JPH01300893A - Production of human kallikrein-like protein - Google Patents

Abstract

PURPOSE:To obtain the title protein for hypertension therapy, etc., in an industrially advantageous manner, by making a culture of microorganisms transformed with a manifestation vector containing the DNA coding human kallikrein or matters having physiological activity comparable to that for human kallikrein to collect the resulting product. CONSTITUTION:A DNA fragment coding human kallikrein or analogous matters having physiological activity comparable to that for human kallikrein is linked to an appropriate manifestation vector to prepare a human kallikrein-secreting manifestation plasmid followed by incorporating this plasmid into microorganisms such as Escherichia coli to transform said bacteria, thus producing a transformant. Thence, this transformant is put to culture in a culture medium to collect the human kallikrein or analogous matters, thereby obtaining the objective human kallikrein-like protein.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing a human kallikrein-like protein, ie, a human kallikrein-like substance containing human kallikrein or a physiological activity equivalent thereto.

(Prior art and problems to be solved by the invention) Kallikrein research was conducted in 7926 when Frey discovered a non-dialyzable substance in human urine that continuously lowered blood pressure when administered intravenously to dogs. Starting with C E, Fr
ey, + Arch, K11n, Chir, ,
IQ2゜443-1. AI, /924). After that, 7
In 930, Kraut et al. discovered that a similar substance existed in the pancreas, and named it Kallikrein.
H. Kraut, E., Frey and
E., Werle: Z.

Physiol, Chem, , / g, q7
-106, /930).

Kallikrein can be broadly classified into plasma kallikrein and kallikrein in solid form, and currently, kallikrein in solid form prepared from the pancreas of mammalian animals is mostly used as a medicine. Both plasma kallikrein and kallikrein in the form of kallikrein act on kininogen present in the α2-globulin fraction in blood as a substrate to produce kinin. All of these produced kinins exhibit effects such as smooth muscle contraction, vasodilation, and improved vascular permeability.

Therefore, clinically, based on these effects, (1)
Circulatory disorders in old age (cerebral arteriosclerosis, hypertension), (2)
It is used to improve tissue malnutrition due to blood circulation disorders, (3) intractable wounds and 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. In addition, the amino acid sequence of kallikrein includes
There are differences depending on the species, and there are some differences in the amino acid sequences 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 exists in urine, the amount is so small that it has not been possible to use it as a preparation.

Therefore, in order to mass-produce human kallikrein by genetic engineering techniques, the present inventor has not yet been able to construct an expression vector that can efficiently express human kallikrein while containing a DNA portion encoding human kallikrein. However, it has not been possible to obtain human kallikrein at low cost and in large quantities that can be used as a medicine.

(Another Means to Solve the Problem) 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 a DNA encoding human kallikrein-like protein
Although sufficient expression efficiency cannot necessarily be obtained when only A is directly integrated into the vector, or when the preprokallikrein gene containing DNA encoding the human prokallikrein-derived signal peptide is integrated into the vector, OmpF the coding DNA
By linking the DNA encoding the signal peptide of the outer membrane protein of E. coli and further combining it with a specific promoter, we were able to construct an expression vector that can efficiently express human kallikrein using genetic engineering techniques. We succeeded in producing a similar protein and completed the present invention.

That is, an advantage of the present invention is to culture a transformant transformed with a DNA encoding human kallikrein or a human kallikrein-like substance having an equivalent physiological activity, and to produce human kallikrein or human kallikrein. The present invention relates to a method for producing human kallikrein-like protein, which comprises obtaining a kallikrein-like substance.

To explain the present invention below, a DNA fragment encoding a human kallikrein-like protein, which is the target protein of the present invention, was obtained from mRNA from human pancreas according to the method described in Japanese Patent Application Laid-Open No. 1983-1989/Co/, No. 9A'θ. was prepared using the Okayama-Barb method CMo1ular and Cellular Bi.
ology, 2, /4/-/70.79g2)
A cDNA library is obtained using the above method, and a cDNA of a human kallikrein-like protein is screened from the cDNA library using, for example, rat kallikrein cDNA as a probe, and the cDNA is amplified.

c of human kallikrein-like protein obtained as above
DNA usually has a DNA portion that encodes a signal peptide consisting of /3 amino acids and a prosegment consisting of 7 amino acids, but in the present invention,
When an expression vector is constructed using a DNA fragment containing a signal peptide portion derived from such a human kallikrein-like protein, or only a DNA fragment of a human kallikrein-like protein from which the DNA portion encoding the signal peptide and prosegment has been removed. If an expression vector is constructed using this method, good expression efficiency cannot be expected in E. coli.

