JPH02190194A - New process for producing protein and transformant to be used in the process - Google Patents

Abstract

PURPOSE:To easily and stably produce a protein in a mass in high efficiency by transferring a specific recombinant DNA into a chromosome DNA of a host cell and culturing the obtained transformant of the host cell in a medium. CONSTITUTION:Plural gene units (A) containing a gene coding an extrinsic protein such as cell growth factor (EGF) is prepared by synthesizing a prescribed oligonucleotide and linking both terminals of the obtained gene with sequences of plural gene units linked with each other in such a manner as to direct the bases in the sequence in same directions. The component A is inserted into plasmid pSK107 to obtain a recombinant DNA pSK108 (B). The component B is inserted into a chromosome DNA of a host cell such as yeast to transform the host cell and obtain a transformant (C). The transformant C is cultured in a medium containing galactose, etc., for about 48hr to produce the extrinsic protein such as human EGF in the cultured product.

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION Technical Field The present invention relates to a method for producing a desired protein by recombinant DNA technology using chromosomes, and a transformant used for the production.

BACKGROUND OF THE INVENTION It has been recognized in numerous papers, patent publications, etc. that a technology for producing large quantities of desired gene products using recombinant DNA technology is being established.

These methods include methods of introducing genes onto plasmids and methods of incorporating genes into chromosomal DNA. The advantage of the method of introducing genes on plasmids is that by using a plasmid that is retained in cells in large numbers of copies, it is possible to easily introduce a large number of genes into one cell. However, the disadvantage of this method is that the plasmid is generally unstable, and cells that have lost the plasmid appear during culture.

On the other hand, the method of integrating genes into chromosomes has the advantage that the integrated genes are stably retained, but it is difficult to introduce a large number of genes into one cell, and it is difficult to introduce a large number of desired proteins into a single cell. The current situation is that it is not possible to make this happen.

[Summary of the invention]

Summary The present invention aims to solve the above-mentioned problems and develop the technical possibilities of genetic manipulation and chromosome manipulation. The purpose is to achieve the above object by providing an expression method and a transformant for use therein.

Therefore, the method for producing proteins according to the present invention is characterized by comprising the following steps.

b) A gene unit containing at least a gene encoding a desired foreign protein, the ends of which are designed so that a plurality of the units can be interconnected with their base sequences facing the same direction. Prepare multiple items.

(1) Prepare a vector capable of transferring the plurality of gene units prepared in step (a) into the chromosomal DNA of the host.

c) Multiple gene units prepared in step a),
Insert or link into the vector prepared at the process entrance),
To prepare a recombinant DNA that can be replicated while being included in the chromosomal DNA of the intended host cell as the cell grows.

2) Transferring the recombinant DNA obtained in step c) into chromosomal DNA in a host cell and transforming the host cell to prepare a transformant.

e) Cultivating the transformant obtained in Step 2) to produce the desired exogenous protein in the culture.

Furthermore, the transformant according to the present invention is characterized by carrying out steps a) to 2) in the above-mentioned production method.

Effects Since the present invention makes it easy to link a large number of arbitrary genes and introduce them into chromosomal DNA, the following effects can be expected.

1. Since the gene to be expressed is stably maintained, there is no need to perform further selection using drugs or the like on the transformants once obtained.

2. When introducing a gene into a chromosome, there is no limit to the length of the gene that can be introduced. Therefore, any gene can be inserted, and also a large number of genes can be introduced, thereby increasing the amount of expression products of those genes.

3. By simply repeating the operation of introducing genes into chromosomal DNA, it is possible to introduce a large number of genes of various types into one cell. For example, using this method, all the genes that carry out a series of enzymatic reactions, such as the biosynthesis of antibiotics or vitamins, can be transferred to each chromosome D.
By introducing it into NA, a series of enzymatic reactions can be carried out efficiently within one cell.

