JPS59198979A - Stabilization of host microbial cell containing specific plasmid - Google Patents

Abstract

PURPOSE:To enable the stable existence of a host microbial cell containing a plasmid having the structural gene of the alkaline phostaphase of Escherichia coli even under the condition to produce a large amount of alkaline phostaphase, by using a specific plasmid vector. CONSTITUTION:The host microbial cell contains tetracycline-resistant gene and trimethoprim-resistant gene as markers and also contains alkaline phostaphase gene. The chromosome DNA of the cell is incised at the PstI site of the restriction enzyme of plasmid vector pTP5-3, and the obtained fraction is combined with pTP5-3 which is incised beforehand with PstI, and bonded together using T4-DNA ligaase. The host bacteria can be survived stably by this process even under the condition to produce a large amount of alkaline phostaphase.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the stabilization of bacterial cells carrying a plasmid containing a structural gene for E. coli alkaline phosphatase with high expression efficiency.

With the recent development of genetic engineering, recombinant DNA techniques 9 are used, for example, to introduce foreign DNA into bacteria such as Escherichia coli and express the gene.

Methods for producing useful proteins encoded in foreign DNA are attracting attention.

When introducing foreign DNA and expressing the gene.

For proteins existing in the cytoplasm, increasing the copy number of their structural genes results in a corresponding increase in production, so it is common practice to inject foreign DNA into a multi-copy plasmid to increase protein production. It became so.

However, in the case of extracytoplasmic proteins, attempts to dramatically increase their production are not achieved due to factors thought to be other than protein synthesis.

E. coli alkaline phosphatase (hereinafter referred to as APase) related to the present invention is also such an example.

APase of E. coli is an enzyme that hydrolyzes phosphomonoesters, and is secreted and produced in the periplasm, which is the extracytoplasmic region of E. coli. The production of APase is influenced by the concentration of inorganic phosphate in the medium, and synthesis is initiated when phosphate becomes deficient. APase is a 2-secreted enzyme, so when it is synthesized, it synthesizes a peptide consisting of about 20 amino acids called a signal peptide.
It is synthesized as a terminal-terminated precursor. Also, APas
The synthesis of e is controlled by five types of regulatory genes 9, such as ph, oB gene, phoR gene, rph, and oM gene.

The production efficiency during expression is high, with an efficiency of approximately 6% of that of the new protein.

Since APase synthesis is efficient and can be regulated, it is expected that the APase structural gene can be used to bind foreign DNA to it and be applied to the production of useful polypeptides. Recently, APase
Gene cloning is underway.

In addition, since APase is a secreted enzyme, APas
This is because it is thought that if the fusion protein of e is produced, purification and isolation of the protein will become easier.

However, according to Yori H1 and 01 Mura [Proteins/Nucleic Acids/Enzymes, vol 26, pr+ 386-39
4 (1984)).

When the $7/I synthetic gene of APase was cloned into the multi-copy plasmid pBR322 and 9 synthesis was induced,
APase activity in the periplasm only increases several times compared to when induced with a single copy, and these cells lose their colony-forming ability with a half-life of approximately 80 minutes. .

Against this background, the present inventors aimed to stably clone the APase structural gene into a multicopy plasmid (
As a result of intensive research, the present inventor has already developed a multi-copy plasmid vector pTP5-3 [Patent Application No. 1983]
-102553 and M, Iwakura et at
.

J.Biochemistry, vol 93.
p'rp5-
They discovered that the restriction enzyme Pst I site of No. 3 can be used, and based on this knowledge, they completed the present invention.

pTP5-3 is a plasmid vector containing genes that confer resistance to three types of drugs, trimethoprim, tetracycline, and ampicillin, to the host, and Ec on the trimethoprim resistance gene.

R1 cleavage site, Bam on the tetracycline resistance gene
There are HI and Hind [■ cleavage sites, and a Pst I cleavage site on the ampicillin resistance gene.

Cloning of the structural gene of APase is carried out using the so-called °
It can be carried out using the 'shotgun' method. That is, the chromosomal DNA of 12 strains of E. coli is cut with Pst I,
This was combined with pTP5-3 previously cut with Pst I and ligated using T4'5 DNA IJ gauze to create various hybrid plasmids.

