or 55 ℃ of (DPPC) lucifuges under nitrogen atmosphere, until obtain settled solution.Lipid-the cholate solution of clarification is cooled to 4 ℃ (POPC) or is stored in 41 ℃ (DPPC).The apolipoprotein that is limiting: the phospholipid ratio adds four of purification at 4 ℃ (POPC) or 41 ℃ (DPPC) and connects albumen-apolipoprotein A-1.Form for lipid granule, under nitrogen atmosphere, at 4 ℃ (POPC) or 41 ℃ (DPPC) and lucifuge reactant mixture is incubated overnight.Finally, remove cholate by a large amount of dialysis (4 ℃/41 ℃) for the lipid buffer.Finally, centrifugal sample removes the material of precipitation.

As above preparation comprises the lipid soln of the cholate dissolving of pure POPC or pure DPPC.By preparing lipid mixture at the ratio of needs combination lipid soln subsequently at T separately _m(T _m=phase transition temperature) stores.As described in for pure lipid soln, carry out, but at the separately T of the lipid mixture of selecting _mCarrying out four lipid granules that connect albumen-apolipoprotein A-1 forms.

After tested following lipid buffer:

1. at pH7.5, use the 250mM arginine hydrochloride, the 50mM kaliumphosphate buffer that 7.5% sucrose replenishes

2. at pH7.5, use the 250mM arginine hydrochloride, 7.5% sucrose, the 50mM dipotassium hydrogen phosphate buffer that the 10mM methionine replenishes

3. at pH7.5, use 140mM NaCl, the 250mM tris-hydroxyl amino methane (TRIS) that the 10mM methionine replenishes

4. at pH7.5, use 250mM arginine hydrochloride, 7% trehalose, the 50mM dipotassium hydrogen phosphate buffer that the 10mM methionine replenishes.

By analyzing the SEC assessment from the uniformity (Figure 11 and 12) of four company's albumen-lipid granules that the apolipoprotein A-1 sample forms.In a word, compare with the selection of phospholipid, the selection of lipid buffer only has small effect.The DPPC-lipid granule is as a main peak eluting, and the POPC-lipid granule shows two peak-mode.The selection of lipid buffer is subject to the impact of the supply of the purge process of apolipoprotein and stable apolipoprotein without lipid.Demonstration can irrespectively form lipid granule with the lipid buffer.In the buffer of various tests, optimal lipid buffer is accredited as 250mM Tris, 140mM NaCl, 10mM methionine, pH7.5.

The lipid mixture comprises respectively the apolipoprotein of limited amount, and corresponding calculating phospholipid, for example amount of POPC.The calculating of the mole of lipid all connects albumen-apolipoprotein A-1 monomer based on four.

B) POPC and cholate

Table 6: use pure POPC to connect albumen-apolipoprotein A-1 with four and form as an example lipid granule.Calculate the apolipoprotein of protein monomer: the mol ratio of phospholipid.Contrast: do not add lipid (pure Apo) incubation apolipoprotein and do not add apolipoprotein (without Apo) incubation lipid.

* in centrifugal rear clarification

In whole process, from 1: 40-1: 160 mol ratio keeps clarification.Do not observe by the muddiness of excessive phospholipid and do not observe albumen precipitation yet.

Analyze lipid granule sample (seeing Figure 13) by non-degeneration PAGE.Find the band pattern of homogeneous with sample 1: 80 (swimming lane 4).In addition, 1x freezes/melts the outward appearance (swimming lane 5) that (80 ℃) do not change sample yet.The band pattern of sample 1: 320 and 1: 160 shows inhomogeneous product, causes a plurality of bands ( swimming lane 2 and 3).Sample 1: 40 and have in addition other band (swimming lane 6 and 7) below the principal product band at 1: 20.The four pure migration models that connect albumen-apolipoprotein A-1 show in the swimming lane 8 of Figure 13.

SEC-MALLS analyzes and is used for acquisition about the more detailed information (albumen-conjugate analysis) of the homogeneity of lipid granule and their apolipoprotein-Lipid composition.Figure 14 shows the chromatogram (UV280 detection) of the sample that SEC differentiates.1: 160 sample is divided into three independent peaks here.1: 80 sample shows the kinds that comprise at least two different sizes, as as shown in bimodal.The peak value that obtains at 1: 20 from sample shows the most uniform product.

In 5 step, use four to connect albumen-apolipoprotein A-1 (3.84mg/ml; The 10mg/ sample) test, and apolipoprotein: the phospholipid mol ratio was increased to 1: 80 from 1: 40.In the mol ratio that is lower than 1: 40, it is incomplete that lipid granule forms.Got rid of by experiment greater than 1: 80 mol ratio, after removing cholate by dialysis, it is muddy that sample becomes.And in higher drugrlipid ratio, it is more inhomogeneous that lipid granule becomes.

Table 7: the lipid granule that uses four of pure POPC to connect albumen-apolipoprotein A-1 forms.Connect albumen-apolipoprotein A-1 monomer based on four and calculate apolipoprotein: the mol ratio of phospholipid.

