JPH0448432B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing human kallikrein-like protein, ie, human kallikrein or a human kallikrein-like substance having physiological activity equivalent to it. (Prior art and problems to be solved by the invention) Kallikrein research began in 1926 when Frey discovered a non-dialyzable substance in human urine that continuously lowered blood pressure when administered intravenously to dogs. Starting with [EKFrey., Arch.Klin.Chir., 142 , 663-668,
1926]. Later, in 1930, Kraut et al. discovered that a similar substance existed in the pancreas and named it kallikrein [H. Kraut, EKFrey and
E. Werle: Z. Physiol. Chem., 189 , 97−106,
1930]. Kallikrein can be broadly classified into plasma kallikrein and glandular kallikrein, and currently glandular kallikrein prepared from mammalian pancreas is mainly used as a medicine. Both plasma kallikrein and glandular kallikrein act on kininogen present in the α ₂ -globulin fraction in blood as a substrate to produce kinin. All of these produced kinins exhibit effects such as smooth muscle contraction, vasodilation, and increased vascular permeability. Therefore, clinically, based on these effects, (1) circulatory disorders in early age (cerebral arteriosclerosis, hypertension), (2) tissue nutritional disorders due to blood circulation disorders, and (3) intractable wounds. It is also used to improve ulcers, etc. Kallikrein, which is currently in clinical use, is mainly prepared from pig pancreas, and it is difficult to obtain it in large quantities. Furthermore, there are differences in the amino acid sequence of kallikrein depending on the species, and there are some differences in the amino acid sequence between human kallikrein and pig kallikrein. For this reason, administering porcine kallikrein to humans is not very desirable due to immunological problems. However, although human kallikrein is present in urine, the amount is so small that it has been impossible to use it as a preparation. Therefore, in order to mass-produce human kallikrein using genetic engineering techniques, the present inventors first obtained the preprokallikrein gene containing the DNA portion encoding human kallikrein (Japanese Patent Application Laid-Open No. 1983-1992-1).
126980), but it has not yet been possible to construct an expression vector that can efficiently express human kallikrein in a host such as E. coli, and it has not been possible to obtain human kallikrein cheaply and in large quantities that can be used as a pharmaceutical. Ta. (Means for Solving the Problems) As a result of further studies to obtain an expression vector that can efficiently express human kallikrein-like protein in a host, the present inventor discovered that only the DNA encoding human kallikrein-like protein Although sufficient expression efficiency cannot always be obtained when directly integrating the preprokallikrein gene containing DNA encoding a human prokallikrein-derived signal peptide into the vector, the DNA encoding the human prokallikrein-like protein of
By linking with DNA encoding the signal peptide of E. coli outer membrane proteins such as OmpF and further combining it with a specific promoter, we were able to construct an expression vector that can efficiently express the protein, making it possible for the first time to use genetic engineering techniques. They succeeded in producing a human kallikrein-like protein and completed the present invention. That is, the gist of the present invention is to encode a signal peptide of an E. coli outer membrane protein upstream of a DNA fragment encoding human kallikrein or a human kallikrein-like substance having physiological activity equivalent to it.
A host microorganism transformed with an expression vector containing a DNA fragment ligated with DNA fragments is cultured, and the human kallikrein or the human kallikrein-like substance produced as an insoluble protein within the host microorganism is obtained. The present invention relates to a method for producing human kallikrein-like protein. In order to explain the present invention below, the present invention will be described below.
DNA fragments were obtained by preparing mRNA from human pancreas according to the method described in JP-A No. 62-126980, and using the Okayama-Berg method [Molecular and Cellular
Biology, 2 , 161-170, 1982] cDNA
It can be obtained by obtaining a library, screening for human kallikrein-like protein cDNA using, for example, rat kallikrein cDNA as a probe, and amplifying it. The human kallikrein-like protein cDNA obtained as described above normally has a DNA portion encoding a signal peptide consisting of 17 amino acids and a prosegment consisting of 7 amino acids, but in the present invention, such human kallikrein-like protein Contains a signal peptide portion derived from kallikrein-like protein
When an expression vector is constructed using a DNA fragment, or when an expression vector is constructed using only a DNA fragment of human kallikrein-like protein from which the DNA portion encoding the signal peptide and prosegment has been removed, Good expression efficiency cannot be expected. In the present invention, human prokallikrein-like protein or human kallikrein-like protein DNA
Upstream of the fragment encodes signal peptides of E. coli outer membrane proteins such as OmpF, OmpC, and OmpA.