It is introduced into 12 mutant E. coli strains that do not produce APase using the transformation method. A bacterial cell carrying a hybrid plasmid containing the APase structural gene.

APas metamorphoses to produce APase.
A strain that is a production strain and has become resistant to trimethoprim and tetracycline, which are the genetic markers of the plasmid vector, is isolated. By isolating a hybrid plasmid from such a bacterial strain, a plasmid containing the APase structural gene can be obtained. When the bacterial cells carrying this hybrid plasmid are transferred to a phosphate-deficient medium containing trimethoprim and tetracycline and APase synthesis is accelerated, the APase concentration before transfer to the deficient medium increases.
The content of Escherichia coli K, which does not have a plasmid and increases 200 to 500 times compared to the content of E. coli K], when two strains were treated in the same way, A
The value is 10 times or more compared to the Pase content.

Furthermore, this hybrid plasmid-carrying bacterium exhibits stable colony-forming ability even when cultured in a phosphate-deficient medium to increase APase production. That is, this hybrid plasmid-carrying bacterium was cultured in advance in a high phosphate medium (for example, TG+20 medium J1!!) containing about 5 μg/mi of trimethoprim.
After culturing until the middle of the logarithmic growth phase, APase production is reduced by 20% by switching the medium to a phosphate-deficient medium (e.g., TG+) containing about 5 μg/ml of trimethoprim.
Although it is possible to increase 0-500 times, 5μg
After converting the medium to a phosphate-deficient medium containing trimethoprim at a concentration of approximately
Spread on a high phosphate agar medium (e.g. TG+20 agar medium) containing about 7 ml of trimethoprim, and further
After culturing for 48 hours and examining the number of growing colonies, no change was observed in the colony forming ability, making it possible to explain the decline in colony forming ability as described for bales and materials.

As a result of careful consideration of this difference, the present inventors found that the knowledge for bales and materials for plasmid vectors
Because BR322 is used and the Hind 111 site is used as a cloning site.

This was thought to be due to the instability of ampicillin, which was used as a selection marker for plasmid introduction.

9 The present inventors also used pTP as a plasmid vector.
5-3 and PstI as a cloning site, it is believed that the plasmid could be stabilized by using trimethoprim resistance, which is a strong selection marker.

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

Example I Cloning of APase Structural Gene Plasmid vector pTP5-3 is a plasmid vector having genes that confer resistance to three drugs, trimethoprim, tetracycline, and ampicillin in the host, and was already produced by the inventors. (Japanese Patent Application No. 57-102553).

5Saito and Miura's method [H9Saito,
K., Miura: Biochim, Bioph
ys, Acta, 72, 619 (1963))
According to EscherichiaooliK 12
I FO3301 to 7! Approximately 1 μg of DNA containing the APase structural gene was transferred to the plasmid.
Approximately 11 tg of p 5-3 was added to 1100 tt (D reaction solution (7mM Tris-1-Cl pH 7.4, 7m
M MgCTo, 7mM 2-mercaptol, 6
5 units of restriction enzyme Pst 1 in 0mM NaCI)
Using.

Digestion was performed at 37°C for 1 hour. The reaction was stopped by adding 100 μe of water-saturated phenol, stirred well, and separated into an aqueous layer and a phenol layer by centrifugation at 3000 rpm for 5 minutes.
50 mM Tris-HCII buffer pH 7.4
i7 analysis overnight. Dialyzed I) NA solution approx. 100
Add 300 μl of cold ethanol to the μl and keep in dry ice-ethanol for 1 hour to precipitate the DNA.
The precipitate was collected by centrifugation at 15,000 rpm for 15 minutes and dried under reduced pressure. To this, add 50μl of ligase reaction solution (50mM Tris-HC
l, pH 7.4, 5mM MgCl,.