* before dialysis and the dialysis after volume 2.6ml

* is in the SD of described method

In-3 ℃ transition temperature incubation process, all samples keep visual clarification.After removing cholate by dialysis, observe sample 1: 40-1: the turbidity of 65 increase.Can be by centrifugal removal precipitation and subsequently sample maintenance clarification.

SEC-MALLS analyzes and is used for acquisition about the details (albumen-conjugate analysis) of the homogeneity of the lipid granule of formation and their apolipoprotein-Lipid composition.All lipid granules are comparable ground homogeneous (SEC on the size exclusion chromatography analyzing; Figure 15), show little rear peak value, this is more significant in lower mol ratio.In addition, exist higher mol ratio to the more significant movement of high molecular in peak-mode.Separately retention time is provided in table 8.

Table 8: size exclusion chromatography result's summary: by the integral and calculating percentage ratio of area under curve (AUC)

Albumen-conjugate analysis (in table 8 sum up) can calculate the albumen (MW albumen) of every kind of lipid granule of eluting from the SEC post and the total molecular weight of lipid components (MW lipid).Based on the molecular weight of four company's albumen-apolipoprotein A-1 monomers (32.7kDa) and POPC (760Da), can calculate the composition (n albumen and n POPC) of lipid granule.Connect albumen-apolipoprotein A-1 trimer corresponding to four of each lipid granule, the molecular weight of the apolipoprotein composition of finding in the lipid granule main peak of all mol ratios is about 100kDa.The ratio of n (POPC)/n (protein monomer) is provided at the number of the POPC molecule of each four company's albumen-apolipoprotein A-1 monomer in the lipid granule.Although used from 1: 40 until 1: 80 mol ratio, each four number that connect the POPC molecule of albumen-apolipoprotein A-1 monomers changes between 54 and 75.The % protein value is the parameter of lipid degree.The percentage ratio of albumen in lipid granule is lower, and the lipid degree is higher.

Table 9: as showing in Figure 16, POPC and four connects the protein conjugate analysis of the lipid granule of albumen-apolipoprotein A-1 and sums up.

C) DPPC and cholate

Before lipid, with the 50mM KH of pH7.5 ₂PO ₄, the 250mM arginine monohydrochloride, 7% trehalose, 10mM methionine dialysis four connects albumen-apolipoprotein A-1.In 5 step, use 1: 60-1: 100 mol ratio (lipid concentration increase) connects albumen-apolipoprotein A-1 (3.84mg/ml, 3mg/ sample) to four and carries out lipid.The lipid buffer is 250mM Tris-HCl, 140mM NaCl, 10mM methionine, pH7.5.

Table 10: the sample summary of the lipid granule of apolipoprotein and DPPC

* calculate for protein monomer

In the lipid granule forming process, do not observe albumen precipitation or the muddiness by excessive lipid.The productive rate of four company's albumen-apolipoprotein A-1s in end product is higher, and the DPPC that is used for lipid is more.

In 1: 20 sample, find on the non-degeneration PAGE the remaining apolipoprotein without lipid (swimming lane 3, Figure 17).On non-degeneration PAGE, 1: 40 and 1: 60 sample look the most even (swimming lane 4 and 5), and 1: 80 and 1: 100 sample also comprise other higher molecule band (swimming lane 6 and 7) on main lipid granule band.

The analysis of SEC-MALLS protein conjugate is used for being characterized in the composition (MW DPPC:734Da) that the DPPC lipid granule forms the lipid granule that obtains afterwards.Obtain uniform SEC peak 1: 80 and following mol ratio.In higher drugrlipid ratio, before occurring-peak (seeing, for example 1: 90 sample in table 11).

Table 11:DPPC and four connects the SEC-MALLS protein conjugate analysis of the lipid granule of albumen-apolipoprotein A-1 and sums up

With 1: 80-1: 90 mol ratios are found the top (lowest percentage of albumen) of lipid.In addition, DLS is disclosed in 1: 80-1: the most uniformly granule of the ratio of 90 (＞98%) forms, and granular size is 14-17nm.

d)75％DPPC/25％POPC

As present embodiment a)-c) in the lipid granule that correspondingly carries out of report form, use following parameter:

Albumen: four connect albumen-apolipoprotein A-1 at 3.84mg/ml, the 3mg/ sample

Lipid buffer: 250mM Tris-HCl, 140mM NaCl, 10mM methionine pH7.5

Lipid: at 34 ℃

Dialysis: at 4 ℃

The mol ratio of test: in 5 step, 1: 60-1: 100, have the lipid of increase

Lipid granule forms simple, and suitable with the method for carrying out with pure lipid.In described method and dialysis procedure, all samples keep clarification.For the ratio (～85%) of all tests, the productive rate of lipid granule is similar.SEC-MALLS analyzes the mol ratio that shows 1: 80 and causes having 90.9% main peak, without the most uniform lipid granule of leading peak and 9.1% postpeak.The protein conjugate analysis is presented in the main species of all samples, 1 four exist (seeing Figure 18 and table 12 and 13) that connects albumen-apolipoprotein A-1 trimer/lipid granule.

