JPH0284195A - Production of natural-type human apolipoprotein e-like protein - Google Patents

Abstract

PURPOSE:To accomplish mass production of the title protein in high efficiency by transforming host microorganisms with a vector incorporated with DNA fragment coding a specific protein and by putting the resultant transformant to culture in a medium. CONSTITUTION:Firstly, e.g., human liver fragments are homogenized and treated with e.g., a restriction enzyme to produce DNA fragment (A) coding human apolipoprotein(HAP)E or HAPE-like protein similar thereto in physiological activity. Second, the DNA stemmed from Escherichia coli is treated with a restriction enzyme to obtain the DNA sequence (B) coding a signal peptide for secretion with such a DNA sequence near DNA initiation codon as that of formula I. Third, two to three of both the components A and B are tandemly linked to a manifestation vector (C) having both tac promoter and ribosome link domain of formula II to obtain a manifestation vector (D) such as pOFAapoE. Fourth, a transformant strain produced by incorporating the component (D) into e.g., Escherichia coli JM 109 strain is put to culture in a medium, thus producing the objective ca.50mg/1 of natural-type human apolipoprotein E-like protein in the medium.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention is directed to the production of natural human apoliboprotein E (Apol 1poprotein E) using recombinant DNA technology.
This invention relates to a method for microbiological production of similar proteins.

(Prior art and problems to be solved by the invention) Major lipids present in plasma include cholesterol, phospholipids, triglycerides, and free fatty acids, of which the first three do not bind to proteins. It exists. This lipid-protein complex, which is made by solubilizing water-insoluble lipids, is called riboprotein, and forms compounds of various weights depending on the ratio of lipid and protein. This riboprotein has a spherical particle structure, with nonpolar triglycerides and cholesterol esters present in the core, and polar phospholipids and free cholesterol together with fluorescence are thought to form the surface layer.

The protein contained in this ribofluorescence is called apoliboprotein, and more than 10 types are currently known. Apoliboprotein is not only necessary for constructing riboproteins, but also plays an important role in riboprotein metabolism. Apoliboprotein E is known as one of these apoliboproteins. This apoliboprotein E is known to serve as a recognition target when various cells in the body take in riboproteins via receptors.

Furthermore, E2, E3, and E4 have been discovered as major subclasses of apoliboprotein E, and while E3 is derived from normal individuals, E2 and E4 have been found from patients with type ■ fossolipidemia. and is considered to be an abnormal type [
The Journal' of Biolo
chemical chemistry), VOI, 255
．． lk5, 1759 1762.1980).

People who lack apoliboprotein E3 or have E2 or E4 often develop hyperlipidemia, which further leads to arteriosclerosis.

In such cases, normal apoliboprotein E function can be restored by administering normal E3 to the patient.

Some of the present inventors previously obtained the above-mentioned full-length cDNA fragment (see JP-A-60-118189) and previously proposed a method for expressing apoliboprotein E in Escherichia coli using this fragment. (Refer to Japanese Patent Application Laid-open No. 61-96997).

However, apoliboprotein E obtained by the above method is
Readout has extra amino acids such as methionine.

Furthermore, until now, there has been no report that a protein with a molecular weight similar to that of apoE could be secreted and produced in large quantities using E. coli as a host. Only the secreted large-scale production of is reported (Hsuing et al.
l, Bio/Technology vol. 4, 9
91.1986).

(Means for solving the problem) The present inventors conducted further studies and constructed a vector into which a DNA fragment encoding a secretion signal peptide was introduced. We were able to secrete and produce a large amount of human apoliboprotein E-like protein, which led to the completion of the present invention.

According to the method of the present invention, since it is no longer affected by the action of proteolytic enzymes, etc. in the host microorganism, it is possible to obtain approximately 1, (11) times more human apolipoprotein E-like protein than with conventional methods. It can be done.

That is, the gist of the present invention is that a DNA encoding a secretion signal peptide is placed downstream of the promoter of an expression vector.
A host microorganism is transformed with a vector into which a fragment and a DNA fragment encoding human apolipoprotein E or a human apolipoprotein E-like protein having similar physiological activity has been introduced, and the resulting transformant is cultured to produce a product. A method for producing a natural human apoliboprotein E-like protein, which comprises obtaining a natural human apoliboprotein E-like protein.

The present invention will be explained in detail below.

First, human apoliboprotein E used in the present invention
A DNA fragment containing DNA encoding human apoliboprotein E-like proteins such as 2, E3, and E4 is prepared, for example, by the following method. That is, human liver sections, small intestinal epithelial cells, blood macrophages, kidney sections, etc. are homogenized with guanidine isothiocyanate, and total RN is isolated by CsC1 equilibrium density gradient ultracentrifugation.
Separate A (Chirgwin et al., Biochemistry, supra 1, 5294-5
299.1979).

Next, this was purified by oligo(dT) cellulose column chromatography in a conventional manner to obtain poly(A)-containing RN.
Isolate A<mRNA raw material).

From this mRNA raw material, Okayama and Berg's method [Molecular and Cellular Biology (Molec
ular and Ce1lular Bjol
ogy), 2.161-170゜1982),
Obtain a cDNA library. That is, pBR322
and SV40 hyperprinted plasmids to obtain vector primers and oligo(dG) tail linkers.

Reverse transcriptase is allowed to act in the coexistence of this vector primer and the above mRNA to synthesize cDNA, and then restriction enzyme H
Digested with indII and then cyclized using the linker described above. Then, replace the mRNA part with DNA,
A plasmid containing a cDNA fragment is obtained.

Next, using this, Escherichia coli (Esche.
richia colt) etc. to be ampicillin resistant. Thereafter, as a probe, a synthetic oligonucleotide containing part or all of the base sequence corresponding to amino acid sequence 218-222 of apoliboprotein E (Met"Glu-Glu-Met-Gly) or at least a synthetic oligonucleotide containing the base sequence is used. Using nucleotides, select a clone containing a sequence complementary to this. Among the clones thus obtained, cut with an appropriate restriction enzyme and select a clone that has a plasmid containing the longest cDNA inserted. .

The cDNA fragment obtained from the clone is Maxa
m and G11bert's method [Methods in Enzymolog
)'), 65.499 560° 1980).

Examples of DNA fragments encoding secretory signal peptides include Omp F, OmpC, OmpA, alkaline phosphatase, and riboprotein.

Escherichia coli-derived products such as A m p C-β-lactamase and TEM β-lactamase, Bacillus subtilis-derived products such as α-amylase, subtilisin, and neutral protease, yeast-derived products such as amylase, and apoE.

Apo A-1, interferon, ISA, etc. derived from animal cells can be used, preferably derived from Escherichia coli, and furthermore, a DNA sequence of 6 bases before and after the start codon encoding the signal peptide can be used. It is preferable to substitute adenine (A) and thymine (T) on the downstream side without changing the amino acids of the signal peptide to increase the adenine and thymine contents to improve expression efficiency.

Specifically, for example, when the signal peptide is OmpF, its DNA sequence is converted to 5'-AAGCTTATGA.
TGAAG-3'5'-AAATTTATGAAG
-3'5'-AAGCTTATGATGAAA-3
' or 5'-AATTTATGAAA-3', etc., and the signal peptide is Om
pC, the DNA sequence is 5'-AAGCTT
ATGGTT-3 '5'-AAATTTATGGT
Examples include those modified to T-3', 5'AAGCTTATGGTA-3', or 5'-AAATTTATGGTA-3'.

