JPH036796B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention provides that when introduced into a host bacterium, the host bacterium
This article relates to a plasmid that can acquire resistance to trimethoprim at a concentration of 200 μg/ml, and also mentions a method for producing the plasmid and a simple method for purifying dihydrofolate reductase from a host bacterium containing the plasmid.

In recent years, with the development of molecular biology and genetic engineering, methods for producing useful substances using recombinant DNA techniques have been attracting attention.

The present inventors have already integrated E. coli dihydrofolate reductase (hereinafter abbreviated as DHFR) into a multicopy plasmid, and by introducing this into the bacterial cells, the intracellular content of DHFR was increased by 10 to 20 times. In addition, trimethoprim is a strong inhibitor of DHFR and is known as an antibacterial agent, but it has been revealed that E. coli becomes resistant to trimethoprim by increasing the content of DHFR through plasmid introduction. . [Patent application, 56-
143520, and M. Iwakura et al, J.
Biochemistry vol. 91, pp. 1205-1212 (1982)] However, the original DHFR content of E. coli is small, ranging from 10 to
Even with a 20-fold increase, 0.5 of the intracellular protein
% or less. Gene expression, ie, protein production, in bacteria is regulated primarily at the initiation stage of RNA synthesis, ie, at the transcription stage. RNA synthesis relies on a special promoter called a promoter.
Starts at a site with a DNA sequence. The protein content within a bacterial cell varies depending on the type of protein, and this is thought to be due to subtle differences in the promoter sequences of each protein gene.

The DHFR gene has a sequence that can be cleaved with the restriction enzyme EcoR in the DNA sequence encoding the protein. By inserting a heterologous DNA into this site, it is possible to produce a protein in which a part of DHFR is bound to a protein encoded by the foreign DNA. By introducing a code that creates a protein, it is possible to separate a part of DHFR from a protein encoded by a foreign DNA by bromosyanolysis. if,
If a plasmid containing the DHFR gene can be modified using genetic engineering techniques to produce DHFR in high yields, it can be applied to other proteins as described above.

Against this background, the present inventor has conducted intensive research in order to increase the content of DHFR, and as a result, the present inventor has already developed a promoter that has promoter activity as a guideline for the emergence of trimethoprim resistance.
We came up with the idea of introducing a strong promoter by applying a DNA fragment detection method (Japanese Patent Application No. 57-102553). As a result of using Escherichia coli chromosomal DNA cut with the restriction enzyme Hinc as a promoter material, we were able to produce a plasmid that could confer resistance to trimethoprim at a concentration of 200 μg/ml. It was discovered that the DHFR content of the microbial cells containing the DHFR reaches approximately 10% of the total protein, and based on this finding, the present invention was completed.

The host bacteria used in the present invention is Escherichia coli, but
Any host in which the plasmid can be replicated autonomously, for example, a bacteria closely related to E. coli, can be used. The following describes E. coli.

The plasmid containing the DHFR gene is the plasmid pTP6-, which the inventor has already created.
6 (described in Japanese Patent Application No. 57-102553) was used. pTP6
-6 is a recombinant plasmid approximately 4300 base pairs in size that confers ampicillin resistance to the host.
Furthermore, pTP6-6 contains a sequence encoding DHFR, but because a portion of the promoter sequence of the DHFR gene is deleted, DHFR gene expression does not occur. Therefore, pTP6-6 cannot confer trimethoprim resistance to the host. pTP6-6 is kept stable in E. coli;
E. coli containing pTP6-6 was sent to FERMP.
Deposited as −8647. pTP6-6 can be isolated and used from E. coli FERMP-8647 according to a commonly used plasmid preparation method. The method for preparing plasmids from E. coli is as follows:
Various methods other than those described in the examples are known, and those skilled in the art related to the present invention will be able to
easily by using FERMP-8647 strain
pTP6-6 can be prepared.

