JPH0528596B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a novel recombinant microorganism. [Prior art] Lysozyme has the ability to lyse various bacteria, so it is used as a preservative in foods and medicines, as an antibacterial agent when used alone or in combination with antibiotics, and also has blood coagulation and hemostasis effects.
It also has anti-inflammatory effects and sputum expulsion effects for people with respiratory diseases, so it is also used as a medicine for those patients. Conventionally, human lysozyme has been isolated and used from human milk, urine, etc. However, it is inefficient and impractical to meet the demands of industrial production for medical purposes and the like. [Problems to be Solved by the Invention] Therefore, the present inventors have conducted research and development to produce pure human lysozyme inexpensively and in large quantities using recombinant DNA technology. We first applied for a patent on a production method for secretory production of human lysozyme using yeast (patent application filed in 1980-
268218). However, in this method, the amount of human lysozyme secreted into the medium is approximately 0.2 to 0.6 mg/, and for industrial implementation, it is necessary to further increase the production amount. Under these circumstances, the present inventors conducted various studies with the aim of extracellular large-scale secretion production of human lysozyme using yeast, and as a result, the present inventors discovered that the human lysozyme secretion expression system gene (promoter, secretion signal A yeast transformant carrying a plasmid into which DNA, the human lysozyme structural gene, and a terminator (hereinafter referred to as the secretory expression unit) was inserted was subjected to mutation treatment, and a large amount of human lysozyme was extracted from the treated cells. We found a method to select mutants that have the ability to secrete lysozyme, used this method to obtain high secretion mutants, and further cultivated this secretion mutant to produce approximately 2 to 6 We have discovered a method that enables twice the secretion production, and have completed the present invention. [Means for Solving the Problems] The present invention provides a microbial mutant strain that carries a plasmid containing the human lysozyme gene and has the ability to highly secrete human lysozyme, and a microbial mutant strain that contains bacterial cells. A method of sorting using an agar plate. A specific example of this microbial mutant strain is the yeast Saccharomyces cerevisiae strain E91.
This has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM BP-1432. In addition, as an agar plate, dried bacterial cells,
A composition consisting of yeast extract, peptone, glucose and agar is used. Next, the present invention will be explained in detail. (1) Simple selection method for high-secreting human lysozyme strains Conventionally, as a relatively simple method for measuring lysozyme activity, an aqueous solution containing lysozyme protein is infiltrated into an agar plate containing bacterial cells, and the bacterial cells are lysed. A method is known to examine the size of the transparent ring (halo) that forms (Osserman, J.Exp.
Med.124:921-951 (1966)). In this method, it was necessary to produce lysozyme in advance in the subject and place a certain amount of it in a hole made on an agar plate. In order to select mutants from yeast, it is necessary to assay the yield of at least several hundred to several thousand independent colonies, and known methods require a great deal of labor and expense. In order to be able to isolate a human lysozyme-high secreting yeast mutant strain, we investigated a very simple method to test the lysozyme production ability of yeast cells. Time, effort, and expense are due to the process of liquid culture, cell removal, and repeated transplantation and dispensing operations. We developed a new method to investigate lysozyme production capacity by omitting these steps and using only agar plates. The principles of the improvement are based on the following three points. Each colony consists of relatively the same number of cells. Yeast colonies carrying the lysozyme gene with a strong promoter and secretion signal secrete and produce lysozyme protein on nutrient-containing agar as well as in liquid medium. Halo formation by lysozyme progresses even in agar plates to which nutrients have been added. (2) Obtaining a high-secretion mutant strain of human lysozyme The present inventors have already developed a hybrid prelysozyme gene consisting of a DNA region encoding the chicken lysozyme secretion signal and a human lysozyme structural gene region into a suitable yeast strain. Human and human
We have proposed a method for secreting and producing lysozyme in its active form into a culture medium (Japanese Patent Application No. 268218/1982). Therefore, this Satsukaromyces cerevisiae KK4 (YEP
−HLYSIG), Satsukaromyces cerevisiae KK4
(pESH) and other transformants were subjected to mutation treatment, and mutant strains that secreted high amounts of human lysozyme were selected using the above-mentioned selection method. The parent strain used for mutation treatment was a multicopy vector (YEp type) containing human lysozyme secretion expression unit.