The expression vector into which the DNA fragment encoding the human apoliboprotein E-like protein and the secretion signal peptide is introduced has a promoter at a position where transcription of these genes can be controlled.

Any known promoter can be used as long as it functions in the host microorganism, including the tac promoter, λP, and P.

Promoter, a promoter consisting of the RNA polymerase recognition region of a phage promoter and the RNA polymerase binding region of an Escherichia coli hyphal promoter (patent application 1986-2)
Hybrid promoters such as No. 68362) are suitable.

In particular, a hybrid promoter (tac promoter) in which the RNA polymerase recognition region has a base sequence represented by the following formula (T) and the RNA polymerase binding region has a base sequence represented by the following formula (n) or (III)
Upper soil! promoter) is preferred.

TOCT AACGA・・・・・・
- (1) TATAATG ATATTAC... (n) TATAATGTGTGG ATATTACACACC... (In) Specific examples of such expression vectors include, for example, pMT[
2 (Japanese Patent Application No. 61-268362).

In the present invention, it is preferable to use two or more of the above promoters, particularly two or three promoters linked in tandem, since this improves expression efficiency.

The secretory signal peptide and the DNA fragment encoding the human apoliboprotein E-like protein can be introduced into the expression vector by combining various known methods, as shown in the Examples below.

In the present invention, two or more genes, especially two or three genes encoding a fusion protein of a secretory signal peptide and human apolipoprotein E or a human apolipoprotein E-like protein having similar physiological activity, are present in tandem. It is preferable to introduce the ligated vector into an expression vector because expression efficiency will be improved.

Furthermore, the expression vector of the present invention has a ribosome binding region, such as lac, 1, ±LL, or λ-CI.
Those derived from I gene etc. are preferred.

Furthermore, an NA sequence that is moderately complementary to the 3' end of ribosomal RNA, such as 5'-AGGAGGTTT
-3',5'-AGGGTTTT-3'5'-AG
GAAAACTGA-3′,5′-TGGATTA
TTC-3' or 5'-AGGAGGTATA-
Those having 3' etc. are also preferred.

Specific examples of vectors obtained in this way include the vector pOFAapoE as shown in FIGS. 1 and 2.
and pNapoE or the vector pOFAap as shown in FIGS. 9 and 10.

E2 and pNapOEz.

In the present invention, a host microorganism is transformed with the above vector.

The host microorganism is E. coli JM109. HB101,
YK537. Escherichia coli such as YA21, Bacillus subtilis Ml 112
．． Examples include Bacillus subtilis such as NP58.

Transformation can be carried out using conventional methods, such as molecular cloning (Cold).
Spring Harbor, pp250.198
2. This can be done according to the method described in .

Next, the obtained transformant was cultured using M9 minimal soil,
This can be carried out using L-pross medium or the like according to a conventional method.

In the present invention, the secretory signal peptide and human apoliboprotein E [protein are expressed in the transformant, and when this passes through the cell membrane of the transformant, the secretory signal peptide part is cleaved, and the secretory signal peptide portion is released into the periplasm or A human apoliboprotein E-like protein having a natural N-terminus can be obtained outside the bacterial cell.

Furthermore, in the present invention, since the expressed target protein is secreted from the bacterial cells, it is less likely to be degraded by intracellular enzymes, and the production amount is even higher.

Specifically, according to the present invention, apoE can be produced at a high rate of about 50 μ/l. Therefore, the expression vector of the present invention
If the transformed transformants are cultured at high density using Jarfer Mentor, etc., the productivity will be further improved, and the productivity will be about 450 μ/cm.
1 of apoE can be produced.

(Examples) Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited by the following Examples unless the gist thereof is exceeded.

Secretory production of human apoliboprotein E in Escherichia coli (see Figure 3) 1) Subcloning of pKCRHAPE to pUC118 2 μg of pKCRHAPE prepared according to the method described in JP-A-61-285990 was added to buffer M (10
mM Tris-HCl (pH 7,5L 60mM sodium chloride and 6mM magnesium chloride) in 2Oplt) (
Complete digestion was carried out using indI[I2u at 37° C. for 2 hours. Next, a buffer solution was added to this reaction solution, and buffer solution H (
1(1mM Tris-HCl (pH 7,5), 1(1
1)mM sodium chloride and 6mM magnesium chloride)
The system was made into a 40 μl system, EcoRI'lu was added, and the mixture was further reacted at 37° C. for 2 hours to complete digestion. After that, 70
The enzyme was inactivated by heating at ℃ for 10 minutes to obtain a liquid.

Separately, 2μg of puc118 under the same conditions as above)
(Digested with indI[I and EC0RI to inactivate the enzyme to obtain a liquid.

After mixing the solution (1) and the solution O, the mixture was dialyzed against water and evaporated to dryness using a vacuum pump. Buffer L (I
QmM) Lis-hydrochloric acid, 6mM magnesium chloride, 1mM
dithiothreitol and 1mM ATP) 10 μj! was added, 1 u of T4 DNA ligase was added, and the mixture was reacted at 4°C for 16 hours.

Using 5 μl of this reaction solution, commercially available JMI09 competent cells (manufactured by Takara Shuzo Co., Ltd.) were transformed according to the instructions.

From the obtained transformant, a plasmid was isolated and purified according to a conventional method, and apized into pUc118.

Plasmid pUC1 with EDNA fragment inserted
18apoE was obtained.

[) Introduction of the cleavage site into the apoE gene (B
IO/TECHNOLOGY, 636-639.198
4) ■ Add 10 μg of pUcl 18apoE to buffer H10 (
Using EcoR110μ and Pst110μ in Lull, each was reacted at 37°C for 2 hours to completely digest.

After subjecting this to 5% polyacrylamide gel electrophoresis, it was stained with 0.05% ethidium bromide, and two DNA band fragments, l-a and r-
b was cut out from the gel and extracted according to a conventional method.

...Fragment I-a, I-b ■ pUcl
18apoE1ug in buffer I] 5ca in 10pi
The reaction mixture was completely digested using llu at 37°C for 2 hours. Then, alkaline phosphatase (E, Co1
1) was added, the mixture was reacted for 1 hour at 65°C, the phosphoric acid at the 5' end was removed, the protein was extracted with water-saturated phenol, the phenol was removed with ether, and the mixture was dialyzed against water. This product was evaporated to dryness using a vacuum pump, and then dissolved in 10 μl of water.

...Fragment ■ ■ Phosphorylation of mutant primer 36 base mutant primer (see Figure 4-1) 150
Buffer K (50mM Tris-HCl (pH 8,0), 10mM magnesium chloride, 5mM
Dithiothreitol and 10mM ATP) were reacted with IU of T4 polynucleotide kinase at 37°C for 1 hour to perform 5' phosphorylation.

■ Formation of heteroduplex Fragments I-a and I-b were added to 0.05 pmol. Fragment II 0.05 pmol and phosphorylated mutant primer 45 pmol/! Polymerase ligase buffer (0.5M sodium chloride, 32.5mM) at 5x concentration Lis-HCl (pH 7.5).