Table 12:SEC result's summary; By AUC integral and calculating percentage ratio

Table 13:75%DPPC/25%POPC and the four even albumen of albumen-apolipoprotein A-1 lipid granule-conjugate analysis sums up.

e)50％DPPC/50％POPC

As present embodiment a)-c) in the lipid granule that correspondingly carries out of report form, use following parameter:

Albumen: four connect albumen-apolipoprotein A-1 at 3.84mg/ml, the 3mg/ sample

Lipid buffer: 250mM Tris-HCl, 140mM NaCl, 10mM methionine pH7.5

Lipid: at 27 ℃

Dialysis: in room temperature

The mol ratio of test: in 5 step, 1: 60-1: 100, have the lipid of increase

All samples keep clarification in described method and dialysis procedure.For the ratio of all tests, the productive rate of lipid granule is similar.

Table 14:SEC result's summary; Integral and calculating percentage ratio by AUC.

Lipid mixture with 50%DPPC and 50%POPC comes to form for four lipid granules that connect albumen-apolipoprotein A-1, obtains the most uniform product (seeing Table 14) 1: 70 mol ratio.Product is 89.9% pure about main peak, and comprises one single four and connect albumen-apolipoprotein A-1 trimers (seeing Table 15).

Table 15: have the protein conjugate analysis summary that 50%DPPC/50%POPC and four connects the lipid granule of albumen-apolipoprotein A-1

f)25％DPPC/75％POPC

As present embodiment a)-c) in the lipid granule that correspondingly carries out of report form, use following parameter:

Albumen: four connect albumen-apolipoprotein A-1 at 3.84mg/ml, the 3mg/ sample

Lipid buffer: 250mM Tris-HCl, 140mM NaCl, 10mM methionine pH7.5

Lipid: at 18 ℃

Dialysis: in room temperature

The mol ratio of test: in 5 step, 1: 60-1: 100, have the lipid of increase

Lipid granule forms simple, and suitable with the method for using pure lipid.In described method and dialysis procedure, all samples keeps clarification.

Table 16:SEC result's summary; Integral and calculating percentage ratio by AUC.

Lipid mixture with 25%DPPC and 75%POPC forms four company's albumen-apolipoprotein A-1s, obtains the most uniform product (seeing Table 17) 1: 60 mol ratio.Product is 90.2% pure about main peak, and comprises one single four and connect albumen-apolipoprotein A-1 trimers (seeing Table 15).

Table 17:25%DPPC/75%POPC and four connects the protein conjugate of the lipid granule of albumen-apolipoprotein A-1 and sums up.

G) use ampholytic detergent to form lipid granule

Correspondingly carry out the formation of lipid granule such as a)-c) report (use has following parameter) at present embodiment, except replacing cholate with the synthetic detergent ampholytic detergent:

Albumen: four connect albumen-apolipoprotein A-1, at 23.5mg/ml

Buffer: 50mM Tris-HCl, 7.2M guanidine hydrochloride, 10mM methionine, pH8

Lipid buffer: 250mM Tris-HCl, 140mM NaCl, pH7.5

100%POPC, apolipoprotein: phospholipid mol ratio=1: 60

Table 18: the whole bag of tricks that lipid granule forms and the sample of observation/parameter summary

Analyze the lipid granule that comprises four company's albumen-apolipoprotein A-1s at non-degeneration PAGE.Connect albumen-apolipoprotein A-1s in 140kDa migration (swimming lane 1 in Figure 19) without four of lipid, and lipid granule shows to the characteristic of the more high molecular between 232kDa and the 440kDa and moves.

Detect without four of lipid in the sample of only 0.1x CMC preparation of useful separately detergent and connect albumen-apolipoprotein A-1s, but do not detect lipid granule (Figure 19, swimming lane 2,8,13, and 19).Yet the detergent of 0.5x CMC concentration is enough to make ampholytic detergent 3-8 and 3-10 can make four to connect albumen-apolipoprotein A-1s and form lipid granule ( swimming lane 3,9, and 14).Use ampholytic detergent 3-12, lipid granule does not occur form, until reach the concentration (swimming lane 21) of 2.0x CMC.

Figure 20 shows use 3x CMC ampholytic detergent 3-8 and POPC (apolipoprotein: the SEC-MALLS chromatogram of the lipid granule that comprises four company's albumen-apolipoprotein A-1s phospholipid mol ratio=1: 60).The result of albumen-conjugate analysis is summarised in the table 18.The lipid granule fraction is by two kinds of different species compositions, such as what show in two overlapping peaks in the SEC chromatogram.Yet these two kinds are closely similar, and difference mainly connects the number (be 4.2 and be 3.5 for peak 2 for peak 1) of albumen-apolipoprotein A-1 molecules at four of each granule.

Table 19: the summary that the albumen of the lipid granule that forms when ampholytic detergent 3-8 exists-conjugate is analyzed.

Figure 21 shows that chromatogram and table 19 that SEC-MALLS analyzes use 2x CMC ampholytic detergent 3-10 and POPC (apolipoprotein: the summaries that albumen-conjugate is analyzed of the lipid granule that comprises four company's albumen-apolipoprotein A-1s phospholipid mol ratio=1: 60).Two peaks comprise and contain respectively 3.5 and 5 four lipid granules that connect albumen-apolipoprotein A-1 molecule.

Table 20: the albumen of the lipid granule that forms when having ampholytic detergent 3-10-conjugate analysis is summed up.