A transformant carrying the inserted plasmid was used. The transformation of yeast can be carried out using known methods (for example, Ito et al., J. Bacteriol. , 153 , 163-168).
(1983)). Furthermore, various yeast promoters can be used as the promoter, and one specific example is a yeast high expression promoter that uses ENO1, a gene encoding the glycolytic enzyme enolase. The structure and DNA base sequence of this promoter have already been proposed by some of the present inventors (Japanese Patent Application No. 120900/1982). In addition, as a specific example of a multi-copy vector, the already proposed
In addition to the YEp type vector (Patent Application No. 1982-268218),
Various known vectors can be used. (3) Analysis and quantification of secreted human lysozyme If the yeast mutant strain containing the human lysozyme expression unit obtained above is cultured according to conventional methods in the fields of molecular biology and applied microbiology, it can be
Lysozyme is secreted and produced. secreted human
Lysozyme can be used for measurements of its enzymatic activity (e.g.
Morsky. Anal. Biochem. 128. 77-85 (1983)), high performance liquid chromatography (e.g. Y. Jigami et al.
Gene, 43 , 273-279 (1980)), as well as gel electrophoresis (Western blotting) using antibodies (for example, Towbin et al., Proc. Natl. Acad. Sci.
USA 76 , 4350 (1979)) and other known methods can be used for analysis and quantification. Details of these methods and results of analysis and quantification will be explained in Examples. [Effects of the Invention] According to the present invention, a microbial mutant strain carrying a plasmid containing the human lysozyme gene and having a high secretion ability of human lysozyme can be selected by a simple method, and this microbial mutant strain can be cultured. As a result, human lysozyme could be produced in high yield. Therefore, the microbial mutant strain of the present invention and the method for producing human lysozyme using the same are suitable as a method for mass production of human lysozyme. [Example] 1. Creation of a multicopy plasmid (pESH) containing a human lysozyme secretion expression unit and a yeast transformant carrying it Plasmid YEp- in which a hybrid prelysozyme gene was inserted downstream of the GAL10 promoter
HLYSIG (Y. Jigami et al., Gene, 43 , 273-279
(1986)), expression can be induced with galactose, but expression is generally suppressed in the presence of glucose. On the other hand, the ENO1 promoter is a strong promoter that shows constitutively high levels of expression in the presence of glucose. As a growth carbon source for the culture medium, glucose is cheaper than galactose and has the advantage of being able to prepare a high-density cell culture solution in a short time. Therefore,
It is better to culture yeast carrying a plasmid expressed under the control of the ENO1 promoter using glucose as a carbon source than to culture yeast carrying a plasmid expressed under the control of the GAL10 promoter using galactose as a carbon source. We can expect an increase in production. Therefore, plasmid
GAL10 promoter part of YEp-HLYSIG
I decided to replace it with the ENO1 promoter. Therefore, by converting the restriction enzyme Sau3AI site present at the 5′ end of the GAL10 promoter into a restriction enzyme BamHI site that does not exist on the plasmid by site-specific mutation, the GAL10 promoter can be digested with the restriction enzymes BamHI and SalI. Made it possible to remove it. Mutations can be introduced using a known method using synthetic oligonucleotides and double-stranded DNA plasmids (Morinaga et al., Biotechnology, 2 , 636-639).