Add 12 μl of 40 mM magnesium chloride and 5 mM β-mercaptoethanol to make a total of 34.8 μl system,
The mixture was boiled at 1 (11)°C for 3 minutes and immediately placed in a constant temperature bath at 30°C for 30 minutes.

Next, the mixture was left at 4° C. for 30 minutes, and further left on ice for 1° to form a heteroduplex.

11.6 μl of an aqueous solution containing this heteroduplex
Add 2 μ2 of 2.5 mM 4-deoxynucleotide-3-phosphate to 21t1.10 mM ATP, and add 2 μ2 of 2.5 mM 4-deoxynucleotide-3-phosphate.
Klenow enzyme, o.

5 u of T4 DNA ligase was added, and a total of 20 ttl was reacted overnight at 12.5°C to circularize the DNA.

E. coli JM109 was transformed using 2 μl of the aqueous solution containing this circular DNA according to a conventional method to obtain a transformant. A plasmid was isolated and purified from this transformant according to a conventional method, cut with restriction enzyme 1<H>, and subjected to 0.8% agarose gel electrophoresis to obtain a cut plasmid as a mutant plasmid. In this case, the resulting mutant plasmid is often contaminated with the original plasmid (wild type), so this mutant plasmid is used again to
109 was transformed and the mutant plasmid was purified.

In this way, plasmid pUc118al)oEB was obtained.

■) Modification of expression vector A, plasmid pMT! 2 (see Figure 5) (11
Creation of Vibe I/Tsudo promoter 1 DNA fragment with the same sequence as the acUV5 promoter region, operator region and transcription start point, and 75P25 promoter.
DNA fragments having the same sequence as the 5 region (■ to ■ below) were synthesized using Nikkaki Co., Ltd. Model 308A DNA synthesizer.
Manufactured.

■ 35 of the 37 base two T5 Phage P25 promoter
Corresponds to a DNA fragment including the head region ■ 37 bases: Complementary to ■ NA fragment ■ 43 bases:
1acUV5 Corresponds to a DNA fragment containing the 106'l region and the operator region 43 bases: NA fragment complementary to
Deprotection was performed by reacting for 6 hours, and purification was performed using a 17% acrylamide-7M urea gel, resulting in 10 μg of each fragment. Then, 1 μg of these DNA fragments
was reacted with 1 u of T4 polynucleotide kinase in 10 μl of a buffer containing IQmM) Lis-HCl (pH 7,6) and I0mM magnesium chloride at 37°C for 1 hour.
phosphorylates the ′ end.

Next, add sodium chloride to a concentration of 50mM, mix the above DNA fragments ■ and ■, and mix ■ and ■, respectively, and mix 1(
11) Annealing was performed by heating at °C for 3 minutes and slow cooling to obtain 2 μg each of double-stranded 376p and 436p DNA fragments.

0.5 μg each of these DNA fragments was added to EC0RI and 1 (
21 μg of plasmid pBR32 treated with indDI was ligated with 10 mM)
Hydrochloric acid (pH 7.5), 6rr+M magnesium chloride,
The mixture is reacted at 4° C. for 16 hours and inserted in 10 μl of a buffer containing 10 mM ATP and 1 mM dithiothreitol.

Calcium chloride was added to this reaction solution to give a concentration of 1 (11) mM, and the mixture was mixed with E. coli HBIOI competent cells.
Transformation is performed at 0°C for 10 minutes and at 37°C for 5 minutes. Transformants acquire tetracycline resistance and can be easily selected by adding 20 gg/ml of tetracycline to the culture medium.

In this way, a supernatant promoter plasmid pBRpac having promoter activity was obtained. The nucleotide sequence of the two-promoter promoter was determined by the dideoxy method in a conventional manner. The dideoxy method is described in the literature Analytical Biochemistry (Anal, Biochem), Vol. 15.
2.232. (For competent cells, collect bacteria in the logarithmic growth phase using a conventional method, suspend in 1/3 volume of 0.1M calcium chloride at 0°C, let stand for 30 minutes, and collect the cells again. , 0.1 of 1/1 (11) volume
Obtained by suspending in M calcium chloride. ) (2) Introduction of a transcription termination sequence into the hybrid promoter Plasmid pBRpac (
4,4kbp) 18g of BamHI, Pヱand■ 1u each of buffer H10μ! The mixture was reacted for 2 hours at 37'C for digestion. The reaction product was precipitated with ethanol and then evaporated to dryness and stored.

On the other hand, plasmid plNI [[A1 (The EMBOJ, LVol,
3,10,2437.1984) 5μg
u, 10mM h lys-hydrochloric acid (pH 7,5) and 6
50μ of buffer containing mM magnesium chloride! 37° inside
Digest by reacting for 2 hours with 330mM Tris-acetic acid (p
H7,9).

Add 5.5μ2 of a buffer containing 660mM potassium acetate and 1 (11)mM magnesium acetate, treat with 10u of T4 DNA polymerase for 15 minutes at 37°C, scrape off the protruding 3' end, and heat at 70°C for 10 minutes. to deactivate the oxygen.

Next, 1 (11) mM 1-lis-hydrochloric acid (pl) was added to this reaction solution.
+7.5), add 6.0 μl of buffer containing 1M sodium chloride and 60mM magnesium chloride, and add BamHI5u.
After digestion at 37°C for 2 hours, 60 μm of water-saturated phenol was added to remove protein, and ethanol precipitation was performed three times for purification. As a result, 1JLP containing the transcription termination sequence
0.5 μg of the L3′ region (˜5(11) bp) fragment was obtained. This DNA fragment was added to the above-mentioned digested plasmid (pBR
pac) using 1 u of T4 DNA ligase, 10 mM
Plasmid pPac was obtained by reacting and inserting the cells at 4°C for 116 hours in 10μ2 of a buffer containing Tris-HCl (pH 7,5), 10mM magnesium chloride, 10mM ATP and 1mM dithiothreitol.

This plasmid is the lac IF strain JMIO
It can be stably maintained with 5 strains or JM109 strain. That is, p
Using 0.1 μg of Pac, JM105 strain was transformed according to the method described in (1) to obtain a transformant, which was then incubated in minimal medium 1 containing 20 μg of ampicillin/mj2.
When the plasmid was grown in β and purified by a conventional method, 1 mg of pPac was obtained.

(3) Introduction of multi-linker Next, a multi-linker linking the SD sequence and starting Met sequence shown in the figure was synthesized using model 308A manufactured by Nikkaki Co., Ltd., and purified according to the method described in (1) above. 10gg D
NA was obtained. 10 μg of the plasmid pPacl
10μ of a buffer containing mM Tris-HCl (pH 7.5), 1(11)mM sodium chloride and 6mM magnesium chloride! The mixture was cut with BamHI lu for 2 hours at 37°C, precipitated with ethanol, and then evaporated to dryness and stored.

This truncated plasmid pPac and the above multilinker-1μ
g was reacted at 4°C for 16 hours using 1 u of T4 DNA ligase in 10 μm of 59r solution containing 10 mM Tris-HCl (pH 7.5>, 10 mM magnesium chloride, 10 mM ATP, and 1 mM dithiothreitol) to remove the multilinker. The inserted plasmid pMT2 was obtained.