In the present invention, a DNA fragment is used in which a DNA fragment encoding a signal peptide of an E. coli outer membrane protein such as OmpF, OmpC, or OmpA is linked upstream of a DNA fragment of a human prokallikrein-like protein or a human kallikrein-like protein. is important.

The expression vector into which such a DNA fragment is introduced has a promoter at a position that can control the transcription of these genes.

As such a promoter, any known promoter can be used as long as it functions in the host microorganism, including the tac promoter (Proc, Natl, A
cad, sci, USA, g/, 2/.

Hybrid promoters such as 79g3'J, λPLPR promoter, Danzo promoter (Japanese Patent Application No. 6/21, g342), and especially tac promoter are suitable. It is preferable to link λ or more of these promoters because expression efficiency improves.

In addition, in the present invention, the expression vector appropriately combines a repressor gene (Iac I) which is a promoter control factor, a liposome binding sequence for initiation of translation, and a transcription terminator sequence (Ipp3' region) for terminating transcription. , those devised to increase the expression efficiency of the target gene 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.

A host microorganism is transformed with the expression vector thus obtained.

Host microorganisms include Escherichia coli JM109, HERO/,
YK! E. coli such as 37, YA2/, Bacillus subtilis M I/
/ Ko, NP! ; Bacillus subtilis such as g.

Transformation can be carried out by conventional methods, for example, molecular cloning + C
oldSpring Harbor, 2nd SO page, /
It can be carried out according to the method described in 9g2.

The thus obtained expression plasmid-carrying bacteria of the present invention were cultured in L broth or M9 medium (M 9
Medium), and by adding inglovir-β-D galactopyranoside, which is an inducer of a promoter such as tac promoter, to the medium, the expression of the target substance, human kallikrein-like protein, is induced. The human kallikrein-like protein can be obtained by producing the human kallikrein-like protein and separating and purifying it according to a conventional method.

In the present invention, it is preferable to express it as a 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.

(Effects of the Invention) According to the present invention, human kallikrein-like protein can be efficiently expressed. For example, in E. coli, about 10
It has become possible to express human kallikrein-like protein at a concentration of approximately 1,000 times more efficiently than the conventional method of preparing it from human urine. .

(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to the following examples unless it exceeds the gist thereof.

Example / 8 Construction of expression plasmid a) Modification of human kallikrein cDNA by site-specific modification method /) Mutation on the N-terminal side ■ Obtained according to the method described in JP-A No. 1986-1/1980-6-0 phKK2! ; (Figure 1-a) 5 tt
y/OmM) Lis-HCl (pH 74), 100 m
10 μl of a buffer consisting of M sodium chloride and 6 mM magnesium chloride (hereinafter referred to as "buffer H")
In the inside, react with Ps't110u at 37℃ for 2 hours and digest. After inactivating the enzyme by heating at 1°C for 3 minutes, the mixture was dialyzed against water and dried.

To this, 33 mM Tris-acetic acid (pH 7,9),
Al, mM potassium acetate, 10mM magnesium acetate and 0. ! ; Add a buffer solution consisting of mM dithiothreitol and 9 types of deoxynucleotide triphosphates (dNTPs) to a concentration of 2 mM, and transfer the total volume to lI.
The 3' protruding ends were blunted with T4' DNA polymerase lIu, the enzyme was inactivated by heating at 70°C for 70 minutes, the enzyme was dialyzed against water, and after drying, S
It was stored as an aqueous solution of 0 μg. ... Fragment I ■ On the other hand, add 0 μl of phKK CoS to the aforementioned buffer H/
In 00μβ, Naji R1 and Nagi I, 20u and 37°C, respectively.
The mixture was reacted for 2 hours and digested.