As the promoter DNA material, E. coli chromosomal DNA cut with the restriction enzyme Hinc was used. The restriction enzyme Hinc recognizes the sequence 5'-GTPyPuAC-3'3'-CAPuPyTG-5' in double-stranded DNA (A, C, G, T, Pu, and
Py is adenine, cytidine, guanine, respectively.
Represents thymine, purine, and pyrimidine residues. ) Just cut the middle part to create blunt ends. E. coli chromosomal DNA is cut with the restriction enzyme Hinc to create fragments of about 1000 base pairs on average. It is thought that there are many fragments in which the promoter sequence remains intact.
Among them, if a fragment containing a strong promoter sequence can be used, it is possible to increase the content of DHFR. Conversely, binding of a strong promoter should increase the content of DHFR, thereby increasing trimethoprim resistance. Therefore, by slightly deleting the promoter of the DHFR gene in pTP6-6 and ligating to this portion a fragment obtained by cutting E. coli chromosomal DNA with the restriction enzyme Hinc using T4 DNA ligase, various genes were generated. Plasmids can be obtained by creating various plasmids into which the fragments have been inserted, introducing them into the host bacterium E. coli, culturing them in a medium containing a high concentration of trimethoprim, and selecting those that can grow. In a bacterial cell containing the target plasmid, DHFR is present in about 10% of the bacterial protein. According to the present inventors' previous findings, it was difficult for bacterial cells carrying a multicopy plasmid containing the DHFR gene to grow in a medium containing trimethoprim at a concentration of approximately 20 μg/ml; The bacterial cells that have
Growth is also possible in a medium containing trimethoprim at a concentration of 200 μg/ml. Furthermore, by performing two steps of purification, namely ammonium sulfate fractionation and DEAE-Sephaadex column chromatography, from the sonicated solution of the bacterial cells, we achieved a high yield of DHFR.
can be purified and a homogeneous protein preparation can be obtained.

Next, the present invention will be explained in more detail with reference to Examples. In addition, isolation of plasmids from bacterial cells in Examples was performed using the method of Tanaka and Weisblum [T.
Tanaka, B. Weisblum; J. Bacteriology, 121,
354 (1975)], the uptake of DNA into the host is
Norgard et al.'s method [MV Norgard, K. Keem, J.
J. Monahan; Gene, 3, 279 (1979)], the method of Saito and Miura [H. Saito, K. Miura; Biochim, Biophys,
Acta, 72, 619 (1963)].

Example 1 Production of plasmid pTP6-10 with enhanced resistance to trimethoprim using plasmid pTP6-6 and Escherichia coli chromosomal DNA.

Plasmid pTP6-6 is a plasmid that confers resistance to ampicillin on the host, and has already been developed by the present inventor. (Patent application 57-
102553) was added to 400μ of a reaction solution [6mM, MgCl ₂ , 0.2mM ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA), and 150μg of pTP6-6.
8mM Tris-HCl buffer (PH
7.6)] with 10 units of the restriction enzyme Sal at 37°C.
After 2 hours of digestion, 8μ of 1M Tris-HCl buffer (PH8.0), 4μ of 1M _MgCl2 , 5μ of 1M _CaCl2 ,
8μ of 5M NaCl and 1μ of 0.25M EDTA were added, kept at 25°C, and treated with 2 units of exonuclease BAL31 for 20 seconds. The exonuclease reaction was stopped by adding 400μ of water-saturated phenol, separated into an aqueous layer and a phenol layer by centrifugation at 3000 rpm, and the aqueous layer was removed and mixed with 50mM Tris containing 50mM NaCl.
Dialyzed against HCl buffer (PH7.4). The dialyzed DNA solution was named Solution A.

Next, about 2 μg of E. coli chromosomal DNA was added to 100 μg of a reaction solution [Tris-HCl buffer (PH7.4) containing 7 mM MgCl ₂ , 1 mM dithiothreitol (hereinafter abbreviated as DDT), and 60 mM NaCl] for 5 minutes. React with unit restriction enzyme Hinc at 37°C for 2 hours.
Add 100μ of water-saturated phenol to stop the enzyme reaction, separate the aqueous layer and phenol layer by centrifugation at 3000 rpm, remove the aqueous layer, and add Tris-HCl buffer containing 50 mM NaCl. (PH
7.4). The dialyzed DNA solution was named solution B.

Take 100μ of solution A and 100μ of solution B and mix both.
Add 20μ of 3M CH ₃ COONa to this, and further,
DNA was precipitated by adding 600μ of ethanol and standing overnight at -20°C. The precipitate was collected by centrifugation at 12,000 rpm, and after removing ethanol under reduced pressure, 20μ of ligase reaction solution (5
mM _MgCl2 , 10mM DTT, 0.5mM ATP,
and 50mM Tris-HCl buffer (PH7.4) containing 50mM NaCl]. To this, 5 units of T4 DNA ligase was added and the mixture was reacted at 4°C for 8 hours to ligate the DNAs and produce a hybrid plasmid. This reaction product was introduced into Escherichia coli K12 C600 strain according to the method of Norgard et al. The treated cells were plated on a nutrient agar medium containing 20 μg/ml ampicillin sodium and 200 μg/ml trimethoprim to obtain one growing cell. Isolate the plasmid from this bacterial body and re-
When introduced into Escherichia coli K12 C600 strain, bacterial cells showing resistance to trimethoprim at a concentration of 200 μg/ml were obtained at a frequency of approximately 10 ⁵ /μg DNA. This plasmid was named pTP6-10. The size of pTP6-10 is approximately 4300 base pairs, and the restriction enzymes EcoR, Pst, Pvu,
It was cleaved at one site each by Pvu, but the restriction enzyme
It was not cleaved by BamH, Hind, or Sal. Figure 1 shows a restriction enzyme cleavage map of pTP6-10.