(apolipoprotein: four results that connect the lipid granules formation of albumen-apolipoprotein A-1 that comprise phospholipid mol ratio=1: 60) are summarised in the table 21 to use ampholytic detergent 3-12 and POPC.The lipid granule fraction is by two kinds of different species compositions, such as what show in two overlapping peaks in the SEC chromatogram.Yet these two kinds are closely similar, and difference mainly connects the number of albumen-apolipoprotein A-1 molecule at four of each granule.

Table 21: the albumen of the lipid granule that forms when having ampholytic detergent 3-12-conjugate analysis is summed up.

(apolipoprotein: four results that connect the lipid granules formation of albumen-apolipoprotein A-1 that comprise phospholipid mol ratio=1: 60) are summarised in the table 21 to use cholate and POPC.The lipid granule fraction is by two kinds of different species compositions, such as what show in two overlapping peaks in the SEC chromatogram.Yet these two kinds are closely similar, and difference mainly connects the number of albumen-apolipoprotein A-1 molecule at four of each granule.

Table 22: the summary that the albumen of the lipid granule that forms when having cholate-conjugate is analyzed

Embodiment 5

Be used for the rapid dilution process that refolding and lipid granule form

A) POPC and sodium cholate

Connect albumen-apolipoprotein A-1 at expression in escherichia coli and according to embodiment 1-3 purification (method 1) with four.Behind the purification, buffer is comprised 250mM Tris, 140mM NaCl, the solution of 6.7M guanidine hydrochloride by what pH7.4 was changed in diafiltration.Protein concentration is adjusted to 28mg/ml.

Comprise 250mM Tris-HCl by in room temperature 100 moles/l POPC being dissolved in, 140mM NaCl prepares the lipid stock solution in the buffer of the pH7.4 of 135mM sodium cholate.With the lipid stock solution room temperature incubation 2 hours.By the 77ml lipid is stored mixture diluted to the 250mM Tris-HCl of 1478ml, 140mM NaCl prepares the refolding buffer among the pH7.4.This buffer in room temperature, was stirred other 7 hours.

Refolding and lipid granule form by will be at 250mM Tris, and 140mM NaCl, 6.7M guanidine hydrochloride, the 162ml four among the pH7.4 connect albumen-apolipoprotein A-1s to add the refolding buffer initial.This causes 1: 10 diluent of guanidine hydrochloride.Solution room temperature incubation 16 hours, is continued to stir simultaneously.Carry out the removal of detergent by diafiltration.

Table 23: the summary of analyzing by the protein conjugate of the lipid granule that obtains with the POPC rapid dilution

Connect albumen-apolipoprotein A-1 at expression in escherichia coli and according to embodiment 1-3 purification (method 2) with four.Behind the purification, buffer is comprised 50mM Tris, 10mM METHIONINE, the solution of 6.7M guanidine hydrochloride by what pH7.4 was changed in diafiltration.Protein concentration is adjusted to 20.4mg/ml.

Comprise 250mM Tris-HCl by in room temperature 100 moles/l phospholipid (POPC: the DPPC ratio is 3: 1) being dissolved in, 140mM NaCl, the 10mM METHIONINE prepares the lipid stock solution in the buffer of the pH7.4 of 135mM sodium cholate.By 3.7ml lipid stock solution being diluted to the 250mM Tris-HCl of 35.6ml, 140mM NaCl prepares the refolding buffer among the pH7.4.This buffer in room temperature, was stirred other 2 hours.

Refolding and lipid granule form by will be at 50mM Tris, and 10mM METHIONINE, 6.7M guanidine hydrochloride, the 9.8ml four among the pH8.0 connect albumen-apolipoprotein A-1s to add the refolding buffer initial.This causes 1: 5 diluent of guanidine hydrochloride.Solution is incubated overnight in room temperature, continues simultaneously to stir.Carry out the removal of detergent by diafiltration.

Table 24: the protein conjugate analysis by the lipid granule that obtains with POPC/DPPC/ cholate mixture rapid dilution is summed up.

B) POPC and DPPC and sodium cholate

Connect albumen-apolipoprotein A-1 at expression in escherichia coli and according to embodiment 1-3 purification with four.Behind the purification, buffer is comprised 250mM Tris, 140mM NaCl, the solution of 6.7M guanidine hydrochloride by what pH7.4 was changed in diafiltration.Protein concentration is adjusted to 30mg/ml.

Prepare two kinds of independent lipid stock solution.Comprise 250mM Tris-HCl by in room temperature 100 moles/l POPC being dissolved in, 140mM NaCl prepares solution A in the buffer of the pH7.4 of 135mM sodium cholate.At 41 ℃, by 100moles/l DPPC is dissolved in 250mM Tris-HCl, 140mM NaCl prepares solution B among the 135mM sodium cholate pH7.4.Lipid stock solution A and B mixed with 3: 1 ratio and room temperature incubation 2 hours.By 384ml lipid stock solution being diluted to the 250mM Tris-HCl of 6365ml, 140mM NaCl prepares the refolding buffer among the pH7.4.This buffer in room temperature, was stirred other 24 hours.