This thing! Polyacrylamide gel electrophoresis (g9
DNA was separated using mM Tris, 19mM boric acid, 2mM EDTA buffer; /θV/'m, S time migration).
After staining the gel with 0.105% ethidium bromide aqueous solution, cut out the fragment with the larger molecular weight under 3 I/Onm ultraviolet light, crush the gel with a glass rod, and add DNA extraction buffer (0.5 M ammonium acetate, 10 m
M Magnesium Acetate,/mM EDTA and 0.7
% sodium lauryl sulfate) qd and incubated at 37°C overnight to extract DNA from the gel. After removing large gel pieces by centrifugation for 1000 r, p, m, / s minutes, remove smaller gel pieces using a glass filter, perform ethanol precipitation three times to purify the DNA, and store as a 200 μl aqueous solution. did. ...Fragment ■■ Synthesized using q9 base DNA consisting of the following base sequence.

Primer: s'-CGCCCT GTCCCT
GGG region This synthesized NA fragment/opmol was added to kinase buffer (tOmM), lithium-hydrochloric acid (pHg, θ), 10mM magnesium chloride and 9mM dithiothreitol) 10
5' phosphorylation was performed with 20 u of TtI polynucleotide kinase in a μl system.

■ Fragment lo, o spmol, fragment 110.0! r pmol and S' phosphorylated primer II! r pmol I/C! ; double concentration of polymerase, ligase buffer (0,!; M sodium chloride 1,? a, s mM Tris-HCl (pHq, s
), q.

Add 72 μl of mM magnesium chloride and μM β-mercaptoethanol to make a total of 3 μl, boil at IOOO℃ for 3 minutes, immediately place in a thermostatic bath at 30℃ and leave for 30 minutes, then leave on ice for 70 minutes. to form a heteroduplex.

This heteroduplex/7.6μlK, 2.5
mM ＜' types of deoxynucleotide tri
Q μll of phosphoric acid (dNTP), /
OmMATP at 2 All, Klenow enzyme at xu and T4' DNA ligase at 0. ! ; The reaction was carried out at 76° C. overnight in a system with a total of 0 μl of u added, and circularization was carried out.

Using this circular substance-μl, Escherichia coli H8101 was isolated according to a conventional method.
Strains were transformed and grown.

Subsequently, the plasmid of the transformant was transformed with PstI according to a conventional method.
A mutant plasmid that was cleaved into ps DNA fragments was obtained.

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 plasmid phKK-8l) shown in Figures 1-6 was obtained.

h) Modification of C-terminus ■ Add phKK-3p j μl to buffer solution Ht 00 t
The enzyme was digested by reacting with 10 u of PStl at 37° C. for 7 hours, followed by heating at qs° C. for 75 minutes to inactivate the enzyme, and then dialyzed against water and dried. To this thing? ,? mM) 'J Susu-acid (pH 7,9),
A buffer solution consisting of 66mM potassium acetate, 10mM magnesium acetate, and 0.3m to 1 dithiothreitol, and t types of deoxynucleotide triphosphates (dNTPs) were added to a concentration of 2mM to make a 1Ioμl system. Next, the 3' protruding ends were blunted with 74 DNA polymerase ldu, heated at 70°C for 70 minutes to inactivate the enzyme, dialyzed against water and dried, and then
It was stored as an Oμl aqueous solution. ...Ming
Specification/Name of the invention Method for producing human kallikrein-like protein Claims (1) A trait transformed with an expression vector containing DNA encoding human kallikrein or a human kallikrein-like substance having an equivalent physiological activity. 1. A method for producing a human kallikrein-like protein, which comprises culturing a transformant and obtaining the produced human kallikrein or the human kallikrein-like substance.

3. Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for producing human kallikrein-like protein, ie, human kallikrein or a human kallikrein-like substance having an equivalent physiological activity.

(Prior art and problems to be solved by the invention) Kallikrein's research was conducted in /921. In 1999, Frey discovered a non-dialyzable substance in human urine that continuously lowered blood pressure when administered intravenously to dogs [E, N, F]
rey, + Arch, K11n, Chir, ,
/172.

A/, 3-648:, /921. ). After that, /q,
In 1930, Kraut et al. revealed that a similar substance existed in the pancreas, and named it calicudiin.
H. Kraut, E., Frey and E.
, Werle: Z.

