JPS5835198A - Novel vector and preparation of useful substance using it - Google Patents

Abstract

PURPOSE:To prepare various kinds of hormones, enzymes, etc. efficiently, by using a vector containing a DNA fragment having a replicating function derived from a specific lambda phase. CONSTITUTION:A vector containing a temperature sensitivity variation irreversible to a cI gene and variation to cro gene(namely variation lacking the gene or having the gene product in an inert type or an extremely inert type), having a DNA fragment with a replicating function derived from lambda phase, preferably a DNA fragment in a lambda NOP range. In cultivating a host bacterium containing a recombined DNA integrating the vector, the cultivation is carried out by maintaining the temprerature at >=42 deg.C and a temperature to enable the growth of the bacterium during the cultivation, followed by setting the culture temperature wherein the desired usefual substance can exist stably, and the desired substance is collected from the prepared culture mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to genetic engineering technology, and more specifically, to the stable production of useful substances by cultivating a vector and a host microorganism containing the vector by genetic engineering technology. It's about how to do it.

Genetic engineering technology can produce replicable NA7 fragments containing specific base sequences, useful substances derived from specific base sequences by artificially introducing them into specific hosts,
In particular, it is a technology for producing all proteins such as enzymes. Restriction enzymes (DNA
A sticky end of DNA produced by restriction enzyme (an enzyme that has the function of selectively cutting into a specific base sequence site of a fragment) and other DNA in a complementary relationship to this.
Enzyme used to join A fragment cohesive ends (
Licase), or enzymes that link DNA ends that are not complementary to each other to form complementary cohesive ends, or ligases that link these ends are already well known, and there are many enzymes available. They are commercially available, and methods using them have also been widely disclosed.

Microorganisms, animals, and plants all have their genetic DNA composed of exactly the same nucleotides, and therefore these foreign DNA fragments can be transferred to the host microorganism, such as the large intestine. g (E, coli) etc., these integrated D
NA fragments) can be replicated and expressed in the genetic information resulting from the integrated DNA fragments. At this time, a so-called vector into which a foreign DNA fragment is integrated and which has replication ability 1 becomes important.

Regarding Escherichia coli, a 2m vector, Nana, has been used in the past, but what is practically important as a vector is the copy number of the vector in the large intestine and the production of stable proteins based on it.

To this day, the techniques of woodcutter have been disclosed to the public in order to artificially increase this number of proteins to a large extent and produce stable protein growth, and research is currently being actively carried out in various places. ing.

In order to achieve this objective, the present inventors used E, col
i-derived DNA fragment and Col EI-derived DNA7
Made by joining lagmen), #! E shown in Figure 1,
coli DNA fragments from pLC-34-44 and pBR-322, which are ColEI-based cilasmids!
Derasmir called pPs-3155 shown in Figure 2.
Vector 1 - Made of Rust [Journal Op. Biochemistry (J, Bio, Chem,
), 2564:2219-2225 Peno (19
81)]. pPs-3155 was thus cleaved.
retains the tetracytalline and ampicillin resistance derived from pBR-322. Also, this pPs-
Is 3155 a site that is cleaved by the restriction enzyme BanHI in the nucleotide sequence that exhibits tetracycline resistance? However, the present inventors have developed a λ7 Arno DNA7. which regulates the transcription and replication of λ phage and contains a temperature-sensitive (tS) mutation in cI at the site cleaved by BarnHI. (By inserting the λNOP region' (rfim I) NA fragment obtained by cutting with BamHI (from pPS-3155-λNO
P? Prepared [Journal op Biochemistry (J, Bio.

Chem, ), volume 256, pages 2219-2225 (
(1981)) (see Figure 3).

Vector pPS-3155-λNOP obtained by sniffing
What are the characteristics of this phase?The vector is introduced into the host microorganism, and the host microorganism grows during the cell growth phase, and the useful product derived from the desired useful gene (for example, a protein such as an enzyme) is produced. Separation [, is at the point. In the method of increasing the cohesive number of a vector bound with useful NA □ fragments in synchronization with the bacterial cell growth phase,
It is known that in many cases it is extremely difficult to stably maintain vectors. Such a joint VC is
Generation of stable gene products becomes extremely difficult.

pPs-3155-λNOP was created by Akiban Kokudama et al. in order to solve all of these problems.

This pPs-3155-λNOP will be explained in more detail.

