JPH0751066B2 - Protease inhibitor gene, microorganism having the gene, and method for producing protease inhibitor using the microorganism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial field of application> The present invention relates to a DNA carrying genetic information of a protease inhibitor, a novel vector incorporating the DNA, a microorganism into which the vector is introduced, and a protease using the microorganism. It relates to a method for producing an inhibitor.

<Prior art and problems> Intracellular proteolysis involves numerous physiological reactions in cells, such as turnover of self-proteins, degradation of foreign proteins / peptides, removal of abnormal proteins, intracellular organelles. It is involved in processing and regulation of vascular and secretory proteins, signal transduction, and enzymatic reactions such as cell proliferation and differentiation. The intracellular structure has a complex structure consisting of many compartments, and proteases directly involved in proteolysis require strict control. The protease inhibitor is extremely effective as a substance that immediately suppresses the action of protease, and is an important element of the regulatory system of protease.

Further, in the microbial industry and the fermentation industry, even if the microorganism secretes and produces the target protein, when the protease is simultaneously produced, the target protein is decomposed by the protease. However, if a protease inhibitor is present in such a system, the action of protease is suppressed, and as a result, the target protein can be efficiently obtained.

Protease inhibitors exist widely in the animal, plant and microbial kingdoms, and in particular, the presence of inhibitors corresponding to trypsin, chymotrypsin and papain is well known. Although its usefulness is being confirmed, the current situation is that it has not yet supplied the amount industrially required.

<Means for Solving Problems> In order to develop an advantageous method for producing a protease inhibitor, intensive studies have been conducted, and as a result, attention has been paid to a method by genetic engineering.

The present invention has been made as a result of further research, and includes a DNA containing a gene encoding a protease inhibitor, a vector incorporating the DNA, a transformant introduced with the vector, and the transformant. It was made for the purpose of developing a method for producing a protease inhibitor using the same.

In order to achieve the above-mentioned object, the present inventors first screened a protease inhibitor-producing bacterium, and as a result, found that Bacillus brevis produced a protease inhibitor extracellularly.
Since this protease inhibitor is novel, it was named protease inhibitor BbrPI.

Specifically, as Bacillus brevis, Bacillus brevis HPD31 (Bacillus brevis H102, FERM BP-108
7) and Bacillus brevis 47 (FERM P-7224) are exemplified.

Based on these findings, the present inventors have found that the protease inhibitor BbrP
The I gene was successfully cloned. Then, the gene was further incorporated into a vector, introduced into a host microorganism, and the recombinant microorganism obtained was cultivated to successfully produce the protease inhibitor BbrPI,
The present invention has been completed.

Hereinafter, the present invention will be specifically described.

1. Cloning of this gene Chromosomal DNA was prepared from microorganisms, animals, and plants capable of producing the proteade inhibitor BbrPI.
NA, a DNA fragment obtained by cutting with an appropriate restriction enzyme, and a vector fragment obtained by similarly cutting the vector,
For example, it is ligated with T4 DNA ligase or the like to form a recombinant DNA containing the protease inhibitor BbrPI gene.

Specifically, for example, culturing a strain belonging to the genus Bacillus,
Chromosomal DNA is extracted from the bacterial cells obtained by centrifugation or the like, and this chromosomal DNA is partially decomposed using a restriction enzyme (for example, Sau3A I). This DNA fragment is subcloned using a plasmid or phage. Vectors for subcloning include, for example, multicopy plasmid type plasmid vector pBR322, pUC series and other single-stranded D
NA13 phage M13 phage phage vector, etc.
Various vectors commonly used for subcloning are appropriately used.

The nucleotide sequence of the insert is determined for the resulting plasmid. Then, a transformant microorganism into which the recombinant DNA has been introduced can be obtained by a conventional method.

As the donor of the protease inhibitor BbrPI gene, in addition to the Bacillus strains described above, a transformed microorganism into which the protease inhibitor BbrPI-producing ability has been introduced by gene recombination can also be used as the donor microorganism.