Example 2 Isolation and purification of DHFR from E. coli harboring pTP6-10.

The bacterial cells obtained in Example 1 were mixed at 20 μg/ml.
3 nutrient medium containing ampicillin sodium
The cells were cultured overnight at 37°C to obtain about 10 g of bacterial cells. 50mM
After washing with Tris-HCl buffer (PH7.4),
ml of the same buffer. Cells were broken by sonicating the cells for about 10 minutes. This liquid
By centrifuging at 20,000 rpm for 30 minutes,
The supernatant and precipitate were separated to obtain about 50 ml of supernatant. When the total protein amount and total DHFR activity of this supernatant were measured, it was found that 524.7 mg of protein and 1996.5
The specific activity of this supernatant was calculated to be 3.8 units/mg protein. To 50 ml of this sonicated supernatant was added solid ammonium sulfate (19.5 g) to 60% saturation, and at 4°C
After stirring for several minutes, the supernatant and precipitate were separated by centrifugation at 20,000 rpm for 30 minutes to obtain about 60 ml of supernatant. Add this to 10mM containing 50mM KCl.
It was thoroughly dialyzed against potassium phosphate buffer (PH7.0).
Dialyzed protein solution containing 50mM KCl
It was adsorbed onto a DEAE-Sephadex A50 column (φ5cm x 50cm) that had been equilibrated with 10mM potassium phosphate buffer (PH7.0), washed thoroughly with the same buffer, and then a linear concentration gradient from 50mM to 0.5M was created.
Elute the protein with KCl, collect 5 ml fractions, and analyze the DHFR activity and
Absorption at 280 nm was measured. DHFR activity at 280nm
The value was calculated for each fraction by dividing by the absorbance of , and all fractions with a value of about 50 were collected (total volume of about 40 ml). For this solution, the total protein amount and
When the DHFR activity was measured, it was found that it contained 17.8 mg of protein and 726 units of DHFR activity, and the specific activity of this preparation was calculated to be 40.7 units/mg protein. Solid ammonium sulfate was added to this preparation to achieve 90% saturation, stirred at 4°C for 30 minutes to precipitate the protein, and the precipitate was collected by centrifugation at 20,000 rpm for 30 minutes.
10mM potassium phosphate buffer (PH
7.0), dissolved in approximately 1 ml. This protein solution
When analyzed by SDS-polyacrylamide gel electrodynamics, only one protein band appeared at a molecular weight of approximately 20,000, and no other protein band was observed. This indicates that the DHFR obtained by the above procedure is a homogeneous protein preparation. Also,
The specific activity of the homogeneous protein preparation is 40.7 units/
mg protein, and the specific activity of the sonicated supernatant is 3.8.
unit/mg protein, pTP6-
It was shown that DHFR was produced at about 9.3% of the total soluble protein in the bacterial cells harboring 10.

[Brief explanation of the drawing]

Figure 1 is a restriction enzyme cleavage map of pTP6-10, where the symbols in the figure represent restriction enzymes and E is
EcoR, P represents Pst, Pl represents Pvu, and P2 represents Pvu. In addition, the unit of number is kilobase, and the unit of number is kilobase.
Although the DNA structure is shown as a straight line for convenience, both ends are originally the same site.

Claims (1)

[Claims] 1. The molecular weight is approximately 4.3 kilobase pairs,
It has a gene that confers trimethoprim resistance and a gene that confers resistance to ampicillin as genetic markers, and the gene that confers trimethoprim resistance is a dihydrofolate reductase gene with enhanced promoter activity, and the dihydrofolate reductase gene with enhanced promoter activity By introducing a recombinant plasmid containing the folate reductase gene into a host bacterium, the host bacterium becomes resistant to 200 μg/ml or more of trimethoprim, which accounts for approximately 10% of the total soluble protein in the bacterium. 1. A recombinant plasmid pTP6-10, which can be transformed to produce dihydrofolate reductase in bacterial cells, and has the restriction enzyme cleavage map shown below. (The symbols in the cleavage map below represent restriction enzymes,
E is EcoR, P is Pst, Pl is Pvu, P2 is Pvu
shows. Also, the unit of numbers is kilobases,
The circular DNA structure is shown as a straight line for convenience, but
Originally, both ends are the same part. )