[Detailed description of the invention]

Gene unit A plurality of gene units will eventually be integrated into chromosomal DNA by the vector described below, but each gene unit must contain at least a gene encoding a desired foreign protein. Structurally, 1) one that contains all the factors necessary for gene expression, such as a bromotor, a coding region, and a terminator, and 2) one that contains only the gene encoding a foreign protein, that is, the coding region (in this case, Regarding the factors necessary for gene expression, chromosomal promoters, terminators, etc. can also be used). Both ends of these gene units are designed so that when a plurality of them are linked, their base sequences are in the same direction. That is, the upstream (or downstream) end of a gene unit is designed to be able to bind only to the downstream (or upstream) end of another gene unit. This is done by chemically synthesizing oligonucleotides with such properties and attaching them to each end, or by placing cleavage sites for appropriate restriction enzymes at each end to form sticky ends. It is something. In this case, it is preferable to use a restriction enzyme site that produces sticky ends that are not self-complementary after cleavage (see the gene fragment (gene unit) in Figure 1. N and N' in the figure). represents an arbitrary base, and the numbers of N and N' can be set arbitrarily). In the experimental example, the cleavage site of the restriction enzyme 5fiI is used.

The open ends of the two gene units located at both ends of the multiple gene unit combination formed by joining the gene units are designed so that they can be linked to one end of the vector described below. be done.

The gene unit preferably includes a promoter,
It is preferable to use one that contains all the factors necessary for gene expression, such as a coding region and terminator.

Further, the desired protein to be expressed may be any desired protein, such as EGF, TGF, and NGF.

Growth factors such as IGF and FGF, calcitonin, interferon, insulin, erythroboietin, interleukin, growth hormone, and viral proteins (
Examples include those that can be used as vaccines), antibiotic synthases, and vitamin synthases.

As mentioned above, a gene unit contains at least a gene encoding a desired exogenous protein, and may also include a plurality of genes, or a gene encoding a fusion protein, and its expression results in the final result. Needless to say, it also includes those in which the desired protein is obtained by decomposing the joint protein obtained by using an enzyme or cyanogen bromide.

Recombinant DNA containing gene unit conjugate The recombinant DNA according to the present invention is obtained by inserting or ligating the above gene unit conjugate into a vector.

The vector transfers the multiple gene unit conjugate into the chromosomal DNA in any host cell, and allows the gene unit to be replicated in the cell as the cell multiplies as the chromosomal DNA replicates. It refers to carrier DNA that has a role.

The vector of the present invention ultimately consists of two fragments, a fragment located upstream and a fragment located downstream of the above gene unit conjugate, and both fragments contain a DNA portion derived from the chromosome of the host to be transformed. Contains. These DNA parts are the chromosomal DNA of the host to be transformed.
It is preferable that they are located adjacent to each other in the same orientation as they were in the chromosomal DNA.

Furthermore, it is preferable that the distance between these adjacent DNA portions on the chromosome be zero or as small as possible.

Specifically, one fragment (upstream fragment) of the vector is linked only to the upstream end of the gene unit such that its downstream end is located at the upstream end of the gene unit conjugate to be expressed. It is designed to be possible. Also, the upstream side of this fragment contains only the DNA sequence part of the chromosome, and is so designed that it cannot be combined with other DNA fragments, that is, other vector fragments or other gene units, or even if it is combined, it is subject to appropriate restrictions. It is sufficient if a restriction enzyme cleavage site is provided so that it can be cut again by an enzyme.

The other fragment of the vector (downstream fragment) is similarly designed to be able to bind only to the downstream end of the gene unit such that its upstream end is located at the downstream end of the gene unit conjugate to be expressed. has been done. Also, does the downstream side of this fragment contain only the chromosomal DNA sequence part and is unable to combine with other DNA fragments?
It is sufficient that a restriction enzyme cleavage site is provided so that even after binding, the vector can be cut again with an appropriate restriction enzyme (see the vector in Figure 1. The definitions and numbers of N and N' in the figure are as described above. ).