In carrying out the present invention, the DNA derived from the donor microorganism is
The donor microorganism can be prepared, for example, by agitating and culturing in a liquid medium for about half to 3 days, centrifuging the obtained culture to collect the cells, and then lysing it. As the lysis method, for example, treatment with a cell wall lysing enzyme such as lysozyme or β-glucanase, ultrasonic treatment, or the like is used. If necessary, other enzyme agents such as protease and ribonuclease and a surfactant such as sodium lauryl sulfate are used together. In addition, freeze-thaw treatment may be applied.

In order to separate and purify DNA from the lysate thus obtained, for example, phenol extraction, deproteinization treatment, protease treatment, ribonuclease treatment, according to a conventional method,
It can be performed by appropriately combining methods such as alcohol precipitation and centrifugation.

The method of cleaving DNA can be performed by, for example, ultrasonic treatment, restriction enzyme treatment, or the like. After cleavage, a modifying enzyme such as phosphatase or DNA polymerase is used if necessary. The nucleotide sequence at the end of the DNA fragment can be changed by using various linkers and adaptors.

From the cleaved DNA fragment, only a fragment having an optimum length can be obtained by sucrose density gradient centrifugation, extraction from a gel subjected to electrophoresis, or the like.

Suitable vectors are phages or plasmids that can autonomously grow in the host microorganism.

Known phages can be appropriately used. For example, when Escherichia coli is used as the host microorganism, λ phage, M13 phage and the like can be used.

As the plasmid, for example, when Escherichia coli is used as the host microorganism, pBR322, pUC18, pCU11
8 and their derivatives can be used, and in the case of Bacillus brevis, pUB110 and their derivatives can be used.

Furthermore, for example, Escherichia coli, capable of autonomous growth in two or more host microorganisms such as Bacillus brevis, for example, in addition to vectors such as pHY300PLK, YIp5, YEp13,
It is also possible to utilize various known shuttle vectors. Such a vector is cleaved with a restriction enzyme or the like in the same manner as the DNA described above to obtain a vector fragment.

The method for ligating a DNA fragment and a vector fragment is known in
A method using NA ligase may be used, and for example, after the DNA fragment and the vector fragment are annealed, a recombinant DNA is prepared in vitro by the action of an appropriate DNA ligase. If necessary, it can be introduced into a host microorganism after annealing and then converted into a recombinant DNA by utilizing in vivo DNA ligase.

The host microorganism may be any one as long as the recombinant DNA can be stably and autonomously propagated and its phenotype can be expressed.

The method for introducing the recombinant DNA into the host microorganism is a known method, for example, when the host microorganism is Escherichia coli, the calcium method (Lederberg, EM and Cohen, SN; J. Bacte).
riol., 119 , 1072, (1974)) can be adopted.

In the case of λ phage DNA, in vitro packaging method (Horn, B .; Methods in Enzymology, 68 , 299, (197
9)) was used to form λ phage particles, and the λ phage particles were added to a culture suspension of Escherichia coli,
It is possible to obtain a special transduction phage having the ability to produce the protease inhibitor BbrPI.

When the host bacterium is Bacillus brevis, Tris-PEG
Law (Takahashi, W, et al; J. Bacteriol., 156 , 1130-1134
(1983)) or the Electroporation method (Takagi, H., et al; Agric. Biol. Chem., 53 .
(11), 3099-3100 (1989)).

The method for selecting the transformed microorganism into which the recombinant DNA has been introduced is
The cells are cultured in a liquid selective medium, and the protease inhibitor BbrPI activity in the culture medium is measured. As the liquid selection medium, a minimal medium or an antibiotic-containing medium is appropriately used depending on the marker on the vector.

In addition, a colony immunoassay using a rabbit antibody obtained by using the purified protease inhibitor BbrPI as an antigen (DMHelfman et al; Proc. Natl. Acad. Sci. USA, 80 , 31-3
5 (1983)).

The obtained protease inhibitor BbrPI-producing bacterium was cultured in a liquid medium at 30 ° C., and a known method, for example, an alkali extraction method (Birnoboim.HC and Doly, J .; Nucleic Acid Re
s., 7 , 1513, (1979)).

Recombinant DNA containing the protease inhibitor BbrPI can be appropriately cleaved with a restriction enzyme according to the above-mentioned method, and incorporated into another vector or introduced into another host.