(1984)). i.e. plasmid
Obtained by cutting YEp-HLYSIG with restriction enzyme PstI
5.75kb obtained by double digestion of 6.7kb fragment (0.03pmole) and YEp-HLYSIG with restriction enzymes SalI and ClaI
(0.03 pmole) was mixed in the presence of a synthetic oligonucleotide (5'GATGATTTTG*phosphorylated at the 5' end of GATCC3', 12 pmole, *mark indicates the site where the mutation was introduced). The method for synthesizing and purifying DNA oligomers is disclosed in Japanese Patent Application No. 60-268218 by the present inventors. After heating this mixture at 100℃ for 3 minutes, heating at 30℃ for 30 minutes,
By leaving the DNA at 10°C for 30 minutes or at 4°C for 10 minutes and slowly cooling it, the DNA that has dissociated into single strands returns to double strands, forming a heteroduplex with a gap at the site of restriction enzyme cleavage. Can form DNA.
This was combined with E. coli DNA polymerase (Klenow fragment, 2 units) and _T4 -DNA ligase (5 units).
After repairing the DNA using the DNA oligomer as a primer, E. coli strain C600 was transformed. Plasmid DNA can be introduced into E. coli using known methods (e.g., Maniatis et al., Molecular
Cloning, 249-253 (1982)). Extract plasmid DNA from the obtained transformed strain,
As a result of analyzing the restriction enzyme cleavage pattern by electrophoresis using 0.7% agarose gel, it was found that only one strain possessed a plasmid (YEp-HLYSIG-BamHI, see Figure 1) containing the target restriction enzyme BamHI site. Separated. In addition, the introduced
The BamHI site is at the desired location and there are no mutations in other regions using the dideoxy DNA sequencing method (Sanger et al., Proc. Natl. Acad. Sci. US).
A., 74 , 5463-5467 (1977)). Next, we added only the ENO1 promoter part to GAL10
In order to replace the promoter portion, a restriction enzyme SalI site was introduced between the ENO1 promoter transcription start point and translation start point. That is, by cutting the plasmid pBR-M-ENO (see Figure 1) containing the ENO1 promoter with the restriction enzyme PstI and further excising the protruding single-stranded portion with S1 nuclease,
A fragment (0.03 pmole) in which the ampicillin resistance gene was disrupted was prepared. On the other hand, the same pBR-M
-ENO was double digested with restriction enzymes EcoRI and SalI to prepare a large fragment of 4.3 kb (0.03 pmole). Both of these were mixed in the presence of a synthetic oligonucleotide (phosphorylated at the 5' end of 5'ACTGCTTG * TCG * ACACACAAAC3', 12.5 pmole, * mark indicates the site of mutation introduction), and in the same manner as above. A plasmid with site-specific mutations introduced was created. Target plasmid pBR-M-ENO
-SalI, it was confirmed by examining the DNA base sequence that there were no mutations in the ENO1 promoter other than this SalI site, as described above. Next, pBR-M-ENO-SalI was added to the restriction enzyme
A DNA fragment (1.0 pmole) containing the ENO1 promoter was recovered by double digestion with BamHI and SalI. On the other hand, plasmid YEp−HLYSIG−BamHI
was double digested with restriction enzymes BamHI and SalI,
Large fragment excluding GAL10 promoter (7.2kb,
0.5pmole) was recovered. The two were ligated in the presence of _T4 -DNA ligase (14 units) to create plasmid pESH (7.9 kb). The plasmid pESH thus obtained was prepared using the known lithium method (Ito et al.