It is important to use DNA fragments that are ligated together. The expression vector that introduces such a DNA fragment is
It has a promoter at a position that can control transcription of these genes. As such a promoter, any known promoter can be used as long as it functions in the host microorganism, but the tac promoter [Proc.
Natl.Acad.Sci.USA, 81 , 21, 1983], λP _L P _R promoter, Pac promoter (patent application 1983-
Hybrid promoters such as No. 268362), particularly the tac promoter, are preferred. It is preferable to link two or more of these promoters because expression efficiency improves. In addition, in the present invention, the expression vector appropriately combines a repressor gene ( lac I) which is a promoter control factor, a ribosome binding sequence for initiation of translation, and a transcription terminator sequence (lpp3' region) for terminating transcription, Those devised to increase the efficiency of genetic expression are used. Furthermore, a drug (ampicillin) resistance gene may be incorporated into the expression vector to facilitate selection of bacteria transformed with the expression vector. The expression vector thus obtained is used to transform a host microorganism. Host microorganisms include Escherichia coli JM109, HB101,
Escherichia coli such as YK537 and YA21, Bacillus subtilis MI112,
Examples include Bacillus subtilis such as NP58. Transformation can be carried out using conventional methods, such as molecular cloning, Cold Spring
Harbor, p. 250, 1982. The thus obtained expression plasmid-carrying bacteria of the present invention are cultured in a medium such as L broth or M9 medium, and the promoter, for example isopropyl-β, which is an inducer of the tac promoter, is -D-galactopyranoside is added to the medium to induce expression of the target substance, human kallikrein-like protein, to produce human kallikrein-like protein, and human kallikrein is isolated and purified according to a conventional method. similar proteins can be obtained. In the present invention, it is preferable to express the human prokallikrein-like protein in terms of expression efficiency, but in that case, the desired human prokallikrein-like protein can be obtained by treating the produced human prokallikrein-like protein with trypsin. Can be done. (Effects of the Invention) According to the present invention, human kallikrein-like protein can be efficiently expressed. For example, approximately 10 mg of human kallikrein-like protein can be expressed in Escherichia coli, and human kallikrein-like protein can be obtained at a rate approximately 1000 times higher than the conventional method of preparing it from human urine. became possible. (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 it exceeds the gist thereof. Example 1 A Construction of expression plasmid (a) Modification of human kallikrein cDNA by site-specific modification method (1) Difference in N-terminus phKK25 ( Figure 1-a)
5μg of 10mM Tris-HCl (PH7.5),
A buffer consisting of 100mM sodium chloride and 6mM magnesium chloride (hereinafter referred to as
Pst in 100μ (referred to as “Buffer H”)
React with I10u at 37℃ for 2 hours, digest
After heating at 75°C for 15 minutes to inactivate the enzyme, the mixture was dialyzed against water and dried. Add this to 33mM Tris-acetic acid (PH
7.9), a buffer consisting of 66mM potassium acetate, 10mM magnesium acetate and 0.5mM dithiothreitol, and 2mM
Four types of deoxynucleotide triphosphates (dNTPs) were added to give a concentration of 40μ, and the 3' protruding ends were blunted with T4 DNA polymerase 4U, and the enzyme was deactivated by heating at 70℃ for 10 minutes. After dialysis against water and drying
It was stored as a 50μ aqueous solution. …Fragment Meanwhile, 20μg of phKK25 was added to the above buffer.
Eco R and Apa each in H100μ
Digestion was performed by reacting with 20 u at 37°C for 2 hours.
This was subjected to 5% acrylamide gel electrophoresis (89mM Tris, 89mM boric acid, 2
DNA was separated using mMEDTA buffer; 10v/cm, 1.5 hours electrophoresis), and the gel was 0.05%
After staining with ethidium bromide aqueous solution
The larger molecular weight fragment was excised under 340 nm ultraviolet light, the gel was disrupted with a glass rod, and DNA extraction buffer (0.5M ammonium acetate, 10mM magnesium acetate, 1mM EDTA and 0.1
% sodium lauryl sulfate) and left overnight at 37°C to remove the gel.
DNA was extracted. a large piece of gel
After removal by centrifugation at 10,000 rpm for 15 minutes, smaller gel pieces were further removed using a glass filter, ethanol precipitation was performed three times to purify the DNA, and the DNA was stored as a 200μ aqueous solution. ...Fragment 49base DNA consisting of the following base sequence
The fragments (primers) are synthesized using a DNA synthesizer (Nikkakisha; Applied Biosystem MODEL).