2. Preparation of Protease Inhibitor BbrPI Protease inhibitor BbrPI can be prepared as follows.

That is, the protease inhibitor prepared as described above
A transformed microorganism that has acquired the ability to produce BbrPI is subjected to liquid culture. The medium for culturing the transformed microorganisms may be any as long as it can be used for ordinary culture of microorganisms.For example, carbon sources include starch, liquid starch, glucose, glycerin, molasses, molasses, etc., Examples of the nitrogen source include various protein decomposition products, soybean flour, meat extract, peptone, urea, nitrates, ammonium salts, yeast extract, corn steep liquor and the like. In addition, nutrients such as biotin and trace metals are appropriately used.

After culturing, if the protease inhibitor BbrPI is present in the microbial cells, the culture solution is centrifuged to obtain the microbial cells, which are treated with ultrasonic waves or cell wall lysing enzyme, etc. Use the solution containing the inhibitor BbrPI.

When the protease inhibitor BbrPI is present in the medium, the culture solution is centrifuged to remove the cells, and the subsequent purification is performed.

The protease inhibitor BbrPI can be obtained from the obtained solution containing the crude protease inhibitor BbrPI by a general protein purification method such as salting out, dialysis, ion exchange resin, affinity chromatography treatment.

Next, the present invention will be described in detail with reference to Examples.

Example 1. Cloning of protease inhibitor BbrPI gene Bacillus brevis strain HPD31 (FERM BP-1087) was prepared in a liquid medium (1% glucose, 1% peptone, 0.2 yeast extract).
%, Meat extract 0.5%, pH 7.0), shake culture overnight, collect cells by centrifugation, wash, and remove the cells from Saito, Miur
Method a (Saito, H. and Miura, K .; Biochem. Biophys. Act
a, 72 , 619 (1963)) to separate the chromosomal DNA, which is then dissolved in Tris-HCl / EDTA buffer and the restriction enzyme Sau3
After adding AI (Takara Shuzo) and partially decomposing at 37 ℃,
The degradation product was subjected to agarose gel electrophoresis, and a chromosomal DNA fragment of about 2 kb or more was separated and extracted from the gel.

Vector cut pUC118 (Takara Shuzo) with BamHI, treated with alkaline phosphatase, ligated with T4 DNA ligase after mixing with the chromosome fragment of about 2 kb or more obtained above,
E. coli XL1-Blue was transformed. The obtained transformant was subjected to colony immunoassay using a rabbit antibody prepared by using the purified protease inhibitor BbrPI as an antigen to obtain a strain that reacts with the antibody.

The plasmid pYAS01 obtained here was further treated with restriction enzymes EcoR I, BamH I and Pst I as shown in FIG. I got pYAS04. It was confirmed that Escherichia coli introduced with the plasmid pYAS04 produced the protease inhibitor BbrPI. This strain is called Escherichia coli pYAS04.

Then, the 1517 bp nucleotide sequence of the pYAS04 fragment BamHI-PstI was determined. The base sequence is shown in FIG. In Figure 1, 1446 from position 832 (TTC: phenylalanine)
Up to the position (GAA: glutamic acid) shows the proteage inhibitor BbrPI activity.

The nucleotide sequence corresponding to the amino acid sequence, from position 778 (ACC: threonine) to position 1446 (GAA: glutamic acid) similarly shows protease inhibitor BbrPI activity. The nucleotide sequence corresponding to the amino acid sequence, the position from 541 (ACC: threonine) to 1446 (GAA: glutamic acid) is the longest nucleotide sequence corresponding to the amino acid sequence exhibiting the same protease inhibitor BbrPI activity. From the translation start point at the position (ATG: methionine) to the position 1446 is the base sequence corresponding to the structural gene for the protease inhibitor BbrPI.

In addition, in the base sequence of FIG. 1, 392 to 397 (TTGAAA), 41
Transcriptional regulatory region in 4-419 (TCGTCC), 454-458 (GGAGG)
And a translational regulatory region of 469-471 (ATG:
Methionine) contains the translation start point.