λ no anno μ, as already known s? d Proliferate in the soil and finally U host all rupture bath - Seshime heart, so-called M
The bacterium is integrated into the host chromosome! As the host cell divides, it reproduces and becomes a descendant, IIal-, in one of the so-called Yubara cycles. λ7ArnoDNA VCf@
There are five cleavage sites by BamHI,
The NOP region is one of the fragments created by cleavage by Bam1LI, and its size is 7.3 Kb (kilo pace), and there is one Fcgco RI &C↓ cleaved n;b site in the fragment. (See Figure 4, 8).

It is well known that cI of λ phage DNA is mutated to be temperature sensitive, and the replication and transcription control mechanism of λ phage having such eIt is as follows.

That is, when the λ phage DNA is in a lysogenized state at 30C, transcription from the promoter is completely suppressed. CI repressor is an orthorepressor that also suppresses its own transcription when its intracellular concentration increases;
Far uDNA is maintained within the cell at a constant amount necessary to maintain the lysogenized state.

On the other hand, when the temperature tube is raised to 42C, the temperature-sensitive repressor is deactivated and the suppression of P and PL is released, but cro-! J Delesser was produced and it was P
Transcription 1 repression from TtM and PL, intracellular cro-
Higher repressor concentrations also inhibit self-transfer from P. In this way, if the temperature of the system is raised to -142°C or higher, at which the host can grow, and the cI repressor is inactivated, even if the temperature is lowered afterwards, the product of the Jl-gene, that is, the phantom 1 -Repressor is PILM
Since it remains bound to f, the -aI repressor is not produced, and transcription proceeds to some extent uninhibited by f (
(See Figure 5).

Therefore, even if this pPs-3155-λNoPYr vector is used, it is possible to produce the target gene product fairly efficiently, but in this case, since the N gene product is not produced, tRI can significantly reduce transcription. It is thought that the generation of g, p, ' gene products will end at a certain level, and furthermore, ? , P. Transcription of useful genes following the gene is also suppressed to a certain extent.

The present inventors conducted intensive research to solve the drawbacks of the conventional technology, and as a result, the present inventors discovered that λ-
The present invention has been completed by demonstrating that products derived from the 9 o, p, genes can be efficiently produced by using the NOP region.

The present invention provides a novel vector tm which incorporates a gene fraction containing a replication function (DNA7 fragment) derived from λ-77-, which has an irreversible temperature-sensitive mutation in the cI gene and a mutation in the v1 gene. This is what we provide.

In the present invention, 42C has a mutation in the so-called -91- mutant cI, which has a temperature-sensitive (tS) mutant tube, and 42C has a clear plaque (
It was discovered that lysogenization occurs at 130°C (transparent part due to bacteriolytic action), but the state of lysogenization at 3°tS 10°C is almost the same as that of (cI, cro). Seems a (cItS%cro-)
In the vector containing the λ-NOP fragment fM, the hacltS repressor is inactivated at 42°C, and the suppression of P and P is released. In this case, since the regulatory action of the cro-gene does not work, transcription from all promoters proceeds. Furthermore, when the N gene product is produced, the termination of transfer in tRI by the β factor does not occur, and products derived from the o, p, and genes are produced in large amounts. Furthermore, at this time, transcription of useful genes subsequent to the o, p, and genes also progresses strongly (see Figure 11N6).

In the present invention, "the history gene (having a mutation)" refers to a person who lacks the μ gene, or the history gene child may be in an inactive type, or may have an extremely low activity type. Describe the mutation that has occurred.

Winter, DN having a replication function derived from λ phage of the present invention
The A fragment may be of any type as long as it has the function of exhibiting the above-mentioned properties and can be joined to other or foreign DNA fragments by some means. λNO
The DNA fragment derived from λ phage in the present invention includes the λNOP region used in P.
Using a restriction enzyme tube such as mHI, a certain DNA fragment)? It may be used after cutting out the DNA of the rλ phage, or it may be used by cutting out a DNA fragment by using a chemical tube instead of a restriction enzyme and attaching sticky ends to it with an enzyme.

Preferred DNA fragments [7] include, for example, Ba
One example is the λNOP region cut out by mHI. The DNA 7 fragment thus obtained can be conjugated with another (or foreign) DNA fragment having a cohesive end complementary thereto according to a conventional method to form the vector of the present invention.

The vector of the present invention incorporates a gene fragment that expresses drug resistance (e.g., ampicillin resistance, tetracycline resistance) or drug susceptibility to fully determine that the ug7 fragment derived from λ phage has been inserted. is preferable.