Physiol, Chem, 97
-101. , /930].

Kallikrein can be broadly classified into plasma kallikrein and kallikrein, and currently kallikrein, which is prepared from the pancreas of mammalian animals, is mostly used as a medicine. Both plasma kallikrein and kallikrein in the form of kallikrein act on kininogen present in the α2-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 old age (cerebral arteriosclerosis, hypertension), (2)
It is used to improve tissue malnutrition due to blood circulation disorders, (3) intractable wounds and ulcers, etc.

Secretion expression vector 0.3 μy and O,/MATP/μ
l buffer (/o mM Lis-HCl, 6 mM magnesium chloride and/mM dithiothreitol) 7
Add 10 u of T4' DNA ligase in 0 μl,
The fragments were reacted for 76 hours at 0.degree. C. and the fragments were ligated to produce pOFhKK (Fig. 2).

C Human prokallikrein secretion pOFhKK in Escherichia coli YK! ;3'/ transformed;
The obtained transformants were treated with L gloss (/o y/l Bactodorypton, !; I!/l Bacto yeast extract, /θg/l sodium chloride, and 2 o q/l
! ampicillin) at 30°C/overnight. This culture was transferred to new L gloss to give a 115o dilution,
The cells were cultured at 30°C for S hours. Then, the inducer of the upper promoter on the plasmid, isopropyl-β-D-
Galactopyranoside was added to a concentration of 2 mM to induce the expression of human prokallikrein, and the cells were further cultured for 3 hours.

After culturing, human prokallikrein-producing bacteria were collected by centrifugation at A, 000r, p, m for io 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 granules. ,/OmM) Lis-HCl, pHg, 0 to 5mM
The cells were lysed with EDTA solution 100- and completely destroyed by ultrahigh waves. Afterwards, insoluble proteins are removed by centrifugation (A
! r, 000 r, p, m, / 0 min) as a precipitate. This precipitate was dissolved in 5M guanidine hydrochloric acid, 50m
The mixture was suspended in M Tris-hydrochloric acid solution and stirred overnight at U°C to dissolve. After removing insoluble matter by centrifugation, the protein concentration was adjusted to A2gO/, and the final concentration was 7M guanidine hydrochloride. r OmM) Lis-HCl, 0.005% T
ween g O, 2mM oxidized glutathione, 0.0
Diluted to 2mM reduced glutathione pHg, o. After leaving this thing at 4<'C for half a night, j Om
M Sodium chloride! r OmM) Lis-HCl, o,
After removing guanidine and glutathione by dialysis against o s% Tween g OpHg, 0, insoluble materials were removed by centrifugation to obtain a supernatant. 5DS-
This was confirmed by polyacrylamide electrophoresis and Western blotting.

After treating this with trypsin to remove the propeptide and making human kallikrein (active form), trypsin inhibitor was added to suppress trypsin activity, and Morit
a et al. J, Biochem.

g2(/97'7')/Qq! -/Kallikrein activity was measured according to the method described in Dade. That is, Pro
-Phe-Arg-MCA (manufactured by Sigma) was used as a substrate and human kallikrein was applied to release M
CA was excited with 3tOnm light and measured by the emission of 1leOnm light.The results are shown in Table 1.

Table / /u: /nmol of substrate (Pro-P
Reference example of the amount of enzyme (kallikrein) that can hydrolyze he-Arg-MCA (preparation of pOF'A) ■ Patent application 1986-20g3! The plasmid phMAVD-LacI/Ottl, obtained as described in No. Push-H1 and Δ! Using 70 μl of each I, after incubation and digestion at 37°C, fiD was analyzed by 5% acrylamide gel electrophoresis.
The NA fragments were separated and stained with 0.05% ethidium bromide, and large DNA fragments were cut out and extracted according to a conventional method. ...Fragmen) 1■
phMAVD-1aci/3g in buffer HiO
After digestion with PstI/μJ in μm, 70°C
The enzyme was inactivated by heating for 10 minutes. This was then dialyzed against water and evaporated to dryness.
? mM Tris-acetate, 10 mVi magnesium acetate and 0. ! ;rnM dithiothreitol) 20μ
T at l! Using DNA polymerase 1. ? ? 'C in l
React for S minutes, then heat at 70°C for 10 minutes, dialyze against the extracted water to deactivate the enzyme, evaporate to dryness, and dissolve in 10 μl of water.
Phosphorylation of mutant primers 30 pmol of the following 36-base mutant primers were added to 0 μl of buffer K (/mM EDTA, !; Then, S' phosphorylation was performed using T polynucleotide kinase/μl at 37° C. for 7 hours.