(4) Introduction of the lac repressor gene and plasmid pMC9 (Procedure of the National Academy of Sciences of the United States of America)
i, USA)+■, 3015.1983) 10μg
1 (11) mM) Lis-HCl (pH 7.5), 10
Buffer 1 (11) containing mM sodium chloride and 6mM magnesium chloride. Using EcoRrlOu in czf, it was reacted at 37°C for 2 hours, cut, precipitated with ethanol, and then evaporated to dryness and stored. As a result, a 1.7 kbp DNA encoding the repressor gene 1acl was obtained.
2.5 μg of fragment was obtained. Then, the plasmid pM
72 1 μg to 1 (11) mM) Lis-HCl (pi!7
．． 5′), EcoRI lu in 10μβ buffer containing 10mM sodium chloride and 6mM magnesium chloride.
was used to react at 37°C for 2 hours, cut, precipitated with ethanol, and then evaporated to dryness and stored.

This truncated plasmid pMT2 and the above 1.7 k bpD
lμg of NA fragment was added to 10mM l-Lis-HCl (9117,
5), 20μ2 of buffer containing 10mM magnesium chloride, 10mM ATP and lrnM dithiothreitol
ligation using 1 u of T4 DNA ligase to obtain plasmid pMTI2.

B. Modification 1 μg of pMTI2 prepared in A above was added to buffer S (1 μg).
Sm
Digestion was carried out using a I1 u at 37°C for 2 hours.

Next, the mixture was heated at 70°C for 10 minutes to deactivate oxygen, and then dialyzed against water and evaporated to dryness. To this thing,
Approximately 50 pmof of the 5' end was phosphorylated.
[Add I linker (5' pCCAAGCTTGG: manufactured by Zenshuzo Co., Ltd.), buffer ej, L101! T in the system of
Using 1 u of 4DNA ligase, the reaction was carried out at 4°C for 16 hours. This was used to transform E. coli JM109 competent cells, and DNA was isolated from the transformants.
Purify and cleave with HindIII to obtain linker-D
A plasmid pMTI2H containing NA was obtained.

Next, 1 μg of pMT12H was reacted with AvaIlu in 10 μ2 of buffer M at 37°C for 2 hours, and after digestion, the enzyme was treated at 70°C for 10 minutes to inactivate the enzyme. This material was dialyzed against water and then evaporated to dryness.

To this, buffer P (33mM Tris-acetic acid (pH 7,9)
1 u of 74 DNA polymerase was added in a 20 μ2 system (66 mM potassium acetate, 10 mM magnesium acetate, 2.5 mM 4-deoxynucleotide triphosphate), and the mixture was reacted at 37°C for 15 minutes. After inactivating the enzyme by treating at 70°C for 10 minutes, dialysis was performed against water.
Evaporated to dryness. This product was added to T in buffer LIOpl system.
1 u of 4DNA ligase was added and reacted at 4°C for 16 hours to transform E. coli JM109 competent cells. From the obtained transformant, DNA was isolated and purified, and A
The product was digested with vaI to obtain plasmid pMTI2HA in which the Ava I cleavage site had been deleted.

IV) Introduction of apoED DNA fragment into pMTI2HA 10 μg of pUcl 18apoEB was added to buffer M1 (11
)1! Hind III, LLLI! 2 each
After reacting and digesting at 37°C for 2 hours,
D containing a gene encoding approximately 1 kbp of ApoE
The NA fragment (apoE fragment) was separated by 5% acrylamide gel electrophoresis, stained with 0.05% ethidium bromide, cut out, and extracted according to a conventional method.

Separately, 1 μg of pMTI2HA was hydrated in buffer MIOμ2.
indm,LLLI! After reaction and digestion with 1 U of each product at 37°C for 2 hours, the protein was extracted with water-saturated phenol, and the phenol was further extracted with ether, followed by dialysis against water and evaporation to dryness. this thing a
p.

Add the E fragment lpmof and add 11 mL of 10μ2
system using 1 u of T4 DNA ligase at 4°C.
The reaction was allowed to proceed for 6 hours.

Using this product, E. coli J, M109 competent cells were transformed, and the plasmid was isolated and purified from the resulting transformant, and examined by Hindm and ln digestion to determine the plasmid into which the apOE fragment had been inserted. pMTapoEO was obtained.

(2) Creation of secretion vector A, Creation of plasmid phMAVD/1acI (Japanese Patent Application No. 61-220834) (1) Human heart fragments were crushed in liquid nitrogen, and then an aqueous solution of guanidinium thiocyanate was added and homogenized. The obtained homogenate was subjected to the method of Chirgwin et al.
, 18. 52945299.1979) by cesium chloride equilibrium density gradient ultracentrifugation.
A was isolated. Next, this was converted into oligo(dT) by a conventional method.
) The poly(A)-containing RNA was purified by cellulose column chromatography and used as a raw material for m RNA.

(2) On the other hand, Okayama and Berg's method [Molecular and Cellular Biology]
and Celular Bi.

1ogy), 2, 161-170, 1982), p.
B, A vector primer and oligo(dG) tail linker were obtained using a hybrid plasmid of R322 and SV40.

That is, pBR322 and 5V4Q (Ma, Buuniisoto
Hybrid plasmid 4 (0.71-0.86)
11) μg was digested with restriction enzymes using -z technology at 37°C for 4 hours in a buffer containing bovine serum albumin.

Next, the DNA was recovered by ethanol precipitation using a conventional method, dissolved in Ili solution containing dTTP, terminal deoxynucleotidyl transferase was added, the reaction was carried out at 37°C for 30 minutes, and the DNA was digested with restriction enzymes. After adding >60 dT details to the digestion site of ``'', the DNA was recovered by ethane precipitation. This [)NA was then digested with 1 ul of restriction enzyme in a buffer containing bovine serum albumin (15 hours at 37°C). The larger DNA fragment was purified by agarose gel electrophoresis and subjected to glass powder method (Voge
lsLein et al. [Proceedings of the National Academy of Sciences of the USA (Proc. Nat I, Acad.

Sci, U, S, A, ), -u, 615 6
19.1979). Then this DN
A was applied to an oligo(dA) cellulose column with O'C,
Yoshikawa (with water), recovered with ethanol, oligo(d
A vector primer with a T') tail was obtained.

On the other hand, pBR322 and 5V40 (manp unit 0.1
Hybrid plasmid 1 (1
1) μg was digested with the restriction enzyme Pstl in a buffer containing bovine serum albumin (1.5 hours at 37°C).

Then, the DNA was collected and dissolved in a buffer containing dGTP, and terminal deoxynucleotidyl transferase was added and reacted at 37°C for 20 minutes to add approximately 10 to 15 dG tails. Recovered DNA
and the restriction enzyme Li! in $11i solution containing bovine serum albumin. Digested with Ld[[(1 hour at 37°C),
This was then subjected to agarose gel (1.8%) electrophoresis, and small oligo (dG) tail linker DNA was extracted and recovered.

(3) Okayama and Berg's method [Molecular and
Cellular biology
d Ce1lural Biol.

gy), 2,161-170.1982), cD
Obtained NA library.