Example 2. Transformation into B. brevis Escherichia coli pYAS04 was cultured overnight at 37 ° C, the culture solution was centrifuged, the cells were collected, washed, and then alkali-extracted from the isolated cells (Birnoboim.HC and Doly, J. .; Nucleic Acids
The plasmid pYAS04 was isolated by Res., 7 , 1513, (1979)).

BamHI and HindIII (manufactured by Takara Shuzo Co., Ltd.) were added to this plasmid, and the mixture was cleaved by reacting at 37 ° C to obtain BamHI-Hi.
A fragment of nd III 1.5 kb was obtained.

Separately, the vector pNH300, which can be expressed in Bacillus brevis, is
After cutting with amH I and Hind III (Takara Shuzo), it was treated with alkaline phosphatase (Takara Shuzo), and BamHI-Hind III containing protease inhibitor BbrPI gene 1.
The 5 kb fragments were mixed, ligated with T4 DNA ligase (Takara Shuzo), and electroporation (Takagi, H., et.
al; Agric.Biol.Chem., 53 (11), 3099 (1989))
Bacillus brevis HPD31 (Bacillus brevis H102 FERM B
P-1087) and transformed into Bacillus brevis H
PD31-S5 / pYS01 was obtained.

The vector pNH300 was prepared as follows. pN
U200 (Yamagata, H. et al ,; Proc.Natl.Acad.Sci.USA 86 , 3
589 to 3593, 1989) was treated with the restriction enzyme EcoRI, and the cleavage site (sticky end) was filled with Klenow enzyme (fill i).
n) Treatment with the restriction enzyme HindIII gave a 350 bp fragment containing the promoter of B. brevis 47 MWP and part of the signal peptide. pBAM101 (Tsukagoshi, N. et al .; J. Bacterio
l., 164, after treatment with a restriction enzyme BamH I to 1182～1187,1985)
Klenow enzyme is used to fill in the cleavage site, then the restriction enzyme
Treatment with nd III yielded a 3.3 kb fragment, which is the same as the 350 bp obtained above.
The fragments were ligated using T4 DNA ligase and the B. brevis HPD31
Was transformed to obtain a plasmid pNH300 in which two fragments were ligated.

Example 3 Production of Protease Inhibitor BbrPI Bacillus brevis HPD31-S5 / pYS01 was transformed with neomycin (60
200 ml of T2 liquid medium (μg / ml) (glucose 1%, peptone 1%, yeast extract 0.2%, meat extract 0.5%, pH 7.
After culturing at 0) for 3 days at 30 ° C., the cells were removed by centrifugation to obtain 180 ml of a culture supernatant. The protease inhibitor BbrPI activity of this culture was measured to be 1100 u / m.
l activity was demonstrated.

Protease inhibitor BbrPI activity is 600μ in 0.1M Tris-HCl (pH7.5) buffer containing 0.01M calcium chloride.
, A sample containing the protease inhibitor BbrPI 10
Mix 5μ of trypsin (200μg / ml solution) and 5
After a minute, the substrate (0.02MN-benzoyl-L-arginine-p-ni
troanilide) 10 μm is added, and the absorbance at 405 nm is immediately measured.

As a control, the activity (increase of 0D value) when the protease inhibitor BbrPI was not contained was measured, and the protease inhibitor BbrP when the activity of 1 μg trypsin was suppressed by 50%
The amount of I is shown as 1 unit.

Ammonium sulfate was added to the supernatant to 0.8 saturation, and the precipitate was collected by centrifugation. This precipitate fraction
It was dissolved in 20 mM Tris-HCl (pH 7.5) buffer and dialyzed against the same buffer. The sample after desalting by this dialysis is DEAE
-After adsorption on cellulose, elution was carried out with a salt concentration gradient of 0 to 0.5 M, and the active fraction of the protease inhibitor BbrPI was collected. Ammonium sulfate was added to this active fraction to further saturate 0.8,
20mM Tris-HCl buffer (pH 7.5)
The protease inhibitor BbrPI active moiety was collected by gel filtration chromatography on Toyopearl-HW-55, which was dissolved in E. coli and equilibrated with 20 mM Tris buffer containing 0.1 M NaCl. The collected active fraction was purified from the culture supernatant about 300 times to obtain almost pure protease inhibitor BbrPI.