380A). 150 pmol of this synthetic DNA fragment was added to a kinase buffer (50 mM Tris-HCl (PH).
8.0), 10mM magnesium chloride and 5
(mM dithiothreitol) in a 10μ system.
5' phosphorylated with 20u of T4 polynucleotide kinase. 0.05 pmol of fragment, 0.05 pmol of fragment and 45 pmol of 5' phosphorylated primer, 5x concentration of polymerase,
Ligase buffer (0.5M sodium chloride,
32.5mM Tris-HCl (PH7.5), 40mM
Add 12μ of magnesium chloride and 5mM β-mercapotoethanol for a total of 34.8μ
The system was boiled at 100°C for 3 minutes, immediately placed in a constant temperature bath at 30°C for 30 minutes, and then left on ice for 10 minutes to form a heteroduplex. This heteroduplex 11.6μ
Add 2.5mM of 4 kinds of deoxynucleotide triphosphates (dNTPs) to 4μ, 10mM
2μ of MATP, 2u of Klenow enzyme and
Total plus 0.5u of T4DNA ligase
The mixture was reacted overnight at 16°C in a 20μ system to form a circular product. Using 2μ of this circular product, Escherichia coli strain HB101 was transformed and grown according to a conventional method. Subsequently, the plasmid of the transformant was subjected to a conventional method to obtain a mutant plasmid that can be cleaved into two DNA fragments by Pst . Since the mutant plasmid thus obtained was contaminated with the original plasmid (wild type), transformation was performed again to purify the mutant plasmid. In this way, the first
The plasmid phKK-sp shown in Figure-6 was obtained. (2) Modification of the C-terminus 5 μg of phKK-sp was added with 100 μg of buffer H.
The enzyme was digested by reacting with Pst10u at 37°C for 2 hours, then heated at 75°C for 15 minutes to inactivate the enzyme, and then dialyzed against water and dried. Add to this 33mM tris-acetic acid (PH7.9), 66mM potassium acetate, 10
A buffer solution consisting of mM magnesium acetate and 0.5 mM dithiothreitol and four types of deoxynucleotide triphosphates (dNTPs) were added to give a concentration of 2 mM to form a 40μ system. Next, the 3' protruding ends were blunted with T4 DNA polymerase 4u, heated at 70°C for 10 minutes to inactivate the enzyme, dialyzed against water, dried, and stored as a 50μ aqueous solution. ...Fragment' Meanwhile, 20 μg of phKK-sp was added to the buffer solution.
Digestion was performed by reacting with Pvu 5u in H100μ at 37°C for 2 hours. This was subjected to 5% acrylamide gel electrophoresis (89mM Tris,
DNA in 89mM boric acid, 2mM EDTA buffer; 10v/cm, 1.5 hours electrophoresis)
After separating the gel and staining the gel with a 0.05% ethidium bromide aqueous solution, the larger molecular weight fragment was cut out under 340 nm ultraviolet light. After crushing this gel with a glass rod, add the DNA extraction buffer 4 described above.
ml and left overnight at 37°C to extract DNA from the gel. a large piece of gel
After removing the gel by centrifugation at 10,000 rpm for 15 minutes, remove small gel pieces using a glass filter and remove the ethanol precipitate for 3 minutes.
The DNA was purified twice and stored as a 200μ aqueous solution. …Fragment′ 40base DNA consisting of the following base sequence
The fragment was synthesized using the DNA synthesizer described above. 150 pmol of this synthetic DNA fragment was 5' phosphorylated with 20 u of T4 polynucleotide kinase in the above-mentioned system containing 10 μ of the kinase buffer. The plasmid phKK-spB shown in Figure 1-c was obtained in the same manner as in (1) above except that fragment', fragment' and 5' phosphorylated primers were used. B Construction of human prokallikrein secretion expression plasmid DNA encoding human prokallikrein
Preparation of a fragment 10 μg of phKK-spB was reacted with Pst 20u in a buffer H100 μ system for 2 hours and cleaved. This product was heated at 75°C for 10 minutes to inactivate the enzyme, then dialyzed against water, desalted, and dried. followed by T4 DNA polymerase buffer as described above.
After adding 50 u of T4 DNA polymerase and reacting with 10 u of T4 DNA polymerase at 37°C for 30 minutes to scrape off the protruding 3' end, the enzyme was heated at 70°C for 10 minutes to inactivate the enzyme. Then add 5.5μ of 10x buffer H (0.1M Tris-HCl, 1M sodium chloride and 60mM magnesium chloride).
Cut with Bgl10u and perform approximately 5% acrylamide gel electrophoresis in the same manner as above.
