JPH083198A - Production of apolipoprotein a-1 from human plasma - Google Patents

Abstract

PURPOSE:To produce apolipoprotein A-1 useful e.g. for the prevention and treatment of ischemic heart diseases in high efficiency by contacting a human plasma containing apolipoprotein A-1 with a lower aliphatic alcohol at a low temperature, collecting the supernatant by centrifugation and subjecting to chromatography with hydrophobic chromatogel. CONSTITUTION:This apolipoprotein A-1 expected to be usable in the field of the prevention, treatment, etc., of ischemic heart diseases is produced in high efficiency by fractionating a human plasma containing apolipoprotein A-1 by Cohn fractionation comprising the incubation with a lower aliphatic alcohol such as ethanol at a low temperature, using the centrifugal supernatant fraction of the obtained Cohn fractions II+III as a raw material, directly adding the supernatant fraction to a hydrophobic chromatogel column equilibrated with a buffer solution containing 90mM sodium chloride in the presence of a lower aliphatic alcohol such as ethanol, developing the chromatography and eluting the adsorbed component from the gel using a chaotropic agent composed of 6M aqueous solution of urea.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a plasma fractionation preparation. More specifically, it relates to a method for producing apolipoprotein A-1 from a human plasma fraction.

[0002]

BACKGROUND OF THE INVENTION Ischemic heart disease is now the leading cause of death in industrialized countries. The cause of this disease is considered to be mainly due to coronary atherosclerosis, and the underlying lesion is atherosclerosis.
(Atherosclerosis). Atherosclerosis is mainly caused by macrophages derived from monocytes that infiltrate the subendothelium of the blood, which is a low-density lipoprotein (denatured LDL) that has undergone degeneration, foamed and deposited, and is characterized by atheroma (Atheroma). I am trying. Risk factors for ischemic heart disease include hypertension, obesity, diabetes, hyperlipidemia, smoking, and heredity, and some of these are inevitable, but due to lifestyle improvement or drug therapy. It can be improved to some extent.

At present, drugs for lowering blood cholesterol (therapeutic drugs for hyperlipidemia) are widely known, and those that lower LDL cholesterol, simultaneously raise HDL cholesterol, and the key to cholesterol biosynthesis. The enzyme hydroxymethylglutaryl-Co
There are drugs based on various mechanisms, such as those that inhibit A-reductase (HMG-CoA reductase) and reduce cholesterol synthesis. However, these drugs have to be taken for a long period of time, and their side effects are unavoidable. Further, they have not been observed to have a fundamental treatment for ischemic heart disease, that is, to reduce the arteriosclerosis generated in the coronary arteries.

[0004] Lipoprotein is well known as a protein deeply involved in lipid metabolism in blood, and among them, high density lipoprotein (HDL) is a pathway for transporting cholesterol accumulated in peripheral tissues to the liver, that is, It plays a central role in the cholesterol reverse transfer pathway. It is a pathway for esterifying cholesterol in peripheral tissues by activating the enzyme lecithin-cholesterol acyltransferase (LCAT), taking it up in the form of cholesteryl ester in HDL particles, and transporting it to the liver. H is responsible for the activation of this enzyme, LCAT.
It is apolipoprotein A-1 which is a major constituent protein of DL.

Apolipoprotein A-1 was reported by Baker et al. (Journal of Biological Chemistry: J. Biol.
Chem., 250 , p. 2725- (1975)), 24
It is a single polypeptide molecule having a linear structure of 5 amino acids and has a molecular weight of about 28,000. The apparent isoelectric point that can be determined by isoelectric focusing is in the range of 5.4 to 6.5. Looking at the secondary structure of apolipoprotein A-1, the hydrophobic group is arranged on one side parallel to the molecular structure and the hydrophilic group is arranged on the other side.
It has a mphipathic structure and is known to cause concentration-dependent self-association in a water solvent. The function of apolipoprotein A-1 is, firstly, the function as an activator of LCAT, which is a cholesterol esterifying enzyme in plasma, as described above. The second is binding to lipids (cholesterol, phospholipids, cholesterol esters). It is considered that this is due to the amphipathic helix structure of apolipoprotein A-1. The third function is considered to be a function of stabilizing prostacyclin (PGI ₂ ) which has a vasodilatory action and a platelet aggregation inhibitory action. Due to these functions, apolipoprotein A-1 has been attracting attention as an anti-atherogenic protein.