Furthermore, a gene fraction having a replication function derived from Cog EI may be incorporated into the vector of the present invention, and it is preferable to contain a gene fraction derived from Cog EI.

Into the thus obtained vector of the present invention, a gene fraction that expresses the production of a useful substance (for example, enzyme protein) is further incorporated, and it is introduced into a host microorganism such as Escherichia coli, and the host is propagated. Recombinant DNA? By copying, the desired useful substance can be produced within the host body.

In order to efficiently produce useful substances using the vector of the present invention, since the vector of the present invention described above is temperature sensitive at temperatures above 42°C, the culture temperature should be adjusted during the entire culture of the host microorganism containing the recombinant DNA association. After maintaining the temperature at 42°C or higher at which the microorganism can grow, the culture mixture is obtained by setting the culture temperature to a temperature at which the desired useful substance can stably exist, continuing the culture, and smelling it. Collect more targeted useful substances.

The lamp of the present invention relates to a method for producing such a useful substance, and the host microorganisms used in the method of the present invention include bacteria such as Escherichia coli, etc. Bacteria, 1Ill
iA, among others, Escherichia coli is preferred. It varies depending on the temperature at which the desired useful substance exists stably, the temperature, and the target substance, but for example, when using phos-7 atidylserine synthase as the target substance,
When the target substance is a hormonal substance or the like which is 37C or lower and is unstable to heat, the temperature is relatively low at which the substance stably exists.

The method of the present invention using the vector of the present invention? %, it is possible to efficiently produce the useful substances expressed by a specific gene.

These useful specific genes include many DNAs derived from microorganisms such as bacteria, filamentous fungi, and yeast, higher animals and plants, and various phages. Examples include synthetic materials such as antibiotics and amino acids.

Hereinafter, the present invention will be further explained with reference to Preparation Examples, Comparative Examples, and Examples.

Example I E, Co11 K-12-0600k% polypeptone 1
%, NaC1α2%, the number of solutes is 8.
'' About 2 x 10 ke/w vcft, btl?, culture 11! 1:Culture at 40C, then MtC1*t10
Add λcI857 cro 27 im, o
, 11, infected at 40'C for 3-4 Fi
The cocoons were gently shaken for a minute. Still image-'[kDNas
・After treatment with ■, centrifuge and place 7) on top, then cesium chloride - ultracentrifugation and phenol extraction to remove 7-age DN.
Atl17'j.

(21 Derasumi) 'DNA preparation medium 1jA (KHz POa O-45%, Nag
HPO41, 05%, NH4Cl 0.1
%, Casamlino acids (Difco
) 1.5% Geratin 0.01%, MfSO4
0-4%, CaC/2 0.3mM, Glucos
E. coli containing a plasmid (vector) community of pPs-3155 was cultured with shaking at 30°C in a medium containing 5% O, 5%), and the culture was completed at a bacterial count of 5 x 108 cells/sj.
L Rizochi A place 11LIIIN 7'j, next [4
After centrifugation at
Remove the DNA 7 fragment for 1&L, then remove Echizome gromoid V and cesium chloride. Next, 1 portion of ethanol was added and 30 molecules of DNA t-4 were added at 6×10'G.

(Preparation of 31pPs-3155-λNOP5i 7't pPs-3155 and λeI 857
Cro 27 DNA was cut using AtBamHIt, mixed, and religated using T4-DNA ligase.

Agarose gel electrogram after recombination? This is shown in the 7th cause. Bam HI is thermally unstable and T4-DN
A ligase uses the Bam HI cleavage site as a substrate and releases it (DN
As the A concentration increased, the rate of recombination reaction increased. Therefore, it is against cutting! I: x tyo 30 It was better to perform CVC and lengthen the Pfr time by about 30 minutes.

DNA1&min't E, Co11-J after the above recombination
A-200(λl[) was transformed. Mukuzono First, add a plate medium containing Anpinlin 1r50 μf/liter (Tori 1
) N (Difco) 1%, NaC 10.5%, yeast extract (1) ifco) 0.5%, agar 1.5%)
Next, culture at 30C and isolate resistant colonies.
Similarly, strains with both tetracycline resistance and colicin EI resistance (ApR, Tc', Cot E
I “””-:Ap...Ampicillin, Tc・
・Tetracycline, Cod EI...colicin EI
) was selected [7. From these strains, D
When NA was extracted and the molecular weight 1r was measured by Aga P-SR electrophoresis, it was found that a new DNA fraction with the same molecular weight as pPs-3155λNOP was prepared. Next, a strain of Derasmir containing this almost identical new DNA fraction was isolated, cut with BamHI,
Analyzes were made in the same manner (Fig. 8). This allows pPs-315
5-λNOP cro-(D N A (D II
Made 'tiiiu friend.