Encodes 739bp human prokallikrein
The DNA fragment (fragment pKK) was isolated and purified. Preparation of expression vector 2μg of pOFA obtained in the reference example described below was mixed with buffer (10mM Tris-HCl (PH7.5) and 6mM
Magnesium chloride) Kpn 5u in 20μ system
The mixture was digested by reaction at 37°C for 2 hours. To this, 2μ of 10x concentrated T4 DNA polymerase buffer was added, four types of deoxynucleotide triphosphates (dNTPs) were added to a concentration of 1mM, and the mixture was reacted with 4μ of T4DNA polymerase at 37°C for 30 minutes. After cutting off the 3' end, the enzyme was inactivated by heating at 70°C for 10 minutes. Add 2.5μ of the above 10x buffer solution H to this.
Add Bgl 4u and react at 37℃ for 2 hours.
After cutting the DNA, extract the protein with 25μ water-saturated phenol, separate the supernatant (aqueous layer), remove the phenol with ether, dialyze against water, desalt, and dry. Dissolved in 10μ of water. Preparation of human prokallikrein secretory expression plasmid: 1 μg of fragment pKK, 0.5 μg of the above B- secretory expression vector, and 1 μg of 0.1MATP
10μ of T4 DNA ligase was added to 10μ of buffer (10mM Tris-HCl, 6mM magnesium chloride, and 1mM dithiothreitol) and reacted at 4°C for 16 hours to ligate each fragment to create pOFhKK (Figure 2). did. C Secretion of human prokallikrein Escherichia coli YK537 was transformed with pOFhKK, and the resulting transformant was washed with L broth (10 g/bactotryptone, 5 g/bacto yeast extract, 10 g/sodium chloride, and 20 mg/bacto yeast extract).
ampicillin) at 30°C overnight. This culture was transferred to fresh L broth at a dilution of 1/50 and cultured at 30°C for 5 hours. Then,
Isopropyl-β-D-galactopyranoside, which is an inducer of the tac promoter on the plasmid, was added to a concentration of 2 mM to induce expression of human prokallikrein, and the cells were further cultured for 3 hours. After culturing, human prokallikrein-producing bacteria were collected by centrifugation at 6000 rpm for 10 minutes. When the obtained bacterial cells were observed using an electron microscope, it was found that human prokallikrein was secreted into the periplasm of the bacterial cells and formed into granules. 1mg/ml of this human prokallikrein-producing bacterium.
Lysozyme, 10mM Tris-HCl, PH8.025m
Bacteria were lysed with 100 ml of M EDTA solution and completely destroyed by ultrahigh waves. Thereafter, the insoluble protein was obtained as a precipitate by centrifugation (65000 rpm for 10 minutes). This precipitate was treated with 5M guanidine hydrochloric acid,
It was suspended in 50mM Tris-hydrochloric acid solution and stirred overnight at 4°C to dissolve. After removing insoluble matter by centrifugation, the protein concentration was adjusted to A280<1.
Also, the final concentration is 1M guanidine hydrochloride, 50mM Tris-HCl, 0.005% Tween80, 2mM oxidized glutathione, 0.02mM reduced glutathione PH.
I diluted it to 8.0. 2 at 4℃
After leaving for half the night, 50mM sodium chloride, 50mM
After dialysis against M Tris-HCl and 0.005% Tween 80 PH8.0 to remove guanidine and glutathione, insoluble matter was removed by centrifugation to obtain a supernatant. It was confirmed by SDS-polyacrylamide electrophoresis and Western blotting that human prokallikrein was solubilized in this supernatant. After treating this with trypsin to remove the propeptide and converting it into human kallikrein (active form), a trypsin inhibitor was added to suppress the trypsin activity, and Morita et al. J. Biochem, 82
(1977) 1495-149, kallikrein activity was measured. That is, Pro−Phe
-Arg-MCA (manufactured by Sigma) was reacted with human kallikrein using its substrate, and the liberated MCA was excited with 380 nm light and measured by emission of 440 nm light. The results are shown in Table 1. [Table] Reference example (Preparation of pOFA) 10 μg of plasmid phMAVD・lac I obtained according to the description in Japanese Patent Application No. 61-220835 was added to 100 μg of buffer H.
Inside, Bam H and Ava each 10μ
After digestion at 37°C for 2 hours, 5% acrylamide gel electrophoresis was performed.