Refolding and lipid granule form by will be at 250mM Tris, and 140mM NaCl, 6.7M guanidine hydrochloride, the 750ml four among the pH7.4 connect albumen-apolipoprotein A-1s to add the refolding buffer initial.This causes 1: 10 diluent of guanidine hydrochloride.Solution room temperature incubation at least 12 hours, is continued to stir simultaneously.Carry out the removal of detergent by diafiltration.

Table 25: by using POPC: DPPC=1: the protein conjugate analysis of the lipid granule that 1 rapid dilution obtains is summed up

C) different concentration of guanidine hydrochloride

To connect albumen-apolipoprotein A-1 according to of the present invention four and carry out purification (seeing embodiment 1-3) at expression in escherichia coli and from occlusion body by metallo-chelate affinity chromatograph method.Behind the purification, buffer is comprised 250mM Tris, 140mM NaCl, the solution of 6.7M guanidine hydrochloride by what pH7.4 was changed in diafiltration.Protein concentration is adjusted to 28mg/ml.

Comprise 250mM Tris-HCl by in room temperature 100 moles/l POPC being dissolved in, 140mMNaCl prepares the lipid stock solution in the buffer of the pH7.4 of 135mM sodium cholate.With the lipid stock solution room temperature incubation 2 hours.By the lipid stock solution is diluted to 250mM Tris-HCl, 140mM NaCl prepares the refolding buffer among the pH7.4.This buffer in room temperature, was stirred other 12 hours.Four not commensurability company's albumen-apolipoprotein A-1s are diluted in the refolding buffer: 1: 5,1: 7.5,1: 10,1: 12.5.This causes the different residual concentrations of guanidine hydrochloride in the refolding buffer.Described solution allows to form with initial refolding and lipid granule at stirring at room o/n.Remove detergent by dialysis.

Table 26: the protein conjugate analysis by the lipid granule that obtains with different dilution ratio rapid dilutions is summed up.

POPC when d) having carbamide and sodium cholate

Connect albumen-apolipoprotein A-1 at expression in escherichia coli and according to embodiment 1-3 purification with four.Behind the purification, buffer is comprised 250mM Tris, 140mM NaCl, the solution of 6.7M carbamide by what pH7.4 was changed in diafiltration.Protein concentration is adjusted to 28mg/ml.

Comprise 250mM Tris-HCl by in room temperature 100 moles/l POPC being dissolved in, 140mM NaCl prepares the lipid stock solution in the buffer of the pH7.4 of 135mM sodium cholate.With the lipid stock solution room temperature incubation 2 hours.By the 77ml lipid is stored mixture diluted to the 250mM Tris-HCl of 1478ml, 140mM NaCl prepares the refolding buffer among the pH7.4.This buffer in room temperature, was stirred other 7 hours.

Refolding and lipid granule form by will be at 250mM Tris, and 140mM NaCl, 6.7M carbamide, the 162ml four among the pH7.4 connect albumen-apolipoprotein A-1s to add the refolding buffer initial.This causes 1: 10 diluent of carbamide.Solution room temperature incubation 16 hours, is continued to stir simultaneously.Carry out the removal of detergent by diafiltration.

E) POPC and sodium cholate and wild type apolipoprotein A-1

In exemplary the second method of another kind, will be at the 6.7M guanidine hydrochloride, 50mM Tris, 10mM methionine, human apolipoprotein A-Ⅰ among the pH8.0 (wild type apolipoprotein A-1) dilution 1: 5 (v/v) obtains the protein concentration of 0.6mg/ml in the lipid buffer.Described lipid buffer is by the 7mM cholate, and 4mM POPC and 1.3mM DPPC form, and corresponding lipid: Protein ratios is 240: 1.SEC-MALLS is used for analyzing complex to be formed.In the complex that is formed by about 200 lipid moleculars, find about two apolipoprotein molecules.

Table 27: the protein conjugate analysis is summed up

Embodiment 6

The lipid granule that begins from degeneration or native protein forms

Need natural apolipoprotein to be used for lipid granule such as the method (the first method) of report in embodiment 4 and form, and the method (the second method) of report begins to carry out lipid granule formation with the apolipoprotein of abundant degeneration in embodiment 5.

In the first exemplary method, will be at the 6.7M guanidine hydrochloride, 50mM Tris, the 10mM methionine, degeneration four among the pH8.0 connects albumen-apolipoprotein A-1 and uses by 250mM Tris, 140mM NaCl, the buffer of the pH7.5 that the 10mM methionine forms is extensively dialysed, and protein concentration is 3.46mg/ml.Then, add the mixture of POPC and cholate in solution, to produce the ultimate density of 6mM POPC and 8mM cholate.This connects the ratio (60: 1) of albumen-apolipoprotein A-1 monomer corresponding to 60 molecule POPC/ four.Subsequently, remove detergent by diafiltration.Analyze the albumen-lipid complex that forms by SEC-MALLS.Make in this way, form inhomogeneous product, the kind apoplexy due to endogenous wind that wherein forms have an appointment 60% comprise surpass three kinds four connect albumen-apolipoprotein A-1 monomers.