[0006] Recently, EMRubin et al.
In a study using transgenic mice in which the gene for apolipoprotein A-1, which is a major component protein of L, was introduced into a mouse that is genetically susceptible to arteriosclerosis, fat that appears before complete atherosclerosis It has been reported that regression of plaque tissue was observed (Nature: Nature 35
3 , p.265-267, (1991)). From this, HD
Apolipoprotein A-1, which is a major component protein of L, is a cardiovascular disease associated with atherosclerotic lesion, that is, myocardial infarction,
It is suggested that it may be effective as a therapeutic drug for angina. Apolipoprotein A-1 has an action of regressing atherosclerosis as described above, and its use in the field of ischemic heart disease treatment is considered to be very useful. For that purpose, apolipoprotein A-1 must be efficiently separated and purified from human plasma or serum.

As a method for separating and purifying apolipoprotein A-1,
Ultracentrifugation method of RJ Havel et al. (Journal of Clinical Investigation: J. Clin. Invest. 34 , p. 1
345, (1955)). According to this, solid sodium chloride was added to plasma or serum to a predetermined density (d = 1.063), the upper layer was removed after ultracentrifugation, and solid potassium bromide was added (d = 1.21) again to obtain ultra-high density. The HDL fraction is prepared by centrifugation and collecting the upper layer. In this method, apolipoprotein A-1 is obtained by degreasing with an organic solvent (ethanol, ethyl ether) in order to remove lipids from HDL. However, the ultracentrifugation method is not suitable for obtaining a sufficient amount of apolipoprotein A-1 on an industrial scale. Because the amount of plasma or serum that can be processed at one time by ultracentrifugation is at most several hundred ml
This is because the apolipoprotein A-1 obtained from it is about several hundred mg, which is not a method applicable to an industrial scale.

On the other hand, the method reported by SD Carson (Biochimikato Biophysical Actor: Biochim.
ophys. Acta 750 , p.317-321, (1983))
Is a hydrophobic gel, Phenyl Sepharose (trade name: Pharma
(manufactured by cia), serum is added to a column equilibrated with a Tris-hydrochloric acid buffer solution, and apolipoprotein is eluted with a Tris-hydrochloric acid buffer solution containing 70% propylene glycol to recover the apolipoprotein. This method is based on the hydrophobic functional group of chromatogel.
(Phenyl group) and apolipoprotein A-1 are a production method utilizing a hydrophobic interaction, and compared with a production method using an ultracentrifugation method, apolipoprotein A-1 can be prepared in one step.
The quantity is large and can be applied to industrial scale. However, in such a method in which the serum is directly added to the hydrophobic gel, the protein and the like bound to the lipid in the serum are also adsorbed to the hydrophobic gel, which is a means of effectively using the binding capacity of the gel. However, the yield of apolipoprotein A-1 obtained from a limited amount of gel is low.

Further, according to a patent application published in Japan (Japanese Patent Laid-Open No. 1-294699), the apolipoprotein is purified by a precipitation method using a precipitation fraction of an alcohol fraction which has been discarded so far as a starting material. There is. In this method, the precipitate fraction is first suspended in an aqueous buffer solution, and a lower aliphatic alcohol is added to precipitate and separate impurities to purify apolipoprotein. However, this precipitation method is quite complicated in terms of operation.