<Effects of the Invention> The present invention makes it possible for the first time to produce a large amount of a protease inhibitor.

Therefore, the present invention is not only useful for elucidating the protease control system, but also can be expected to be used as a drug. Further, by using this inhibitor, in the microorganism utilization industry, the target protein secreted and produced will not be destroyed and denatured by the protease, so that a protein that could not be obtained in the past or could not be obtained in a sufficient amount is free. It is possible to obtain it.

Further, by introducing the protease inhibitor BbrPI gene according to the present invention into various microorganisms, the protease produced by the microorganisms is inactivated, and thus the expression of the structural gene is performed extremely efficiently. As a result, not only the target protein can be produced in a large amount, but also the protein that has been destroyed by the protease can be produced, and the possibility that a previously unknown physiologically active substance is expressed becomes very high.

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence containing the protease inhibitor BbrPI gene, and FIG. 2 shows the physical map of the vector, pYAS01-04.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12P 21/02 C 9282-4B // (C12N 15/09 ZNA C12R 1:08) (C12N 1 / 21 C12R 1:08) (C12P 21/02 C12R 1:08) C12R 1:08)

Claims (9)

[Claims] 1. The position of 832 (T
D: having a base sequence corresponding to the amino acid sequence of the protease inhibitor BbrPI starting from (TC: phenylalanine) and ending at the final position 1446 (GAA: glutamic acid)
NA. 2. The position of 778 shown in the nucleotide sequence of FIG. 1 (A
CC: threonine) and the final position 1446 (GAA:
Glutamic acid) -terminated protease inhibitor Bb
A DNA having a base sequence corresponding to the amino acid sequence of rPI. 3. The position of 541 shown in the nucleotide sequence of FIG. 1 (A
CC: threonine) and the final position 1446 (GAA:
Glutamic acid) -terminated protease inhibitor Bb
A DNA having a base sequence corresponding to the amino acid sequence of rPI. 4. The position of 469 shown in the nucleotide sequence of FIG. 1 (A
(TG: methionine) starting from the translation start point and ending at 144
A DNA having a nucleotide sequence corresponding to the structural gene of the protease inhibitor BbrPI ending at position 6 (GAA: glutamic acid). 5. The 392 to 397 (TT shown in the base sequence of FIG.
GAAA), 414-419 (TCGTCC) transcriptional regulatory region, 454-458
(GGAGG) and including 469-47
A DNA having a base sequence corresponding to the amino acid composition starting from the 541 position and ending at the 1446 position, including a translation start point at the 1 (ATG: methionine) position. 6. A DNA having the nucleotide sequence shown in FIG. 1 from the first codon GGA to the final codon CAG. 7. A plasmid containing a DNA fragment encoding the protease inhibitor BbrPI derived from Bacillus brevis shown in the nucleotide sequence of FIG. 1, which is pYAS01 obtained by treating Bacillus brevis chromosomal DNA with Sau3A I. ,
PYAS02 obtained by treating it with EcoRI, pYAS03 obtained by treating it with BamHI, and / or pYAS04 obtained by treating it with PstI. 8. A plasmid containing a DNA fragment encoding a Bacillus brevis-derived protease inhibitor BbrPI represented by the nucleotide sequence of FIG. 1, which is pYAS01 obtained by treating Bacillus brevis chromosomal DNA with Sau3A I. ,
A transformant comprising pYAS02 obtained by treating it with EcoRI, pYAS03 obtained by treating it with BamHI, and / or pYAS04 obtained by treating it with PstI. 9. A plasmid containing a DNA fragment encoding the Bacillus brevis-derived protease inhibitor BbrPI shown in the nucleotide sequence of FIG. 1, which is pYAS01 obtained by treating Bacillus brevis chromosomal DNA with Sau3A I. ,
Protease inhibitor by culturing a transformant containing pYAS02 obtained by treating it with EcoR I, pYAS03 obtained by treating it with BamH I, and / or pYAS04 obtained by treating it with Pst I A method for producing a protease inhibitor BbrPI, which comprises producing BbrPI.