DNA fragments were separated, stained with 0.05% ethidium bromide, and large DNA fragments were excised and extracted according to conventional methods. …
1 μg of the fragment phMAVD· lac was digested with 1 μg of Pst I in 10 μg of buffer H, and then heated at 70° C. for 10 minutes to inactivate the enzyme. It was then dialyzed against water and evaporated to dryness. This was mixed with buffer P (10mM potassium acetate,
33mM Tris-Acetate, 10mM Magnesium Acetate and 0.5mM Dithiothreitol) at 20μ
Using T4 DNA polymerase, the mixture was reacted at 37°C for 15 minutes, then heated at 70°C for 10 minutes to inactivate the enzyme, dialyzed against water, evaporated to dryness, and further dissolved in 10μ of water. ...Fragment 5' phosphorylation of mutant primer 150 pmol of the following 36 base mutant primer was added to buffer K (1mM MEDTA, 50mM Tris-HCl (PH7.5), 6mM magnesium chloride and 5mM
5' phosphorylation was carried out using 1μ of T4 polynucleotide kinase in 10μ of M dithiothreitol at 37°C for 1 hour. Formation of heteroduplex Fragment 0.05 pmol, Fragment
0.05pmol and 5′-phosphorylated primer
Add 12μ of a 5-fold concentration of polymerase ligase buffer (0.5M sodium chloride, 32.5mM Tris-HCl (PH7.5), 40mM magnesium chloride and 5mM β-mercaptoethanol) to 45pmol.
The system was then boiled at 100°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 then on ice for another 10 minutes to form a heteroduplex. Aqueous solution containing this heteroduplex
Add 2μ of 2.5mM 4-deoxynucleotide triphosphate and 2μ of 10mMATP to 11.6μ, add 2u of Klenow enzyme and 0.5u of T4 DNA ligase, and react in a total of 20μ at 12.5℃ for 16 hours to circularize the DNA. I let it happen. Escherichia coli JM109 was transformed using 2μ 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 a restriction enzyme Kpn , and confirmed by 0.8% agarose gel electrophoresis to obtain a digested plasmid as a mutant plasmid. Since the mutant plasmid obtained in this case is often contaminated with the original plasmid (wild type), this mutant plasmid was used to transform Escherichia coli JM109 again to purify the mutant plasmid. Plasmid pOmpKpn was thus obtained. Obtained in accordance with the description in Japanese Patent Application No. 61-268362
1μg of pMTI2 was added to buffer L (10mM Tris-HCl and 10μ of 6mM magnesium chloride).
Digestion was performed using 1μ of KpnI by reaction 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, 1μ of Pst was used in 10μ of buffer solution H to react and digest at 37℃ for 2 hours, and then at 70℃ for 10μ of Pst.
The enzyme was inactivated by heating for a minute, dialyzed against water and evaporated to dryness. Meanwhile, pOmpKpn was digested with Kpn and Pst in the same manner and evaporated to dryness. Add the above two digested DAN fragments to buffer solution.
It was dissolved in 10μ of L and reacted with 1μ of T4 DNA ligase at 4°C for 16 hours. E. coli JM109 competent cells were transformed using 3μ of this reaction mixture, and the plasmid was isolated and purified from the resulting transformant .
and Sma restriction enzymes.
A plasmid pOFA containing a fragment of pOmpKpn containing lac - Omp F, a replication origin of pMTI2, an lpp transcription termination sequence, and a multicloning sequence was obtained. (Figure 3).

[Brief explanation of drawings]

FIG. 1 is a diagram schematically showing the plasmid produced in Example 1. In the figure, a is a plasmid
phKK25, b represents plasmid phKK-sp and c represents plasmid phKK-spB. FIGS. 2 and 3 are diagrams schematically showing plasmids produced in reference examples. Plasmid pOFhKK respectively
and represents plasmid pOFA.

Claims (1)

[Scope of Claims] 1 Encodes human kallikrein or a human kallikrein-like substance having physiological activity equivalent to it
A DNA fragment encoding the signal peptide of the outer membrane protein of E. coli was ligated upstream of the DNA fragment.
A human kallikrein-like protein characterized by culturing a host microorganism transformed with an expression vector containing a DNA fragment to obtain human kallikrein or the human kallikrein-like substance produced as an insoluble protein within the host microorganism. production method. 2. The production method according to claim 1, wherein the host microorganism to be transformed is Escherichia coli. 3 The DNA fragment encoding the signal peptide of the outer membrane protein of E. coli is OmpF, OmpC, or OmpA.
The production method according to claim 2, characterized in that: 4. The production method according to claim 3, wherein the DNA fragment encoding the signal peptide of the outer membrane protein of E. coli is OmpF.