J. Bacteriol., 153 , 163-168 (1983)) and yeast Sacheromyces.cerevisiae strain KK4 (Nogi et al.
Mol.Gen.Genet., 195 , 29-34 (1984)) and A2-
1-1A strain (Jigami et al., J.Biochem., 99 , 1111~
1125 (1986)). These transformants Satucharomyces cerevisiae KK4 (pESH) and Satucharomyces cerevisiae A2-1-1A (pESH)
secreted and produced lysozyme in the culture medium. 2. Simple selection method for human lysozyme-high secreting mutant strains Micrococcus highly sensitive to lysozyme
Lysodeikticus ATCC4698 freeze-dried cells (manufactured by Sigma) were suspended in distilled water to a concentration of 10 mg/ml, and sterilized by boiling for 10 minutes. 120℃, 10
The heat-sterilized suspension was mixed with an agar aqueous solution heat-dissolved by autoclaving for 1 minute, poured into a shear dish, and solidified to prepare a bacteria-containing agar plate with a final concentration of agar of 2% and bacteria of 0.5 mg/ml. Using agar plates containing various modified bacteria, a known method (Morsky,
Anal.Biochem 128:77-85 (1983)) yeast strains whose ability to produce lysozyme had been demonstrated were tested.
Lysozyme non-producing strains (Satucharomyces cerevisiae A2-1-1A and Satucharomyces cerevisiae
KK4) and lysozyme-producing strains (Satucharomyces cerevisiae A2-1-1A (pESH) and Satucharomyces cerevisiae KK4 (pESH)) were precultured on an agar plate, and then a small amount was placed on an agar plate with the composition shown in Table 1. Transplanted with toothpicks. The amount of agar medium is set to 1 so that the halo can be observed clearly and in a short cultivation time.
The amount was as small as 10 ml per plate. On the agar plate containing no nutrients, there was no growth of yeast and no halo formation was observed (Table 1). SD synthetic medium (glucose 2%, 0.67% Yeast Nitrogen Base w/
o Amino Acids, amino acids and nucleic acids)
(Sherman et al., Methods in Yeast Ganetics,
Although yeast growth was observed on agar plates containing p62 (1983), no halo formation was observed, and 0.1M (final concentration) potassium phosphate buffer (PH7
Alternatively, halo formation was observed only after addition of 8). The non-lysozyme producing strain did not form a halo on this agar plate. YPD natural medium (1% yeast extract, 2% peptone and 2% glucose)
On agar plates containing lysozyme, it was possible to observe halo formation without adding a PH buffer, and non-lysozyme-producing strains did not form halos. Among the seven types of agar plates, the most obvious halo could be observed when using the YPD-added agar plate, making it suitable for examining a large number of specimens. Addition amount of bacterial cells is 0.5mg/ml
-2.0mg/ml. At low concentrations, the diameter of the halo increases, but the contrast between the inside and outside of the halo decreases. On the other hand, at high concentrations, it takes a long time to form a clear halo, so 1 mg/ml
ml concentration was optimal. To summarize the above conditions, 10mg per plate
10 ml of 2% agar containing dried bacterial cells, 0.1 g yeast extract, 0.2 g peptone and 0.2 g glucose was suitable as an assay agar plate. When the yeast suspension was smeared onto the agar plate without using a toothpick to inoculate the agar plate, and colonies were formed from single yeast cells, a one-day delay in halo formation was observed, but this was unusual. A clear halo formation could be observed.

[Table] +: Contained or present, -: Not contained or absent.
3 Obtaining a mutant strain with high secretion of human lysozyme Satucharomyces cerevisiae KK4 using plasmid vector pESH carrying human lysozyme gene combined with ENO1 promoter and secretion signal
S. cerevisiae transformant of strain
After pre-culturing KK4 (pESH) in a synthetic liquid medium that does not contain leucine, 3 million cells were cultured using a known method (Suzuki and Yanagisima, Curr. Genet. 9, 185~
189 (1985)) and mutagenic treatment with ethyl methanesulfonic acid. After the treatment, the bacterial cells were washed and diluted with 6% sodium thiosulfate and then water, and then smeared on the above-mentioned agar plate for screening high lysozyme secretion mutants. The killing rate of bacterial cells by mutagen treatment was 90.5%. Colonies forming a large halo on an agar plate were picked with a toothpick, and the colonies were inoculated onto a new agar plate to examine the reproducibility of halo formation. 31 out of 592 colonies formed a halo significantly larger than the parent strain. In particular, four strains that formed large halos were assayed for lysozyme activity by liquid culture. The culture was performed using 5 ml of leucine-free synthetic medium and shaking at 30°C for 2 days. After removing the bacterial cells from the culture solution by centrifugation, Morsky's method (Anal.