Namely, tris-hydrochloric acid (pH 8.3), magnesium chloride, potassium chloride, dithiothreitol, dATP
, To an aqueous solution containing dTTr', dGTP, and (szp)dcTP, 30 μg of the mRNA obtained in (1) above and 10 μg of the vector primer obtained in (2) above were added, and the mixture was incubated at 37° in the presence of reverse transcriptase. The mixture was reacted at C for 20 minutes to synthesize plasmid c DNA:mRNA, which was precipitated with ethanol and collected in the form of a pellet. This pellet is made of cobalt chloride, dithiothreitol, poly(A)
, (“P) dissolved in a buffer containing dcTP and terminal deoxynucleotidyl transferase, 3
After reacting at 7°C for 10 minutes, the recovered oligo (dC
) Tail plasmid-cDNA: The pellet containing mRNA was dissolved in a buffer containing bovine serum albumin, digested with the restriction enzyme HindI[I at 37°C for 11 hours,
HindI [[digested oligo(d
C) Tail cDNA: mRNA plasmid was recovered.

This was dissolved in a buffer containing the oligo(dG) tail linker DNA obtained in (2) above, incubated at 65°C for 2 minutes, further kept at 42°C for 30 minutes, and then
Cooled to °C. Next, in the presence of βNAD (cotinamide adenine dinucleotide), Escherichia coli (cotinamide adenine dinucleotide)
) Add DNA ligase and incubate overnight.

Then dATP, dTTP, dGTP, dCTPl
β-NAD, E, Co I 1 DNA ligase, E, c
oliDNA polymerase and dan.

E. coli, RNa5e H was added and the mixture was incubated at 12°C for 1 hour, then at room temperature for 1 hour, then cooled to stop the reaction and release the desired cDNA.
A plasmid containing the A fragment was obtained.

Next, using this plasmid, E. coli (
E. coli) HBIOI was transformed.

(4) On the other hand, Met-AspAr of cardionathrin
Two groups of nucleotides complementary to the DNA sequence encoding g-11cmGly were synthesized.

T CT T CT Next, a cDNA library is screened using this oligo I and ■ as a probe, and the 4o and ooo transformants hybridize with the same probe.
Two clones were selected. cDNA fragments were extracted from these 12 clones, a restriction enzyme map was created, and based on the map, the Maxam-Gilbert method [Methods in Enzymology] was used.
n Enzymology), 65.499-560
, 1980) was used to determine the base sequence of the fragment (pHANF48) [Nature (Natu
r c), , 31.023 699.1985).

In addition, the DN of AVD (Atrial Batho Dei Latin)
The A sequence has 72 amino acids estimated from its molecular weight of 1.5 (11), and was estimated from the position of arginine, which is easily processed in vivo.

The above-mentioned human-derived AVD is the above-mentioned pig-derived AVD C.
Anatomy and Embryon
y and EmbryolOgy, 168,30
7-313.1983), there are 4 differences in the 30 amino acid sequences on the N-terminal side.

Preparation of plasmid phAJD> (1) Digest PHANF4B with pvulI and Rsal to obtain a 301 bp fragment containing AVD. This was combined with a methionine linker and T4 DNA gauze (14 hours at 8°C), and then the restriction enzyme Hind
Digest with 1I [37°C for 12 hours] to obtain a 333 bp fragment. This fragment and a linker having a ribosome binding sequence were ligated with T4 DNA ligase at 8°C for 14 hours) and further digested with BamHI at 37°C for 2 hours to obtain a 365 bp fragment.

On the other hand, plasmid pUc8 was digested with the restriction enzyme BamHI (at 37°C for 2 hours), and the above 365 bp fragment was introduced into this BamH1 site. Then, E. coli JM105 was transformed and white colonies on XgaE were selected to obtain plasmid pUCcd8.

Partially digest the above plasmid with the restriction enzyme Aval.
30 min at °C), the linker described above containing a stop codon (
ligation linker) in the presence of 74 DNA polymerase to create plasmid pUCcd8te
I got rm.

Furthermore, plasmid pDR5 with tac promoter
Digest 40 with restriction enzymes EcoRI and BamHI 37
2 hours at °C), an approximately 370 bp fragment was obtained. On the other hand, the above plasmid pUccd8 term was
Digest with IS BamHI at 37°C for 12 hours), 26
A 0bp fragment was obtained.

-, pHANF48 was digested with AJLLI and EcoRl to obtain a large fragment containing the ampicillin resistance gene.

After ligating the three fragments thus obtained with T4 DNA ligase (14 hours at 8°C), the resulting plasmid was transformed into Escherichia coli JM1058, and the plasmid phAVD was obtained from the transformed strain.

Plasmid pHFOO6C Nucleic Acids
Research (Nuclicic Ac1d Re5ea)
rch) Sat U, 6957.1982] with the restriction enzyme Pst
A 178 bp PstI fragment was obtained.

On the other hand, plasmid psP64 (Boehringe
rManhcim) was digested with restriction enzyme t1↓I, then treated with alcal phosphatase, and ligated with the above PstI fragment to create plasmid pSP64・Om.
pF was obtained.

This plasmid psP64・OmpF was treated with restriction enzyme Hi.
After digestion with ndI[[ and Ba131, T4D
The ends were blunted with NA polymerase. Then HindI
[I linker was ligated, further digested with HindlI[, and then closed with ligase.

Next, E. coli was transformed with this plasmid, and plasmid DNA was isolated from the resulting transformed strain. This plasmid was digested with HindI [[, Pstl,
A fragment smaller than 0 bP was selected, and clone Nα33 was selected (Pstl-Hindlll digested fragment is 130 bp
).

The resulting plasmid psP64・ompFN.

33 was digested with Pstl, blunted with T4 DNA polymerase, and a lJ'' linker was integrated, followed by ■''''.
The plasmid psP64.o was digested with
mpF-Hpa I was obtained.

<Construction of plasmid pUc-OmpF-3+Met> (1) Plasmid pUC8 was digested with EcoRI and then with 1ndl[[, and the Amp' gene was extracted with EcoRI.
The RI-Hindl[I fragment was obtained.

(2) Plasmid psP64・OmpF-Hpa
Digest I with EcoRI and then Hindm,
A fragment of approximately 2 (11) bp was obtained. Further digestion with Hha I yielded an 85 bp fragment containing the OmpF signal peptide region.

The two fragments obtained in (1) and (2) above were ligated with a synthetic linker using T4 DNA ligase to create plasmid pUC-
OmpF-3+Met was obtained.

<Construction of plasmid phMAVD> Plasmid pUC-OmpF-3+Met was converted to HindI
[l, digested with FnuDn to obtain an approximately 70 bp fragment containing the OmpF signal peptide region.

On the other hand, the plasmid pHANF48 obtained above was transferred to Al
The fragment was digested with UL to obtain a 269 bp fragment containing the AVD gene region, and further digested with human soil ③ to obtain an approximately 220 bp fragment.

These two fragments were ligated, and this was further combined with the p
ligated with the larger fragment obtained by digestion with hAVDeHindm and 8llI using T4 DNA ligase,
Plasmid phAVD・ΔST was obtained.

The obtained plasmid was 71. The larger fragment obtained by digestion with y-E I and plasmid phAVD were
The smaller fragment obtained by digestion was ligated with T4 DNA ligase to obtain plasmid phMAVD-8P.