In the second exemplary method, will be at the 6.7M guanidine hydrochloride, 50mM Tris, the 10mM methionine, four of the degeneration among the pH8.0 connects albumen-apolipoprotein A-1 and directly dilutes 1: 10 (v/v) in the lipid buffer, obtains the protein concentration of 2.5mg/ml.Described lipid buffer is comprised of 6mM cholate and 4.5mM POPC, and corresponding lipid and Protein ratios are 60: 1.Make in this way, form uniform product, comprise surpassing 90% single formation kind, wherein per four connect albumen-apolipoprotein A-1 molecule in conjunction with the lipid (seeing Figure 22) of 60 molecules.

Table 28: the protein conjugate analysis is summed up

Embodiment 7

With cholate-and the POPC/DPPC of ampholytic detergent-dissolving lipid that insulin-F is carried out

The insulin that select to be used for albumen that lipid granule forms and to be being purchased (

Insulin Lispro, Lilly).The molecular weight of this albumen is 5808Da.In order to increase the detectable limit of insulin in lipid granule, with albumen with NHS-fluorescein (6-[fluorescein-5 (6)-carboxyamino] caproic acid N-hydroxy-succinamide ester, Sigma Aldrich#46940-5MG-F) labelling.

1: 1 mixture that uses POPC and DPPC carries out ampholytic detergent-and the insulin of the NHS-fluorescein-labelling of cholate mediation (lipid of insulin-F) such as report in embodiment 4.The 0.5mM lipid mixture is dissolved in 1x CMC cholate in PBS pH7.4, among 2x CMC ampholytic detergent 3-8 or the 5x CMC ampholytic detergent 3-10.Being dissolved in the ultrasonic bath at 45 ℃ of lipid realized in 1 hour.Insulin-F is added in the lipid of dissolving, albumen: the mol ratio of lipid is 1: 2 (ampholytic detergent 3-8) or 1: 1.2 (ampholytic detergent 3-10 and cholate).In room temperature, with lipid mixture incubation 1 hour, use subsequently PBS pH7.4 extensively to dialyse to remove detergent.

Using fluoroscopic examination (494nm ext., 521nm em.) and UV280 to absorb analyzes the lipid granule and the control sample that form at SE-HPLC.On SE-HPLC to three kinds of different samples of every kind of lipid methods analyst: be dissolved in the insulin-F among the PBS, the liposome of insulin-containing F and comprise the lipid granule of insulin-F not in PBS.Insulin-the F of non-lipid is at the elution time of about 40min eluting from the post, and detects detected peaks by fluorescence and UV280.The insulin F-sample of lipid is with two independent peaks eluting from the post, and described peak detects by fluorescence and UV280.Peak (maximum at peak is at about 40min) moves jointly with insulin-F control sample late.Peak morning in the 15min elution time has higher molecular weight than pure insulin-F, and is comprised of the insulin-F of lipid.Do not contain the albumen of lipid granule at the elution time eluting of 15min.

Embodiment 8

The application of apolipoprotein

A) DPPC and POPC are on the impact of LCAT activity

The ability that inspection comprises POPC (POPC) or dipalmitoyl phosphatidyl choline (DPPC) and the cholesterol esterification is carried out in the lipid granule supports of recombinate wild type apolipoprotein A-1 or four company's albumen-apolipoprotein A-1s by LCAT.

The cholesterol (4% of tritiate; With respect to the phosphatidylcholine content based on mole) be combined in the lipid granule by adding the cholesterol alcoholic solution.Exist at 125 μ l (10mM Tris, 150mM NaCl, 1mM EDTA, 1mM NaN ₃PH7.4; 2mg/ml HuFAF albumin; When 0.2 μ g/ml the 4mM beta-mercaptoethanol) recombinates LCAT enzyme (ROAR biochemistry), support the ability of the cholesterol esterification of LCAT catalysis at the 37 ℃ of albumen that carry out testing in 1 hour obtaining-lipid complexes.By adding chloroform: methanol (2: 1) comes cessation reaction and extracts lipid.After separating cholesterol-cholesteryl ester and scinticounting by TLC, calculate " percentage ratio " esterification.Be incorporated in the ester of formation because be less than 20% tracer, reaction rate can be thought constant under reaction condition.Use XLfit software (IDBS), data fitting is in michealis-Menton equation (Michaelis Menten equation).For these results' demonstration, see Fig. 3.

B) the DPPC/POPC mixture is on the impact of LCAT activity

By will recombinate the wild type apolipoprotein A-1 with ³H cholesterol, DPPC/POPC mixture and cholate were with 1: 4: 80: after 113 mixed in molar ratio, prepare lipid granule with cholate as detergent.The DPPC/POPC mixture comprises arbitrary 100%POPC; 75%POPC; 50%POPC; 25%POPC.