[0010]

Human plasma is now collected in large quantities and separated by alcohol fractionation into individual fractions. Since the method for producing apolipoprotein A-1 according to the present invention uses the centrifugal supernatant fraction in the alcohol fractionation step as a starting material, it can be easily introduced into the current fractionation step. The present invention is particularly directed to fractions II + II obtained by alcohol fractionation of plasma.
A step of directly adding a centrifugal supernatant fraction represented by I to a hydrophobic chromatographic gel equilibrated with a buffer solution containing alcohol and sodium chloride having the same composition as that of the supernatant fraction to develop chromatography Is characterized by.

The hydrophobic chromatogel has functional groups such as an octyl group, a phenyl group and a butyl group.
l) group, phenyl ether (Phenyl ether), ether (Ethe
r) and the like are preferably bonded, such as octyl-Sepharose, butyl-Toyopearl (Butyl-To
yopearl), Poros HIC phenyl ether (Poros HIC Phe
Nylether) and other commercially available products can be used. Washing of the gel after addition of the supernatant fraction was performed with an alcohol / sodium chloride buffer solution of the same composition, and 1M was used for elution of apolipoprotein A-1 from the gel.
Aqueous solution containing chaotropic agent at the above concentration or 2
It is carried out using a buffer solution containing a lower aliphatic alcohol at a concentration of 1% (V / V) or higher, or a combination thereof. Preferred examples of the chaotropic agent in this case include urea and guanidine hydrochloride, and preferred examples of the lower aliphatic alcohol include methanol and ethanol. Further, the fraction that was not adsorbed to the gel and passed through could be subjected to alcohol fractionation subsequently, and had no effect on the plasma proteins (albumin globulin, etc.) purified from each fraction fraction thereafter. Absent.

The method for producing apolipoprotein A-1 provided by the present invention is described in the above-mentioned patent application (Japanese Patent Laid-Open No. 1-294699).
The greatest feature is that the centrifugal supernatant fraction present during the alcohol fractionation step is used as the starting material, instead of the discarded precipitate fraction used in 1. Further, the centrifugal supernatant fraction can be directly added to a hydrophobic chromatographic gel without any special treatment, and apolipoprotein A-1 can be very selectively eluted and recovered from the gel. Therefore, it is superior to the conventional method for producing apolipoprotein A-1 on an industrial scale in that it can be purified in a simpler and higher yield than the above-mentioned precipitation method. Furthermore, when the apolipoprotein A-1 purified by the method of the present invention is used as a medical drug, other biochemical purification means,
For example, it is preferable to further improve the purity by carrying out purification by an ion exchange chromatography method or the like. Further, it is also a preferred embodiment to carry out a heat treatment for inactivating contaminating infectious virus and freeze-dry with an additive having a composition administrable in vivo. As an additive at this time,
Sodium citrate, sodium chloride, amino acids or salts thereof, sugars such as sucrose and sugar alcohols such as mannitol are preferable.

Apolipoprotein A-prepared as described above
In the field of prevention or treatment of ischemic heart disease, 1 is highly expected as a therapeutic and preventive agent for arteriosclerosis in the cardiovascular system.

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0015]

Example 1 The centrifugal supernatant of Fraction II + III obtained by ethanol fractionation of plasma known as the Cohn method was used as a starting material, and the composition was preliminarily equilibrated with a buffer solution of 20% ethanol / 90 mM sodium chloride. Hydrophobic chromatography gel
(Butyl Toyopearl: (trade name) manufactured by Tosoh Corporation) was added while cold. Immediately after the addition of the centrifugation supernatant to the gel, the gel was thoroughly washed with a buffer solution of the same composition, and then apolipoprotein A-1 was eluted from the gel with an aqueous solution containing 6 M concentration of urea. As a result of SDS-PAGE analysis, this eluted fraction contained apolipoprotein A-1 in a purity of 90% or more (see FIG. 1). The flow-through fraction that was not adsorbed on the gel was further subjected to ethanol fractionation to obtain globulin and albumin, but no qualitative change from those of the conventional one was confirmed.