Recombinant DNA can be prepared by ligating such a vector to both ends of the gene unit conjugate.

In order to adjust this recombinant DNA, that is, to link a large number of the gene units and to link the vector to both ends thereof, the gene units and the vector should be mixed in an appropriate ratio and linked. good. For example, if it is desired to make 100 gene units white, the gene units and the vector may be mixed at a ratio of 100 to 1 and combined. In addition, if the concentration of these DNA fragments, i.e., gene units and vectors, is low, the gene unit conjugate or recombinant DNA will form a ring, making it difficult to achieve the binding of multiple genes. There must be.

If a restriction enzyme recognition site is provided at the end of the vector that does not combine with the gene unit, and this site can be cut with a restriction enzyme, the end of the vector will be cut after the gene unit and the vector are combined. Even if they bind to each other, the restriction enzyme can completely cleave them to form a linear recombinant DNA.
It can be A.

Furthermore, a selection marker for selecting transformants in which the gene unit conjugate has been introduced into the chromosome can also be introduced into the vector.

The selection marker may be inserted into either fragment of the vector. At this time, it is desirable that the marker be closer to the end that binds to the gene unit conjugate. The selection marker is obtained by repeatedly introducing recombinant DNA into the chromosome,
When it is desired to introduce another gene unit conjugate or the same gene unit conjugate into a chromosome, it is preferable to design it so that it can be removed from the chromosome with a high mutation rate after being introduced into the chromosomal DNA. For example, the same DNA fragments are ligated in the same direction on both sides of the marker, such that self-complementary recombination at this site removes the marker. Further, as the selection marker, any commonly used selection marker may be used, but it is preferable to use one that allows selection even when the marker is removed, as will be described later. For yeast, UR
These include A3 and LYS2. In the experimental example, the URA3 gene is used.

In order to obtain this recombinant DNA, in addition to mixing multiple gene units of one type with the necessary vectors, it is also necessary to mix multiple gene units of multiple types with multiple different types necessary for each. Chromosome D
It is also possible to obtain recombinant DNA containing multiple gene units of multiple types that can be introduced into NA.

In addition, the chromosomal DNA referred to in the present invention refers to the original chromosome D.
In addition to NA, it also includes those modified by the introduction of any foreign gene, or those modified partially in base sequence (substitution, addition, deletion, etc.).

Transformant The transformant according to the present invention is a transformant obtained by inserting or ligating the gene unit conjugate into an expression vector.
can be obtained by transfecting into appropriate host cells. When the expression vector used has a marker gene, for example, a gene related to auxotrophy, drug resistance, etc., transformants can be easily identified using this marker gene as an indicator.

Any host to be transformed can be used, including bacteria such as Escherichia coli, animal or plant cells, and fungi such as mold and yeast.

Preferably, it is a yeast strain, and a specific example thereof is Saccaromyces cerevisiae (Sacc) shown in the experimental example below.
haromyces eerevlslae).

In order to introduce a linear recombinant DNA prepared as described above and having a structure like (upstream vector fragment - gene unit conjugate - downstream vector fragment) into a specific position of the host's chromosomal DNA. , using complementary recombination (
Literature: 5science, 240°1439 (1988
)). The region of the chromosome used for this purpose is chromosomal DNA.
Any region of the body may be used, but it is preferable to use a region where the introduction of the gene will not cause any harm to cell proliferation. In the experimental example, the yeast HIS4 gene is used.

It goes without saying that the vector used contains a DNA fragment of this region.

The transformant thus prepared can be isolated as a cell that has lost the marker by culturing it in the absence of selective pressure, ie, without additional conditions such as the addition of a substance corresponding to the marker. It is easier to operate if you use something that can be selected when the marker is removed. In the experimental example, the URA3 gene is used, but in this case cells were treated with 5F OA (5-Fluor
o-orotlcacld) on a plate containing
Just choose one that will grow (only URA hosts will grow).