10
Insert the 4bp tac promoter and create the plasmid ph
Got MAVD.

This was digested with EcoRI and treated with BAP, while plasmid pMC9 was digested with EcoRI to obtain a 1.7 kbp fragment containing the lacl gene, and both were ligated to obtain the plasmid phMAVDlacl shown in Figure 6. Ta.

2B, ■ Plasmid p-hMAVD created in A above.
lacllOIlg was incubated at 37°C in buffer H1(11)pE using 10u each of HI and AvaI.
After 2 hours of reaction and digestion, the DNA fragments were separated by 5% acrylamide gel electrophoresis and 0.0
Large DNA was stained with 5% ethidium bromide.
A fragment (fragment ■) was cut out and extracted according to a conventional method.

■ 11 μg of phMAVD-1ac was added to 10 μg of buffer H.
After digestion with Pstllu in F, the enzyme was inactivated by heating at 70°C for 10 minutes. Next, it was dialyzed against water and evaporated to dryness.

This was reacted with T4 DNA polymerase in buffer P20μ2 at 37°C for 15 minutes, heated at 70°C for 10 minutes to inactivate the enzyme, and then dialyzed against water and evaporated to dryness. After that, the fragment was further dissolved in 10 μl of water to obtain a solution of fragment ①.

■ 5' phosphorylation of mutation primer 36 base mutation primer (Figure 4-2) 150 pmo
f was 5' phosphorylated using 1 u of T4 polynucleotide kinase in 10 μf buffer at 37° C. for 1 hour.

■ Heteroduplex formation fragment I0.05pmof, fragment II
0.05 pmol and 45 pmol of 5'-phosphorylated primer, 5x polymerase ligase buffer (0.5M sodium chloride, 32.5mM), Lis-HCl (pf 17.5), 40mM magnesium chloride. and 5mM β-mercaptoethanol) at 12
μ! Add to this a total of 34° 8 μl system, and dilute with 1 OO'C for 3
The mixture was boiled for a minute and immediately placed in a constant temperature bath at 30°C for 30 minutes. Next, the mixture was left at 4°C for 30 minutes and then on ice for another 10 minutes to form a heteroduplex chain.

11.6 pl of an aqueous solution containing this heteroduplex
Add 2μE of 2.5mM 4-deoxynucleotide triphosphate, 2μE of 10mM ATP, and add 2μE of 2.5mM 4-deoxynucleotide triphosphate to
Klenow enzyme and 0.5 u of T4 DNA ligase were added, and the reaction was carried out at 12.5° C. for 16 hours in a total of 20 μ2 to circularize the DNA. Escherichia coli JM109 was transformed using 2 µm of the aqueous solution containing this circular DNA according to a conventional method to obtain a transformant.

The plasmid was isolated and purified from this transformant according to a conventional method, cut with restriction enzyme . The mutant plasmid obtained in this case often includes the original plasmid (
This mutant plasmid was used to transform Escherichia coli JMI09 again to purify the mutant plasmid. In this way, plasmid pOmpKpn
I got I.

Add pMTI2 Iug to buffer LIOμn:
Digestion was performed using n1lu at 37°C for 2 hours. After further heating at 70°C for 10 minutes to inactivate the enzyme, the mixture was dialyzed against water and evaporated to dryness. Next, use ■1↓-r lu in buffer HIOμl to 37°
After digestion at C for 2 hours, the enzyme was inactivated by heating at 70°C for 10 minutes, dialyzed against water, and evaporated to dryness.

Meanwhile, pompKpnl is added to 1nl and P in the same procedure.
digested with stl and evaporated to dryness.

The above two digested DNA fragments were added to ll + i solution L 1
0 u 1? The mixture was dissolved and reacted with 1 U of T4 DNA ligase at 4°C for 16 hours. E. coli JM109 competent cells were transformed using 3 μl of the second one, and the plasmid was isolated and purified from the resulting transformant.
Cut with HindI[I and -5-aI restriction enzymes to create a fragment containing the IacT-dandan and F of pompKpnl and the replication origin of pMTI2, -1.
- A ligated plasmid pOFA with a fragment containing a transcription termination sequence and a multicloning sequence was obtained.

VI) 1 μg of pMTapoEO was digested with KLLlIu in 10 μf of buffer L at 37° C. for 2 hours, and further heated at 70′C for 10 minutes to inactivate the enzyme. After dialysis against water, it was evaporated to dryness.

Thereafter, digestion was performed at 37°C for 2 hours using 3ca Iu in buffer HIO μl, and the protein was extracted with water-saturated phenol, and then the phenol was extracted with ether.
It was dialyzed against water and evaporated to dryness.

On the other hand, x, LlI and S
Digested in car and evaporated to dryness.

The above two digested DNA fragments were added to Hime Wataru i solution L10.
4 using 1 u of T4 DNA ligase in uN.
After reacting for 16 hours at °C for ligation, 2μ of the reaction solution was
2 to transform JM109 competent cells,
Isolate and purify the plasmid from the obtained transformant7.
After digestion with EcoRV, pOF″A was purified by 611E′2 electrophoresis on a 0.8% agarose gel (7).
A plasmid pOFapoEo in which a fragment containing the I a c-I -1 screaming IF and a fragment containing the apoE gene of pMTapoE○ and a replication origin are linked.
Tokunoko.

1 μg of pOFapoE0 was digested with buffer 'a, L10al by reaction with -[LlI I u at 37''C for 2 hours, and the enzyme was inactivated by further heating at 70'C for 10 minutes. After dialysis, it was evaporated to dryness.Next, it was reacted at 37°C for 15 minutes using 1 u of T4 DNA polymerase in a system containing 20 μr of buffer P.
By LLLI digestion, the protruding portion less than 3' was removed to give a blunt end. The enzyme was inactivated by heat treatment at 7°C for 15 minutes, followed by dialysis against water and evaporation to dryness. This was digested by reacting with Aval lu in buffer solution M10uf for 2 hours at 37°C, then the protein was removed with water-saturated phenol, the phenol was further removed with ether, and then the mixture was dissolved in water. Dialysis was performed and evaporation to dryness yielded fragment (■).

D the following DNA linker corresponding to the N-terminus of apoE.
NA synthesizer (manufactured by Nikkaki Co., Ltd.: Appiied Bios)
system (Ma2O) and purified according to conventional methods.

TTT CTA CTT GTT CG^ C
AT CTT This synthetic NA fragment 150 pmon
was phosphorylated at the 5-end using T4 polynucleotide kinase IU in buffer KIOμ°C. Next, 2(11
) Add 10 μl of mM sodium chloride to 1(11)'
Annealing was performed by boiling at C for 3 minutes and slow cooling.

Add 15 pmof of the 5' phosphorylated DNA linker to the fragment (2) above, add 10 u of buffer L! At T
Using 1 u of 4 DNA ligase, ligate by reacting at 4'C for 16 hours. Using this 5 μ2, E. coli JM1
09 competent cells were transformed, plasmids were isolated and purified from the resulting transformants, and Dankodan HI and Av
The desired plasmid pOFAa was confirmed by 5% acrylamide gel electrophoresis.
Obtained poE.