13mM dithiothreitol, Q, 5mM ATP, 5
After adding QmMHa(?l) and dissolving the DNA, 1
Unit 74 DNA ligase was added and the DNA ligation reaction was carried out at 10°C for 16 hours. This reaction product is
The method of gard et al. M, V, Norgard
, K. Keem, J. J. Monahan:
Gene, 3, 279 (197B)) into Escherichia coli K12E15 strain. The treated bacterial cells were placed on a TG+20 agar medium (composition: 0.I M
Tris-HCI! , pH7,4,80mMNa
C11+ 20mM KCI + 20mM NH,C
1l, 3mM Na.

So, 1mM MgC1!2.0.2mM Ca
C1!2.21℃MFeC113, 2μM ZnCl,
, 0.64mM KH, PO4, 0.2% glucose, 1.5% agar), and two blue (colored colonies) were obtained from among the growing bacterial bodies.

A plasmid with a size of approximately 17.2 kilobase pairs was isolated from one of these colonies. This plasmid was adjusted to pH
It is called 0A2. pH0A2 restriction enzyme BamHI,
EcoRI, Hindll, HpaI, Ps
tI, 5ail.

The cleavage map by XhoI is shown in FIG.

(7) Escherichi pHOA, 2 to 1
The A. coli K12E15 strain produced approximately 0.027 units/mg protein of APase. This value converts the APase structure gene into a needle Escherich
ia coli K12 I Fo 3301 strain A
Pase production was approximately 20 ia 4 of 0.00011 units/mg protein. Esthe
richia col 1K12 E] 5 strains are AP
1 because there is a mutation in the ase structural gene.
No APase was produced. Also, this pI-TOA
Escherichia coli K12 with 2
The EIFI strain grew on media containing trimethoprim or tetracycline, but not on media containing ampicillin. PstT of pTP5-3
The site is an ampicillin-coordinating insertion deactivation site, so
The results on J1 indicate that the APase production ability of pH0A2 is due to Escherj at the PstI site of pH0A2(7)
It is concluded that the fragment was obtained by inserting a fragment excised by :Pst I of DNA obtained from chia coli K12 IFO3301 strain.

Example 2 Escherichia col with pHOA2
i Stability of K12 E15 Strain CharacteristicsAccording to Strain Materials, when the structural gene of APase was cloned into the plasmid vector pBR322 and cultured in a low phosphate medium to induce APase synthesis, the cells were It is said that the colony-forming ability is lost over the half-life. Esch with pH0A 2
Erichia coli K]2 E15 strain was induced to synthesize APase, and after induction, the culture solution was appropriately diluted and treated with 27zg/mE of trimethoprim and 40μ
The cells were plated on 7 ml of TG+20 agar medium containing XP and examined for colony forming ability. As shown in FIG. 2, no decrease in colony forming ability was observed even when cultured in a low phosphate medium. Furthermore, we investigated APase synthesis and found that (
J, activity increased approximately 200-fold compared to before initiation.

This value is more than 10 times that of having ■ copies of the gene.
The value was

[Brief explanation of drawings]

Figure 1 is a cleavage map using the pH0A2 restriction enzyme.9
The symbols in the figure represent restriction enzymes, and H is Hin d III.
. P is Pst I, S is Sal I, Hp is Hpa
I and E represent EcoRl, and B represents )3amHI. Also,
The unit of numbers is kilobase pairs, and the circular DNA structure is conveniently expressed as a straight line. Figure 2 shows Escherichia with pH 0A2.
Colony forming ability of E. coli K]2 EI5 strain in low phosphate medium I1. 'I shows a change. The vertical axis represents the colony forming ability immediately before transfer to the low phosphate medium.
The colony forming ability is shown as %, and the #I axis shows the culture time in a low phosphate medium. Figure 3 shows Escherichi with piTOA2.
A coliK]2E15 strain in low phosphate medium
Pase synthesis is shown. The vertical axis is the intracellular content of APase. The horizontal axis shows the culture time in the low phosphate medium. Patent applicant: Agency of Industrial Science and Technology Nagagawa 1) Yube

Claims (1)

[Claims] 1. In a host cell that has a tetracycline resistance gene and a trimethoprim resistance gene as genetic markers and a plasmid containing an alkaline phosphatase gene, the host cell can survive stably even under conditions that produce a large amount of alkaline phosphatase. Stabilization of host bacterial cells.