Example 2 A hydrophobic chromatographic gel (Phenyl Sepharos) equilibrated with a buffer containing 20% ethanol / 90 mM sodium chloride as a starting material, the centrifugal supernatant of Fraction II + III.
e: (trade name) manufactured by Pharmacia Co., Ltd.) was added cold. Immediately after the addition of the centrifugation supernatant fraction to the gel, the gel was thoroughly washed with a buffer solution having the same composition, and then the apolipoprotein A-1 was removed from the gel with a buffer solution having an ethanol concentration increased to 40%. Was eluted and collected. 85 in this elution fraction
The apolipoprotein A-1 was contained in a purity of at least%.

Example 3 A high-purity fraction containing apolipoprotein A-1 obtained by the method of Example 1 was treated with 20 mM sodium citrate /
0.15M sodium chloride / 0.1M glycine, pH 7.
It was dialyzed overnight with a buffer solution of composition 0, and the dialyzed solution was dispensed into a glass vial and freeze-dried. The freeze-dried sample can be rapidly dissolved in sterilized distilled water, and even when compared with the apolipoprotein A-1 solution before freeze-drying, the turbidity (A
450) was not significantly increased.

Example 4 Changes in the activity of apolipoprotein A-1 during the purification process were measured using the phospholipid binding ability as an index. The phospholipid-binding ability is considered to be one of the biological activities of apolipoprotein A-1, and phospholipid caused by the amphipathic helix structure, which is a characteristic of the molecular structure of apolipoprotein A-1. (HJownall et al., Biochemistry: Biochemistry 18 , p. 574-57).
9, (1979)). The principle of measurement is that by adding apolipoprotein A-1 to dimyristoyl phosphatidylcholine (DMPC) / cholesterol liposome, a hydrophobic bond between apolipoprotein A-1 and DMPC is generated, resulting in smaller liposomes. Based on measuring the reduction in turbidity due to the formation of The measured samples between the purification steps are II + III centrifugal supernatant, hydrophobic chromatography elution fraction,
It was an ion exchange chromatography elution fraction, and the results shown in Table 1 were obtained.

[0019]

[Table 1]

From the results shown in Table 1, it is concluded that the activity of phospholipid binding activity is not decreased during the purification process because the purification process progresses and the purity is improved.

[0021]

[Brief description of drawings]

FIG. 1 is an electrophoretogram (drawing-substituting photograph) showing the result of analyzing the purity of apolipoprotein A-1 obtained based on the production method of the present invention.

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Claims (5)

[Claims] 1. A centrifugal supernatant fraction obtained by incubating human plasma containing apolipoprotein A-1 with a lower aliphatic alcohol in a cold state is used as a raw material, and the supernatant fraction is subjected to hydrophobic chromatography in the presence of a lower aliphatic alcohol. A method for producing apolipoprotein A-1 which comprises a step of directly adding to a gel and developing a chromatography. 2. The method for producing apolipoprotein A-1 according to claim 1, wherein the centrifugation supernatant fraction is a centrifugation supernatant of fraction II + III obtained by low temperature ethanol fractionation of plasma based on the Cohn method. 3. From a hydrophobic chromatographic gel to which a centrifugal supernatant fraction has been added, an aqueous buffer solution containing a chaotropic agent at a concentration of 1 M or more or a lower aliphatic alcohol of 21% (V / V) or more. The method for producing apolipoprotein A-1 according to claim 1 or 2, wherein apolipoprotein A-1 is selectively eluted with a combined eluate. 4. The method for producing apolipoprotein A-1 according to claim 3, wherein the chaotropic agent is urea or guanidine hydrochloride. 5. The method for producing apolipoprotein A-1 according to claim 3, wherein the lower aliphatic alcohol is methanol, ethanol or propanol.