Lysozyme activity was measured by a known method according to Biochem. 128:77-85 (1983). As shown in Table 2, the mutant strain secreted significantly more lysozyme into the culture solution than the parent strain. This shows that the above-mentioned simple selection method is useful and that human lysozyme can be secreted at a high level by treating the cells with a mutagen. Table 2 Lysozyme activity (Units/ml) Relative ratio Parent strain KK4 (pESH) 14 1.0 Mutant strain E74 27 1.7 E82 36 2.5 E91 114 8.0 E95 102 7.2 4 Mutants selected by mutation treatment of transformants carrying plasmid pESH , secretory production of human lysozyme by Satucharomyces cerevisiae (E91 strain) Among the mutant strains obtained above, plasmid
Using the mutant Satucharomyces cerevisiae (strain E91) selected by mutagenic treatment of pESH-carrying transformants, we grew it in a medium with glucose as the carbon source, and further investigated the secretion of human lysozyme into the medium. I investigated it in detail. Cultivation was carried out at 30°C in SD minimal medium (SD (-leu)) excluding leucine. As a control, the previously proposed plasmid YEp-HLYSIG containing the hybrid prelysozyme gene downstream of the GAL10 promoter was used.
(Patent Application No. 60-268218)
Saccharomyces cerevisiae strain KK4 (KK4 (YEp
−HLYSIG) or Saccharomyces cerevisiae strain KK4 (KK4
(pESH) was used. Table 3 shows a comparison of the enzyme activity of human lysozyme in the medium at various culture times when transformants containing the various plasmids described above were cultured in the above medium containing glucose (SD (-leu)). Ta. The enzyme activity was measured using Morsky's method (Anal,
Biochem., 128, 77 (1983)). For details, please refer to the patent application filed by the present inventors in 1988-
It is detailed in the specification of No. 268218.

[Table] Next, in order to investigate whether the increase in human lysozyme enzyme activity in the mutant strain Satucharomyces cerevisiae (strain E91) is truly due to an increase in the amount of human lysozyme secreted, high-performance liquid chromatography was performed. Separation and quantification were performed using graphie. The details of the analysis and quantitative method have already been described in detail by the present inventors (Japanese Patent Application No. 268218/1982), but a brief outline thereof will be given below. That is, Klett
The culture that had grown to around 350 units was centrifuged to remove yeast cells and obtain a culture supernatant fraction. This was freeze-dried and thoroughly dialyzed against 50mM phosphate buffer (PH8.0), and the final volume was unified to 40ml. And cation exchange column Mono S HR515
(manufactured by Pharmacia) using an FPLC system (manufactured by Pharmacia). Column 1ml/
Human lysozyme standard (Sigma) was eluted with a 0-1M KCl linear gradient over 20 minutes in the presence of 50mM phosphate buffer (PH8.0) at a flow rate of
It is eluted as a single peak around 0.2M KCl. Similarly, Satucharomyces cerevisiae KK4
(pESH) or the S. cerevisiae E91 strain obtained by mutagen treatment, a peak indicating bacteriolytic activity was detected at exactly the same position. The above is as shown in Figure 2. In addition, in Figure 2,
A is the case when 5 μg of a commercially available standard product was used as a sample, and B is the case where E91 was incubated in SD (-leu) medium for about 80 hours.
This shows the case where the culture supernatant obtained by culturing the strain was used as a sample. The arrow indicates the peak of human lysozyme. Next, the amount of secretion is quantified by cutting out the peak of the recording paper that has been enlarged and copied several times, measuring its weight, and correcting its area using a calibration curve created from a known amount of standard product. This was done by doing this. An example of the results is shown in Table 4.