The host cell transformed with the recombinant DNA according to the present invention is
In addition to unaltered original cells, as mentioned above, this includes cases where the chromosomal DNA has been modified by introducing any foreign gene or whose base sequence has been modified. It is.

A preferred specific example of transforming a host cell modified in this way is to transform an original host cell with the recombinant DNA according to the present invention, and then transform this host cell with the recombinant DNA. Transformation, ie, repeated transformation with recombinant DNA.

In the present invention, repeating this transformation means repeating the above steps 1) to 2).

When repeating the transformation operation using recombinant DNA,
In this way, the combination of the gene unit conjugate and the vector fragment, transformation, and removal of the marker form one cycle, but in the next cycle, the gene unit conjugate (the gene of the gene unit is the same as that of the previous step) forms one cycle. The same operation may be performed using a vector fragment (which may be the same or different) and a vector fragment different from the vector used in the previous step (the marker may be the same). By repeating this operation, a large number of genes can be transferred to one type or many types of chromosomal DNA.
It can be inserted into A.

By culturing the transformant thus prepared according to a conventional method, the desired exogenous protein can be produced in the culture, and if this is recovered by an appropriate protein recovery method commonly used, good. In addition, the above-mentioned "in culture" means in transformant cells and/or in a medium.

[Experiment example]

1. Preparation of plasmid for gene fragment preparation (Second
Figure) Plasmid pUc118 (1 μg) was digested with restriction enzyme Hinc.
ll and Sma I, and added an 8g1m linker (CAG
ATCTG) and T4 DNA ligase. E. coli HBIOI was transformed using this reaction solution according to a conventional method, and an ampicillin-resistant strain was selected to obtain the target clone (pUC-Bg l II).

Plasmid pUC-BglII (1 μg) was cut with restriction enzyme Bg1m and ligated with a 5fiI linker (27mar) using T4 DNA ligase. Using this reaction solution, Escherichia coli HB 101 was transformed according to a conventional method, and an ampicillin-resistant strain was selected. From the obtained strain, a clone in which two copies of the 5fil linker were inserted in tandem was obtained by restriction enzyme analysis (pUC-3fi I
).

2. Preparation of selection marker (Fig. 3) and (Fig. 4) The yeast URA3 gene was transferred to the plasmid YEP 24 (1μ
g) was excised with restriction enzyme HindIII, and the DN
A polymerase Klenow fragment was used to make the ends blunt, and Xhol linkers (CCTCGAGG) were added to both ends of the T4 DNA.
After ligation with ligase and cleavage with XhoT, the plasmid pUC12, which had been cleaved with 5all, was ligated with T4 DNA ligase. E. coli HB101 was transformed using this reaction solution according to a conventional method, and an ampicillin-resistant strain was selected to obtain the desired clone (pUc12-U
RA3) (Figure 3).

The URA3 gene was excised from the plasmid pUc12-URA3 using Hindm and EcoRI. Next, Hindm-Eco containing the downstream region of URA3 gene
RV fragment was excised from YEP24 and 8gln
The EcoRV end was converted to a BglII end using a linker. These two fragments were digested with the restriction enzyme Bgl.
Plasmid pUC-Bgl cut with n and EcoRI
n and T4 DNA ligase.

E. coli HBIOI was transformed using this reaction solution according to a conventional method, and an ampicillin-resistant strain was selected to obtain the desired clone (pSK79) (Fig. 3).

The partially overlapping URA3 gene was excised from plasmid pSK79 using restriction enzymes Bgln and BamHI, and this fragment was ligated to plasmid pUC-8fi I*2, which had been cut with restriction enzyme BamH1, using T4 DNA ligase. E. coli HBIOI was transformed using this reaction solution according to a conventional method, and an ampicillin-resistant strain was selected to obtain the desired clone (pSK93) (
Figure 4).