After removing cholate by dialysis, the albumen that test obtains-lipid complex is supported the ability of the cholesterol esterification of LCAT catalysis.Will by adding the cholesterol alcoholic solution ³H cholesterol (4%; With respect to the phosphatidylcholine content based on mole) be combined in the lipid granule.Exist at 125 μ l (10mM Tris, 150mM NaCl, 1mM EDTA, 1mM NaN ₃PH7.4; 2mg/ml HuFAF albumin; When 0.2 μ g/ml the 4mM beta-mercaptoethanol) recombinates LCAT enzyme (ROAR biochemistry), support the ability of the cholesterol esterification of LCAT catalysis at the 37 ℃ of albumen that carry out testing in 1 hour obtaining-lipid complexes.By adding chloroform: methanol (2: 1) comes cessation reaction and extracts lipid.After separating cholesterol-cholesteryl ester and scinticounting by TLC, calculate " percentage ratio " esterification.Be incorporated in the ester because be less than 20% tracer, reaction rate can be thought constant under reaction condition.Use XLfit software (IDBS), data fitting is in michealis-Menton equation and be presented among Fig. 4.

Table 3a: apparent kinetics parameter

C) outflow of the cholesterol foam cell of deriving to THP-1

Obtain macrophage such as Human THP-1 cells by THP-1 monocytic leukemia cell being exposed to phorbol-12-myristinate-13-acetas (phorbol myristate acetate).Subsequently, by cell is comprised in existence ³Further cultivate load during the acetylizad LDL of H cholesterol tracer.Then, these model foam cells are exposed to cholesterol receptor 4h-8h test compounds (seeing lower).

The harvesting culture supernatants is also cut cell in 5%NP40.OF is calculated as cholesterol radioactivity in supernatant with respect to the ratio that adds the radioactive summation in the supernatant at cell.Deduct the outflow of the cell that is exposed to the medium that does not comprise receptor and calculate the rate of outflow by linear fit.The outflow of use from cell comes the rate of outflow is carried out standardization (the relatively rate of outflow) with respect to as a reference 10 μ g/ml wild type apolipoprotein A-1s.The relative rate of outflow mapping that will in two independent experiments, obtain for the function of cholesterol acceptor density and with data fitting in michealis-Menton equation.

The cell that use is exposed to the RXR-LXR agonist carries out parallel laboratory test, the known rise of described RXR-LXR agonist ABCA-1 transport protein, and the outflow that cholesterol transportation deflection ABCA-1 is mediated.

In test series, only observe the minor impact (Fig. 5 and table 29) of lipid mixture.

Table 29: different samples.

The strong outflow that increases to non-lipid compound matter of RXR-LXR pretreatment foam cell is compared with untreated cell, and maximal rate increases by 6 times.Impact on lipid granule is then much smaller, has 2 times of increases, has reflected the less contribution (Fig. 6) of ABCA-1 transport protein to Cholesterol Efflux.

D) research in the body

Studied 5 kinds of lipid granule variants:

I) POPC only

Ii) DPPC only

iii)POPC∶DPPC3∶1

iv)POPC∶DPPC1∶1

v)DPPC∶SM9∶1

At 80mg/kg, in 0.5h, rabbit is carried out intravenous infusion (n=3 rabbit/test compounds), behind infusion, carry out subsequently serial blood sampling in the 96h.

Use the elisa assay Apolipoprotein Levels:

-levels of drugs

-about the data of the blood plasma value of liver enzyme, cholesterol, cholesteryl ester.

For all test composition, plasma concentration is very similar, shows extremely unconspicuous initial " distribution " phase, follows by the log-linear of concentration descend (Fig. 7, table 3).

Table 3: pharmacokinetic data.

For all test compounds, the pharmacokinetics of mensuration (PK) parameter is similar.In addition, also found transmutability in the low individuality.The half-life of measuring was namely compared increase with the wild type apolipoprotein A-1 close to 1.5 days.The volume that distributes similar to the blood plasma volume (in rabbit, about 40ml/kg).

F) cholesterol is mobilized

In blood plasma, mobilize and esterified cholesterol.Even after four company's albumen-apolipoprotein A-1s had begun to reduce, plasma cholesterol ester level continued to increase really.Reduce to 0.5mg/ml (about normal wild type apolipoprotein A-1 about 50%) when blood plasma four connects albumen-apolipoprotein A-1 levels, still can detect the cholesteryl ester level (Fig. 8) of increase.

G) the liver enzyme discharges

Comprising four lipid granules that connect albumen-apolipoprotein A-1 that contain POPC does not induce the liver enzyme to discharge (Fig. 1).Be similar to rabbit, it is safe in mice that the interior injection of azygos vein connects albumen-apolipoprotein A-1 according to four of the POPC of comprising of the present invention or POPC/DPPC mixture.Comprise mol ratio and be 1: 3 DPPC: the apolipoprotein compositions of POPC and independent POPC are suitable (Fig. 9).

Do not observe obvious haemolysis, until in any of five prepared products behind infusion 2 hours.By the photoelectric colorimetric assay haemolysis for use four redness that connect in the plasma sample that obtained in 2 hours behind albumen-apolipoprotein A-1s at intravenous.Whole blood haemolysis with 100% (producing by 0.44%Triton X-100-ultimate density) is used for calibration (Figure 10).

H) four connect albumen-apolipoprotein A-1 to the antiinflammatory action of Human umbilical vein endothelial cells

5-10 four was connected in albumen-apolipoprotein A-1 prepared product incubation 16 hours for HUVECs (Human umbilical vein endothelial cells) at each, and stimulated last 4 hours with TNF α.By FACS, with detection of specific antibody VCAM1 surface expression.