Furthermore, the structure and quantification of secreted human lysozyme was confirmed by Western blotting. That is, a certain amount (2 μ, 5 μ, 10 μ
) was separated by SDS-boryacrylamide gel electrophoresis and transferred to a membrane filter (15cm x 9.2cm manufactured by BIO-RAD).
Electrical transfer was performed using an ATTO horizon blot device (10V, 1 hour), and this was transferred using a commercially available human lysozyme antibody (Alpha Therapeutic,
After reacting with Lot No. TTOO5GU) as the primary antibody,
¹²⁵ I-Protein A (Amersham
A product that reacts with human lysozyme was detected by autoradiography using a product that reacts with human lysozyme (Code No. IN 144). The amount was measured using a densitometer (manufactured by Beckman, OU) to measure the degree of blackening of the X-ray film.
-8 type). As shown in FIG. 3, the product of Satucharomyces cerevisiae strain E91 was also detected at a position with exactly the same mobility as the commercially available standard product. In Figure 3, lane 1 contains 10 ng of the commercially available standard product, lane 2 contains 20 ng, and lane 3 contains 40 ng.
g, lane 4 is 80ng, lane 5 is 160ng.
Lane 6 shows the pattern obtained by electrophoresing 240 ng. In addition, lane 7 is about SD (-leu) medium.
Lane 8 shows a pattern obtained by electrophoresing a 2.5μ sample of a concentrated medium in which the E91 strain was cultured for 80 hours, lane 8 shows a pattern using 5μ of the same sample, and lane 9 shows a pattern using 10μ of the same sample. Therefore, human lysozyme secreted by these strains is already expressed in S. cerevisiae KK4.
(YEP-HLYSIG) (Patent application 1986-
268218), the secretion signal of chicken lysozyme is correctly cleaved in yeast, and commercially available human lysozyme
It is thought to be secreted as a molecule with an N-terminus that is exactly the same as lysozyme. The quantitative results obtained by the various methods described above are summarized in Table 4.

[Table] Obtained by mutation treatment of KK4 (pESH)
There is a possibility that a mutation has been introduced into the E91 strain into the host yeast chromosome, or a mutation may have been introduced into the plasmid pESH carried by this strain, or both. However, it has been confirmed that no mutations have been introduced into the human lysozyme structural gene portion on the plasmid pESH present in the E91 strain using known methods (Suzuki and Yoshida,
Plant Cell Physiol., 27 , 801-808 (1986)).
This was confirmed by examining the sequence method. Therefore, human lysozyme secreted by S. cerevisiae strain E91 is already known as S. cerevisiae KK4 (YEP-HLYSIG).
As in the case shown in (Japanese Patent Application No. 60-268218), it is thought that the molecule is exactly the same as human-derived lysozyme.

[Brief explanation of the drawing]

Figure 1 shows the method for creating plasmid pESH.
Figure 2 shows human lysozyme secreted and expressed in yeast.
The analysis and quantitative method using MonoS column is shown. Third
The figure shows the results of separating proteins secreted into the medium by yeast by SDS-polyacrylamide gel electrophoresis, and specifically detecting and quantifying only human lysozyme using an antibody specific to human lysozyme. This is what is shown.

Claims (1)

[Scope of Claims] 1. Mutational treatment of Satucharomyces cerevisiae into which a plasmid containing the human lysozyme gene has been introduced, and the resulting mutant strains are selected by the size of transparent rings on an agar plate containing bacterial cells. A mutant strain belonging to Satucharomyces cerevisiae that has the ability to highly secrete human lysozyme. 2. The mutant strain belonging to Satucharomyces cerevisiae having a high secretion ability of human lysozyme according to claim 1, characterized in that the mutant strain belonging to Satucharomyces cerevisiae is the Satucharomyces cerevisiae E91 strain.