3. Preparation of a vector for introduction into the chromosome (Fig. 5) The EcoRI fragment containing the downstream region of the yeast HIS4 gene was excised from the plasmid YIP310, ligated with the EcoRI-cleaved plasmid pUc19 using T4 DNA ligase, and the reaction solution was Escherichia coli HBI according to standard methods using
The target clone was obtained by transforming OI and selecting an ampicillin-resistant strain (psK81). This plasmid was cut with restriction enzyme Bgln and a fragment made blunt-ended with DNA polymerase Klenow fragment, and a fragment cut out from plasmid pSK93 with restriction enzymes BamHI and 5phI and made blunt-ended with T4 DNA polymerase were combined with T4 ligase. The desired clone was obtained by transforming Escherichia coli HBIOI using the reaction solution according to a conventional method and selecting an ampicillin-resistant strain (
psK127) (Figure 5).

4. Preparation of hEGF expression unit (Figure 6) The BglU-Hindm fragment containing the transcription termination region of the yeast PGK gene was cut out from plasmid pGK5-65, and Hinall
ligated with plasmid pUc19 cut with T4 ligase, and using this reaction solution to incubate Escherichia coli HB10 according to a conventional method.
1 was transformed and an ampicillin-resistant strain was selected to obtain the desired clone (psK61) (Fig. 6).

Plasmid pFN8-52 containing the promoter region of yeast HIS4 gene was cut with restriction enzyme Xhol, and UAS
It was ligated with oligonucleotides containing g (21 cr) using T4 ligase. E. coli HBIOI was transformed using this reaction solution according to a conventional method, and ampicillin-resistant strains were selected. From the obtained strain, a clone in which two copies of the oligonucleotide (21 mar) containing UASg were inserted in tandem was obtained by restriction enzyme analysis (pFN8-52-
8) (Figure 6).

Hinf containing the promoter region of yeast HIS4 gene
I fragment was excised from plasmid pFN8-52-8,
Make blunt ends with DNA polymerase Klenow fragment and Hi
Plasmid pUC19 cut with ncn is ligated with T4 ligase, and this reaction mixture is used to incubate E. coli H according to a conventional method.
The desired clone (pSK86) was obtained by transforming BIOI and selecting an ampicillin-resistant strain (6th
figure).

A fragment obtained by cleaving plasmid psK61 with restriction enzyme Sma I and a fragment excised from plasmid pSK86 with restriction enzymes Xhol and Hindm and blunt-ended with DNA polymerase Klenow fragment were ligated with T4 ligase, and using this reaction solution. E. coli HBIO according to standard methods.
The target clone was obtained by transforming I and selecting an ampicillin-resistant strain (psK106) (Fig. 6)
.

A fragment obtained by cutting plasmid pUc118 with the restriction enzyme Sma I and a chemically synthesized DNA fragment containing the base sequence encoding the signal of the yeast 1nVOrtaSO gene were ligated with T4 ligase, and using this reaction solution, the reaction was carried out according to a conventional method. The desired clone was obtained by transforming E. coli HB 101 and selecting ampicillin-resistant strains (
pSK66) (Figure 7).

This plasmid was cut with restriction enzyme N5il, and T4DN
The fragment made blunt-ended with A polymerase and the hEGF gene fragment excised from plasmid pTA1502 with restriction enzymes EcoRI and 5all and made blunt-ended with DNA polymerase Klenow fragment were ligated with T4 ligase, and this reaction solution was used to E. coli HBIO according to standard methods.
The desired clone (pSK84) was obtained by transforming I and selecting an ampicillin-resistant strain (FIG. 7).

Plasmid pSK84 was cut with the restriction enzyme EcoRI,
A hEGF gene fragment that was made blunt with DNA polymerase Klenow fragment and cut out with restriction enzyme 5all, and a fragment that was obtained by cutting plasmid psK106 with restriction enzyme Xbal, making blunt ends with DNA polymerase Klenow fragment, and then cutting with XhoI. and E. coli HBIOI using T4 ligase and using this reaction solution according to a conventional method.
The desired clone (psK107) was obtained by selecting an ampicillin-resistant strain (Fig. 7).