Embodiment 9

Lipid granule stability

To comprise the wild type apolipoprotein A-1 in N-end histidine-labelling and IgA protease cutting site at expression in escherichia coli and by the column chromatography purification, as reporting in the above-described embodiments.Prescind except histidine-labelling by the IgA proteolytic cleavage.Use albumen and the Lipoid S100 soybean phospholipid mixture assembling lipid granule (HDL granule) of 1: 150 ratio.Described granule is stored in comprises 5mM sodium phosphate and 1% sucrose, pH value is in 7.3 the buffer.SE-HPLC is disclosed in behind the lipid incubation and incubation, and after 10 days three peaks independently.Behind 40 ℃ of incubations, can detect at the main peak of 10.8 minutes retention times (47% total protein), this does not exist in being stored in 5 ℃ sample.10.8 minute the peak show because albumen is unstable, form soluble large molecule amount assembly.

From POPC: the initial acquisition of DPPC mixture (POPC and DPPC ratio are 3: 1) such as the HDL granule that comprises four albumen-apolipoprotein A-1 of this paper report also at 5 ℃ and 40 ℃ of incubations.Cause the forward of slight extent to form at the incubation that heats up, but not have significantly to the movement (in 11 minutes＜2% increase) at 10.8 minutes high molecular assembly.This explanation is with the Particle Phase ratio that comprises the wild type apolipoprotein A-1, the HDL granule stability of raising.

Embodiment 10

Cholesterol is mobilized

After using apolipoprotein in vivo, discharge and apolipoprotein concentration can be measured cholesterol and mobilizes efficient in the blood by each time of T-CHOL relatively.About qualitative assessment, calculate T-CHOL baseline correction the merchant of area under the concentration-time curve (AUC) with the area under concentration-time curve of apolipoprotein.

In this experiment, analyze following substances:

-will comprise the wild type apolipoprotein A-1 in N-end histidine-labelling and IgA protease cutting site at expression in escherichia coli and by the column chromatography purification, as reporting in the above-described embodiments; Prescind except histidine-labelling by the IgA proteolytic cleavage; Use albumen and the Lipoid S100 soybean phospholipid mixture assembling lipid granule (HDL granule) of 1: 150 ratio;

-apolipoprotein A-1 Milano variant: use albumen and the POPC assembling lipid granule (HDL granule) of 1: 40 ratio,

-connect albumen-apolipoprotein A-1 such as four of this paper report; Use the albumen and (POPC and DPPC) (POPC and DPPC ratio are 3: 1) assembling lipid granule (HDL granule) of 1: 60 ratio.

Three kinds of HDL granules are put on rat.See that the value that obtains about each AUC ratio is presented in the table 30.

Table 30: cholesterol is mobilized

Claims (14)

1. for generation of the method for the lipid granule that comprises polypeptide, described method feature is to form described lipid granule in the presence of ampholytic detergent 3-8 or ampholytic detergent 3-10.

2. according to each method in the aforementioned claim, it is characterized in that described polypeptide was with described ampholytic detergent incubation about 2 hours to about 60 hours.

3. according to each method in the aforementioned claim, it is characterized in that described method comprises the steps:

I) provide the first solution that comprises the degeneration polypeptide,

Ii) described the first solution is added in the second solution, described the second solution comprises at least a lipid and ampholytic detergent 3-8 or ampholytic detergent 3-10, but does not contain described polypeptide, and

Iii) from step I i) remove described ampholytic detergent and produce thus lipid granule the solution that obtains.

According to claim 1 and 2 in each described method, it is characterized in that described method comprises the steps:

I) provide the solution that comprises natural polypeptides,

Ii) at least a lipid and ampholytic detergent 3-8 or ampholytic detergent 3-10 are added i) solution, and

Iii) from step I i) the described ampholytic detergent of solution removal that obtains and produce thus lipid granule.

5. according to each method in the aforementioned claim, it is characterized in that described polypeptide has the SEQ of being selected from ID NO:01,02,04-52, the aminoacid sequence of 66 or 67 aminoacid sequence, or comprise at least a SEQ of comprising ID NO:01,02,04-52, at least 80% continuous fragment of 66 or 67 aminoacid sequence.

6. method according to claim 5 is characterized in that described polypeptide is four company's albumen-apolipoprotein A-1s with aminoacid sequence of SEQ ID NO:01 or SEQ ID NO:02 or SEQ ID NO:66 or SEQ ID NO:67.

7. each described method according to claim 3-6 is characterized in that described at least a lipid is two kinds of lipids.

8. method according to claim 7 is characterized in that described two kinds of lipids are two kinds of different phospholipid.

9. method according to claim 8 is characterized in that the first phospholipid is POPC, and the second phospholipid is DPPC.

10. according to each method in the aforementioned claim, it is characterized in that described detergent is the detergent with high CMC.

11. according to each method in the aforementioned claim, it is characterized in that described removal undertaken by diafiltration or dialysis or absorption.

12. the lipid granule that obtains by each method of claim 1-3 and 5-11.

13. by claim 1,2 and the lipid granule that obtains of each method of 4-11.

14. pharmaceutical composition, it comprises claim 12 or 13 each lipid granules.

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