■) Secretory production of apoE E. coli JM109 carrying pOFAa poE, ■
, broth (10 g of hectotributone in 11 parts of water).

Bacto yeast extract 5g, sodium chloride L
og, ampicillin 20μ) at 30°C overnight. This culture was diluted 50 times in fresh broth and incubated at 30°C for 5 hours, followed by isopropyl-β-D-thiogalactopyranosomes, an inducer of the tac promoter on the plasmid. Acid was added to a concentration of 2 mM to induce secretory production of apoE, and the culture was continued for an additional 3 hours. After that, centrifugation (6,0OOr
, p, m for 10 minutes) to collect bacteria. Furthermore, 1 g (wet) of bacterial cells was added to 33 mM) lithium-hydrochloric acid (p118).
．． 0) and suspended in 20% sucrose solution 20mj2, 25
Shake at °C for 10 minutes. Next, centrifugation (10,0O
After removing the supernatant, 20 ml of cooled water was added, thoroughly stirred in water for 10 minutes, and centrifuged (8.0 OOr, p, m, for 10 minutes).
When the bacterial cells were removed (for 10 minutes) and the supernatant (periplasm fraction) was lyophilized and subjected to SDS polyacrylamide electrophoresis, it was confirmed that a significant amount of human apolipoprotein E was secreted and produced in the periplasm. Ta.

The N-terminus of human apolipoprotein E is LySVa
1-Glu-Gin-Ala-Va I-It was confirmed that it was a natural type.

Example 2 Secretory production of human apolipoprotein E in Escherichia coli using signal peptide 1 of human apolipoprotein E (see Figure 7) I) Creation of tac promoter vector PMTI2 1
μg of EcoRI and B in a buffered HIOμ2 system.
After reaction and digestion with 1 u each of amHI at 37°C for 2 hours, the enzyme was further heated at 70°C for 10 minutes to inactivate the enzyme.

Separately, 1 μg of pDR540 (purchased from Pharmacia)
was digested with EcoRI and BatanHr by a similar procedure.

After mixing the above reaction solution, add 2μ of 10x buffer solution! In addition, 1 u of T4 DNA ligase was added and the mixture was reacted at 4'C for 16 hours to perform ligation. Escherichia coli JM109 competent cells were transformed using 1oμ2 of this product, and the plasmid was isolated and purified from the resulting transformant.
It was digested with oRV and examined by 0.8% agarose gel electrophoresis to obtain plasmid pUs2 into which the tac promoter fragment of pDR540 was inserted. This P
US2 was converted to Ec containing ±unil by EcoRI digestion.
Since the oRI fragment was missing, pMTI2 was digested with EcoRr to create a 1.7 kb fragment containing 1acl.
The pEcoRI fragment was separated and purified by acrylamide gel electrophoresis in a conventional manner to obtain plasmid pUsI2 which was reintroduced into the EcoRI site of pUs2.

■) 1 μg of pMTapoE0 was incubated at 37°C using 1 u each of BamHI and Sca I in 10 μff of buffer H.
After digestion for 2 hours at 7°C,
The enzyme was inactivated by heat treatment for a minute.

Separately, 1 μg of pUSI2 was added to Sca by the same procedure.
Digested with T and BamHT.

After mixing the above reaction solution, add 10 times the concentration of buffer [7 to 2μ!
Add 1 u of T4 DNA ligase and incubate at 4°C.
:J Reacted for 6 hours and ligated. Transform E. coli JM109 competent cells using 10μ2 of the second
Plasmids were isolated and purified from the obtained transformants, and E.
It was digested with coRV and examined by 0.8% agarose gel electrophoresis to obtain plasmid pUsapoEo into which the tac promoter fragment of pUsI2 was inserted.

■) Introduction of HindlI [site] into the N-terminus of human apoliboprotein E signal peptide by site-directed mutagenesis (1) 10 μg of PUSapoEO was added to buffer H1 (
11) Digest by reacting with 10 u each of BamHI and AvaI at 37°C for 2 hours.

This was subjected to 5% acrylamide electrophoresis, stained with 0.05% ethidium bromide, and the larger DNA fragment (fragment I) was cut out from the gel and extracted.

(2) 1 g of PU S apoE O was dissolved in 5 car lu with 10 μ of buffer solution H.
After digestion by reacting at 7°C for 2 hours, add 1 u of alkaline phosphatase, react at 60'C for 1 hour to remove the 5'-end phosphate group, then remove the protein with water-saturated phenol, and add ether. Yo-rifenol was removed, the residue was dialyzed against water, evaporated to dryness, and dissolved in 10 μ2 of water to obtain a solution of fragment (2).

(3) 5' phosphorylation of the mutant primer A 41-base mutant primer (see Figure 8) 5Qpmof was reacted at 37°C for 1 hour using 10 μl of buffer solution with T4 polynucleotide kinase. Phosphorylated.

(4) Formation of heteroduplex fragment I0.05pmoffi, fragment 1
10.05pmof and 45p of 5' phosphorylated blymer
Polymerase ligase buffer (0
, 5M sodium chloride, 32.5mM) Lis-HCl (p
H7,5), 40 mM magnesium chloride and 5
Add 12μ2 of mM β-mercaptoethanol) for a total of 3
4.8 μ2 system, boiled at 1 (11) °C for 3 minutes,
Immediately, it was placed in a constant temperature bath at 30°C and left for 30 minutes. Next, the mixture was left to stand at 4°C for 30 minutes and further left on ice for 10 minutes to form a heteroduplex.

Aqueous solution 11,6 t containing this heteroduplex
At t 1, add 2μ of 2.5mM 4-deoxynucleotide triphosphate! 2με of 10mM ATP were added, and 2u of Klenow enzyme and 0.5u of T4 DNA ligase were added, and the reaction was carried out at 12.5°C for 16 hours in a total of 20ul of the system to circularize the DNA.

E. coli JM109 was transformed using 2 μl of the aqueous solution containing this circular DNA according to a conventional method to obtain a transformant. A plasmid was isolated and purified from this transformant according to a conventional method, cut with restriction enzymes, and confirmed by 0.8% agarose gel electrophoresis to obtain a mutated plasmid. The mutant plasmid obtained in this case often contains the original plasmid (
Since the mutant plasmid was contaminated with wild type, E. coli JMI09 was transformed again to purify the mutant plasmid.

In this way, plasmid pUsapoEB) was obtained.

TV) Using 10 μg of pOFAapoE, buffer M1 (
11) In the μβ system, using HindI[[10u,
Incubate at 37°C for 2 hours. After subjecting this material to 5% acrylamide gel electrophoresis, it was stained with 0.05% ethidium bromide, and HtndII of approximately 11 Obp was
The I tac promoter fragment is excised and extracted from the gel.

Separately, add 1 μg of pUsapoEBH to 10 μg of buffer M! After cleavage using HindII[lu at 37°C for 2 hours, alkaline phosphatase
After 1 hour of reaction at 65°C to inactivate the enzyme with water-saturated phenol, the phenol was removed with ether, dialyzed against water, and evaporated to dryness. Add HindlII tac promoter fragment o, 1pmoj2 to this, and add buffer LI
Oμjl! Add 1 u of T4 DNA ligase in the system of 4
Ligation was performed by reacting at °C for 16 hours.