Cut plasmid psK107 with restriction enzyme Pstl,
hEGF gene expression unit fragment made blunt-ended with T4 DNA polymerase and plasmid pUC-SfiH2
was cut with restriction enzyme BamHI, and the fragment made blunt-ended with DNA polymerase Klenow fragment was ligated with T4 ligase, and this reaction solution was used to incubate E. coli HB according to a conventional method.
The desired clone (psK108) was obtained by transforming IOI and selecting an ampicillin-resistant strain (8th
figure).

6. Introduction of multiple copies of hEGF expression gene into yeast chromosome 3 l) Preparation of vector fragment Plasmid psKN127 (50 μg) was injected with restriction enzyme E.
A 4.7 kb fragment containing the His4 region was cut with coRI and purified by agarose gel electrophoresis. Next, this fragment was cut with restriction enzyme 5fil.

2) Preparation of expression unit Plasmid psK108 C2C200u and restriction enzyme 5
980bp containing hEGF expression unit after cutting with fil
60 μg of the fragment was purified by agarose electrophoresis.

3) The DNAs of 1) and 2) above were ligated using T4 ligase under the following conditions using a conventional method.

Experiment 1 Experiment 2 Vector 2 μg 2 μg Expression unit 8 μg 26 μg Reaction solution 10 μm 10 μm Molar ratio (unit/30100 vector) After the reaction, the yeast S and cerevislae N Y 6 A strains were transformed according to the conventional method after cutting with the restriction enzyme EcoRI. A large number of transformants were obtained by selecting the URA strain.

The length of the third chromosome of these transformants was analyzed by pulse field electrophoresis. When 19 strains of the transformants obtained from Experiment 1 were selected and analyzed, it was revealed that the third chromosome of 15 of them was longer than that of the parent strain.

8 strains are 1O-20kb longer, 2O-30kb
Five plants were longer, and one plant was longer than 50 kb. Furthermore, when three strains were selected and analyzed from among the transformants obtained in Experiment 2, it was revealed that the third chromosome of all three strains was longer than that of the parent strain. 2O-30kb longer one strain, and 5
Two strains were longer than 0kb.

7. Expression of hEGF A rosary was selected from the above transformants, cultured for 48 hours in a medium containing galactose, and the amount of hEGF secreted was measured.

#19 7 90μg1
9 23 1600μg#7
49 1580 μg.

FIGS. 3 and 4 are explanatory diagrams showing the construction of partially overlapping URA3 markers.

FIG. 5 is an explanatory diagram showing the construction of a plasmid for vector preparation.

FIG. 6 and FIG. 7 are explanatory diagrams showing the construction of an EGF expression unit.

FIG. 8 is an explanatory diagram showing the introduction of an EGF expression unit into a gene fragment preparation plasmid.

By introducing a large number of genes into the chromosome in this way, it becomes possible to increase the expression of the genes.

Claims (1)

[Claims] 1. Characterized by comprising the following steps a) to e),
Protein production method. b) A gene unit containing at least a gene encoding a desired foreign protein, the ends of which are designed so that a plurality of the units can be linked to each other with their base sequences oriented in the same direction. Prepare multiple items. b) Prepare a vector capable of transferring the plurality of gene units prepared in step a) into the chromosomal DNA of the host. c) The multiple gene units prepared in step a) are inserted or linked into the vector prepared in step b), and can be replicated while being included in the chromosomal DNA of the cell as the planned host cell grows. Preparing recombinant DNA. d) Transferring the recombinant DNA obtained in step c) into chromosomal DNA in a host cell and transforming the host cell to prepare a transformant. e) Cultivating the transformant obtained in step d) to produce the desired exogenous protein in the culture. 2. A transformant obtained by performing steps a) to d) in the production method according to claim 1.