Coughing primer: 5'-GGT ACT G
CA AACpn 1 GCG GTA CCCATG TACAAT
GCCGTG-3'■ Formation fragment of heterodiplex l O, Ojpmol, 7 fragments 10.0! pmol and y-phosphorylated primer 4t! r pmol of 5-fold concentrated polymerase ligase buffer (0,!; M sodium chloride, 32
．． 3mM) Lis-HCl (pH 7,!r),'l0m
M magnesium chloride and jmM β-mercaptoethanol) were added in a total of 3'1. g μl system, 1
The mixture was boiled at 00°C for 3 minutes and immediately placed in a constant temperature bath at 30°C for 30 minutes. Next, the mixture was left at 9° C. for 30 minutes and further left on ice for 70 minutes to form a heteroduplex.

Aqueous solution containing this heteroduplex //, 6 μl
Add 2 μl of 2,5 mM I-deoxynucleotide triphosphate, 2 μl of iOmMATP, and 2 u of Klenow enzyme and OA; u of T&D
NA1. Add l gauze and make a total of 20 μl/j, f
c for 76 hours to circularize the DNA.

Using μl of the aqueous solution containing this circular DNA, E. coli JM109 was transformed according to a conventional method to obtain a transformant. The plasmid is isolated and purified from this transformant according to a conventional method, cut with the restriction enzyme Kpnl, confirmed by o, tr% agarose gel electrophoresis, and the digested plasmid is used as a mutant plasmid. Since the mutant plasmid is often contaminated with the original plasmid (wild type), this mutant plasmid is used to reintroduce E. coli JM10? was transformed and the mutant plasmid was purified. In this way, plasmid pompKpnl was obtained.

■ p obtained according to the description in patent application No. 4/-2Ag3 & 2
MTI2/All was mixed with Kpnl in 10 μl of buffer L (/θmM Tris-HCl and 6mM magnesium chloride).
/μl was used for digestion at 37°C for 1 hour. After further heating at 70° C. for 70 minutes to inactivate the enzyme, the mixture was dialyzed against water and evaporated to dryness. Next, buffer H1
After reaction and digestion using 0 μl of Pstl/μl at 37° C. for several hours, the enzyme was inactivated by heating at 70° C. for 7θ minutes, dialyzed against water, and evaporated to dryness.

Meanwhile, pompKpnl was digested with Kpnl and twice I in the same manner and evaporated to dryness.

Add the digested DNA fragments to 10 μl of buffer solution.
and reacted with Tda DNA ligase/μl at 9°C for 6 hours.

Escherichia coli JM109 competent cells were transformed using 3 μl of this reaction, and the plasmid was isolated and purified from the resulting transformant, cut with HindIII and ≦na) restriction enzymes, and lac l of pompKpnl.
A plasmid pOFA containing a fragment containing -Omp F, the replication origin of pMTIJ, the lpp transcription termination sequence and the multi-cloning sequence was obtained (Figure 3).

[Brief explanation of the drawing]

FIG. 1 is a diagram schematically showing the plasmid produced in Example. In the figure, (a) is plasmid phKK, 15
．． (b) represents plasmid phKK-8p and (c) plasmid phKK-BpB. FIGS. 2 and 3 are diagrams schematically showing plasmids produced in reference examples. Each plasmid pOF
h K K and plasmid pOFA. Applicant Tadayo Nakanishi
Mr. Kagawa, Patent Attorney - 7 others, 2 drafts, 3 diagrams

Claims (1)

[Claims] (1) Cultivate a transformant transformed with an expression vector containing DNA encoding human kallikrein or a human kallikrein-like substance having an equivalent physiological activity, and obtain the human kallikrein or the human kallikrein-like substance produced. A method for producing a human kallikrein-like protein.