Large 11% gI incorporating pPs-315 λNOP Gokai
C-600 (λ) tube, trypton (Difco) 1.
2%, yeast extract (Difco) 2.5%, glycerol 0.5% PH7.6, cultured at 30C, and when the number of cells reached approximately 4xlO5ce#/-, by short operation. Raise the temperature to 42C, then 1120
It was held for a minute. Thereafter, the mixture was cooled to 37°C and cultured for 3 hours. After harvesting, 0.1M pl, 6 sm phosphoric acid
After washing the bacterial cells, ultrasonic treatment was performed to destroy the bacterial cells, and PS enzyme activity was measured using a PS enzyme activity tube.
Co., Ltd., CDP-diglyceride.

0.675mM, L-3--aH-serine 0.5m
A solution of M, B8A1mf/d, 0.1 M phosphate buffer p)l 7.4 was used. An example of heat-induced increased activity is shown in Table 1.

wJ1 Table Plasmid Relative Activity Control 1 Production pPs-3155-λN0Pcro f-incorporated colon-
The treatment was carried out in the same manner as in Example 151 except that pPs-3155-λN0Pcro f was used, and the activity was measured, and the relative activity was found to be 110.

[Brief explanation of the drawing]

Figure 1n E, coli DNA 7 lagmen) -
Cal EI 3rd figure line, to/l'-pPs-315
5-λNOP'. RI: Restriction enzyme Eco RI action site () is Kb
Units in Figures 5 and 6 are λ-NOP (C1857S7)
FIG. 7 shows an agarose gel electropherogram of the DNA fragment in Example 1. Figure @8 shows an agarose gel electropherogram of the plus jy cut fragment prepared in Example 1. +01 Figure 5 Figure 6 @ (Induc@d) Figure 7 ■ ■ ■ Figure 8 ■ ■ Continued from page 1 0 Inventor Yoshihiro Takayama 13-15 Oaza Wakaguri, Ami-cho, Inashiki-gun, Ibaraki Prefecture Mitsubishi Yuka Co., Ltd. Company Central Research Institute

Claims (1)

[Claims] 1. Having an irreversible temperature-sensitive mutation 1 in the cI gene,
A vector containing a replication function-containing gene fraction derived from λ phage and having mutation 1 in the cro gene. 2. The vector according to claim 1, which contains a gene sequence that expresses drug resistance or drug sensitivity, as determined by inserting a gene fraction derived from λ phage. λ Claim i in which the drug resistance is ampicillin resistance or tetracycline resistance! ] @ Vector described in item 2. 4. Cod EI Yamaki's Australian-made functional association gene-
Claims containing minutes! The vector described in Section 1. 5 The gene fraction derived from λ phage is DNA in the λNOP region
The vector according to any one of claims S 1 to 5, which is a fragment. 6 Has an irreversible temperature-sensitive mutation 1 in the cl gene,
and a host microorganism containing a recombinant DNA association consisting of a vector completely integrated with a gene fraction gold-containing that has a replication function derived from λ phage and having a mutation in the cro gene [
During culturing, after maintaining the host microorganism at a temperature Ut42C or higher at which the host microorganism can grow, the culture temperature Vr is increased.
1. A method for producing a useful substance, which is characterized in that the culture is continued at a temperature fK at which the desired useful substance can stably exist, and the desired useful substance is collected from the resulting culture mixture. 7. Claims in which the vector contains a gene sequence tube for expressing drug resistance or drug susceptibility for determining drug resistance or drug susceptibility for determining the inserted tube of a gene derived from lambda phage! ! ! lNo.6XJ
Manufacturing method described. & Claim i where the drug resistance is ampicillin resistance or tetracycline resistance! 1. The manufacturing method according to item 7. 9. The production method according to claim S 6, which contains a gene fraction having a replication function derived from Co/EI. 10. The production method according to any one of claims 6 to 9, wherein the gene fraction derived from the λ phage is a DNA 7 fragment of the λNOP region. 11 Claim 6 in which the host microorganism is E. coli
The manufacturing method according to any one of items 1 to 10.