Using 5 μl of this reaction solution, E. coli JMI 09 was transformed, and the plasmid was isolated and purified from the transformant.
(The desired plasmid pNapoE was obtained by cutting with a restriction enzyme such as indln.

When E. coli harboring pNa poE was cultured under the same conditions as in Example 1, it was confirmed that natural human apolipoprotein E was secreted and produced.

Example 3 Plasmid pOFAap created in Example 1.

Cut E with BamHI and jLL soil ■, according to the usual method,
The fusion gene of OmpF signal peptide and apoE gene was separated and purified by 5% polyacrylamide gel electrophoresis. This product was further introduced into the cleavage site of pOFAapoE, and the OmpF signal peptide and a
A plasmid pOFAapoE into which two poE gene fusion genes were introduced was obtained (Figure 9).

Plasmid pNapoE created in the same manner as in Example 2
was cleaved with BamHI and Dankodo, and the fusion gene between the natural apoE signal peptide and the apoE gene was separated and purified by 5% polyacrylamide gel electrophoresis according to a conventional method. Add this to pNapoE
r cleavage site, apoE signal peptide and ap
Plasmid p into which two oE gene fusion genes have been introduced
NapoE2 was obtained (Figure 10).

Example 4 (Pharmacological effect of apoE produced by Escherichia coli) E. coli transformed with pOFAapoEz obtained in Example 3 was cultured, and apoE was purified according to a conventional method.
Conventional method (R, E, 5tephans.

Anal, Biochemi, vol. 65, 369.
Using natural apoE purified from blood, the binding ability to LDL receptors and the ability to form complexes with phospholipids were determined according to the method of T. L. Innerarity et al. (Journal of Biological Chemistry (J. Bi. .

1, Chem, ), vol, 254.4186°197
When measured according to 9), both showed equivalent activity.

(Effects of the Invention) According to the method of the present invention, natural human apolipoprotein E-like protein can be efficiently expressed, and the resulting protein (apolipoprotein E2, E3, or E4, etc.) can be used as an antigen by a conventional method. Antiserum was obtained and used to detect the deficiency of apolipoprotein normal type E3 or abnormal type (E2, E
4) can be detected.

Furthermore, the obtained apolipoprotein E3 can also be used as an antihyperlipidemic agent and an antiarteriosclerotic agent.

[Brief explanation of the drawing]

FIG. 1 shows the vector pOF of the present invention prepared in Example 1.
1 is a diagram schematically showing AapoE. Figure 2 shows the vector pNa of the present invention prepared in Example 2.
It is a drawing showing an outline of poE. FIG. 3 is a diagram schematically showing the construction of pOFAapoE. Figures 4-1 and 4-2 show pOFAap. FIG. 2 is a diagram showing mutant primers used in constructing E. FIG. 5 is a drawing schematically showing the construction of pMTI2 used in Example 1. Figure 6 shows phMAVD・lacl used in Example 1.
FIG. FIG. 7 is a diagram schematically showing the construction of pNapoE. FIG. 8 is a diagram showing mutant primers used in constructing pNapoE. FIG. 9 is a diagram schematically showing the construction of pOFAapoEz. FIG. 10 is a diagram schematically showing the construction of pNapoEz. Applicant Mitsubishi Kasei Corporation

Claims (1)

[Scope of Claims] (1) A DNA fragment encoding a secretion signal peptide and a DNA encoding human apolipoprotein E or a human apolipoprotein E-like protein having a similar physiological activity to the promoter of the expression vector. A method for producing natural human apolipoprotein E-like protein, which is characterized in that a host microorganism is transformed with a vector into which the fragment has been introduced, and the resulting transformant is cultured to obtain the produced natural human apolipoprotein E-like protein. Production method. (2) The method according to claim 1, wherein the DNA fragment encoding the secretory signal peptide is derived from E. coli. (3) The secretion signal peptide is OmpF, OmpC,
OmpA, lipoprotein, alkaline phosphatase,
The method according to claim 1, wherein the signal peptide is AmpC-β-lactamase or TEMβ-lactamase. (4) Secretory signal peptide is OmpF or Omp
The method according to claim 3, wherein C. (5) The method according to claim 1, wherein in the DNA fragment encoding the signal peptide for secretion, a DNA sequence near the start codon of the DNA encoding the signal peptide is rich in adenine and thymine content. (6) The method according to claim 5, wherein the DNA sequence near the start codon of the DNA encoding the signal peptide is expressed as "There is a gene sequence" or "There is a gene sequence." (7) The method according to claim 1, wherein two or more promoters are linked in tandem. (8) The promoter is ¥tac¥promoter or ¥
2. The method according to claim 1, wherein the pac\ promoter. (9) The expression vector is ¥lpp¥, ¥trp¥, ¥la
The method according to claim 1, which has a ribosome binding region derived from c\ or λ-CII gene. (10) The method according to claim 9, wherein the expression vector has a ribosome binding region derived from \lac\. (11) Claim 1 wherein the expression vector has a \tac\ promoter and a ribosome binding region derived from \lac\
Method described. (12) The method according to claim 1, wherein the expression vector has a ribosome-binding region represented by [there is a gene sequence] or [there is a gene sequence]. (13) Two or more genes encoding a fusion protein of a secretory signal peptide and human apolipoprotein E or a human apolipoprotein E-like protein having similar physiological activity are linked in tandem, and encode the fusion protein. An expression vector in which a gene is under the control of a promoter. (14) The expression vector according to claim 13, wherein the promoter is \tac\ promoter or \pac\ promoter. (15) The expression vector according to claim 13, wherein the DNA fragment encoding the secretion signal peptide is derived from E. coli. (16) Secretory signal peptides are OmpF and OmpC
14. The expression vector according to claim 13, which is a signal peptide of OmpA, lipoprotein, alkaline phosphatase, AmpC-β-lactamase or TEM β-lactamase. (17) Secretory signal peptide is OmpF or Om
The expression vector according to claim 16, which is pC. (18) The expression vector according to claim 13, wherein in the DNA fragment encoding the signal peptide for secretion, a DNA sequence near the start codon of the DNA encoding the signal peptide is rich in adenine and thymine contents. (19) The expression vector according to claim 18, wherein the DNA sequence near the start codon of the DNA encoding the signal peptide is expressed as [gene sequence exists] or [gene sequence exists]. (20) The expression vector according to claim 13, wherein two or more promoters are linked in tandem. (21) The expression vector according to claim 13, wherein the promoter is \tac\ promoter or \pac\ promoter. (22) ¥lpp¥, ¥trp¥, ¥lac¥ or λ
- The expression vector according to claim 13, which has a ribosome binding region derived from the CII gene. (23) The expression vector according to claim 22, which has a ribosome binding region derived from \lac\. (24) The expression vector according to claim 13, which has a \tac\ promoter and a ribosome binding region derived from \lac\. (25) The expression vector according to claim 13, wherein the DNA sequence of the ribosome binding region is expressed as [gene sequence exists] or [gene sequence exists]. (26) The expression vector according to claim 13, which is pOFAapoE_2. (27) The expression vector according to claim 13, which is pNapoE_2. (28) An expression vector that is pOFAapoE. (29) An expression vector that is pNapoE. (30) E. coli transformed with the expression vector according to any one of claims 9 to 29.