JPH0638786A - Production of fused protein - Google Patents

Abstract

PURPOSE:To provide a new manifestation vector useful for the production of a protein containing a heterogenic product fused to the carboxy-terminal of DHFR. CONSTITUTION:The manifestation vector pTP104-4 having the DNA sequence of formula. The vector can be prepared by introducing the terminator region of rrnB gene to the downstream of the DHFR gene of pTP70-1.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to Escherichia coli-derived dihydrogen.
Modified folate reductase (hereinafter abbreviated as DHFR) gene
3'end side of the modified DHFR gene containing the gene
In addition, the reading frame of the genetic code is matched so that the heterologous gene
To introduce the heterologous gene product into DHFR.
As a combined protein and having DHFR activity.
A group that enables efficient production of high quality
Fusion tamper using replacement plasmid pTP104-4
It is about how to create quality. Industrial of the invention
Applications include microbial industry, fermentation industry, pharmaceutical manufacturing
Is the field. (Prior Art) DHFR reduces dihydrofolate
An enzyme that catalyzes the reaction that produces lahydrofolic acid
Acid coenzyme is an important enzyme in the biosynthesis system. Trimethoprim
Is an inhibitor of folic acid coenzyme biosynthesis similar to sulfa drugs
However, this drug strongly binds to DHFR and inhibits enzyme activity.
Hurt. Therefore, trimethoprim is present in the medium
And bacteria such as E. coli are unable to grow. When
When the DHFR gene was integrated into a plasmid,
DHFR in cells by increasing the copy number of the gene
When the content is increased, the bacteria are resistant to trimethoprim.
Acquire sex (M. Iwakura et al.,
J. Biochemistry, vol91, p. 12
05 (1982)). Using this property, the DHFR remains
The gene is used as a selection marker for plasmids, and DH
Using a plasmid vector incorporating the FR gene,
In fact, gene cloning has been carried out (Patent No. 13
69288, M.I. Iwakura et al. ，
J. Biochemistry, vol. 92, p. 6
15 (1982)). Trimeth using the DHFR gene
The prim resistance marker has a gene size of about 500 bases.
Small pair, easy-to-use restriction enzyme site in gene
Presence, gene expression level and strong trimethoprim resistance
Excellent because there is a very good correlation with
It is a genetic marker that is expected to be widely used.
It The present inventors have integrated the DHFR gene of Escherichia coli.
We are developing various vectors (Patent No. 136928)
No. 8, 1369291, JP-A-57-11099.
No. 9, JP-A-59-135889, JP-A-58.
-133769 gazette, JP-A-60-184388
Report, JP-A-60-199385, JP-A-62-6
No. 9990, JP-A No. 62-126984.
Do). As a plasmid containing the modified DHFR gene
Is a pTP70-1 created by the present inventors.
63-46193). 3'end of DHFR gene
Heterogeneous inheritance by matching the reading frame of the genetic code to the end side
By introducing the offspring into the DHFR
A method to express and produce a fused protein and
Then, the method using the DHFR gene of Bacillus subtilis is
A fusion tamper with DHFR, which has been conducted by the inventors.
It is already clear that creating quality is meaningful.
It has been made clear (Japanese Patent Laid-Open No. 63-87981,
JP-A-63-102696, JP-A-63-2456
79, Sho 63-245680, JP 6
JP-A-3-258595, JP-A-1-38099
Information etc.). (Problem) However, the DHFR gene of Bacillus subtilis
The 3'end side is different by matching the reading frame of the genetic code.
By introducing a seed gene, a heterologous gene product is DH
Trying to express and produce as a protein fused with FR
In the method, the expression efficiency of the gene is not so high.
The intracellular production of the fusion protein produced is
The quality of the protein is at most about several percent, and the expression efficiency is improved.
As a problem that needs to be solved to raise production
It was left. The present inventors have developed a large amount of Escherichia coli DHFR.
The recombinant plasmid pTP64-1 produced in
(JP-A-62-69990). pTP64-1
In E. coli, which has DHFR
About 15% or more is produced. But Naga
Et al., In the DHFR gene of E. coli,
When a heterologous gene is introduced at the terminal side, DHFR enzyme activity
Whether it is expressed as a fusion protein without losing
Was totally unknown. (Object of the Invention) The object of the present invention is to solve the above problems.
For the purpose of adding a heterologous gene product to the carboxy terminal side of DHFR
To establish a method for efficiently producing a fusion protein
To stand up. As a result of earnest research, the present inventors have found that
In addition, the present inventors have used the DHFR gene as a genetic marker.
Of pTP70-1 developed for the purpose of making it easy to use
The DHFR gene artificially changes the sequence at the 3'end of the gene.
Modified DHFR that has enzymatic activity even if modified
Focusing on the fact that about 20% of the quality is produced,
By modifying pTP70-1, a new recombinant
Construction of Smid pTP104-4 and pTP104-4
It is revealed that the above problems can be solved by using
This invention has been completed and the present invention has been completed. (Structure of the Invention) pTP104-4 used in the present invention,
In order to improve the production efficiency of DHFR, pTP70-1
As an efficient terminator downstream of the DHFR gene
Known as rrnB (ribosome RNA gene
1) To introduce the terminator region of the gene
It is the recombinant plasmid obtained by. Figure 1 is a book
The whole base sequence of pTP104-4 used in the invention is shown.
ing. Fig. 2 shows the restriction enzyme digestion site of pTP104-4.
The figure is shown. pTP104-4 is 4466 base pairs
And the tolerance to Escherichia coli host, trimethoprim
Sex and ampicillin resistance can be imparted. p
TP104-4 is a restriction enzyme AatII, BamHI,
BclI, BglII, ClaI, EcoRI, Hin
dIII, HpaI, PstI, PvuII, and S
by alI, 1 (4318-4323),
1 (532-537), 1 (58-63), 2 (13-
18,538-543), 2 (1-6, 545-55)
0), 1 (471-476), 2 (19-24, 446)
1-4466), 2 (7-12, 4434-443)
9), 1 (3641-3646), 1 (1822-18)
27), and 1 (825-830) recognition cutting parts
Position (the cleavage recognition site of each restriction enzyme in FIG. 1
Are shown in parentheses). introduced in pTP104-4
The generated terminator DNA is the terminator of the rrnB gene.
It is a noether area. terminator of rrnB gene
The plasmid containing the -region is Brosius (J. Br.
house et. al. J. MolBiol. vo
l. 148, p. 107 (1981))
Including the terminator region of the rrnB gene
The plasmid can be purchased from Pharmacia. p
To construct TP104-4, plasmid pKK175-
The one introduced in 6 is used. 16 of FIG.
Sequences 00 to 1824 of the rrnB gene
It is a sequence containing a terminator region. Introduce this array
The production amount of the modified DHFR in E. coli.
It could be increased from 1.0 to 1.4 times. above
pTP104-4 was added to E. coli host
And can confer resistance to ampicillin and ampicillin,
In addition, pTP104-4 is an E. coli C600 strain
And maintained in a stable state, including pTP104-4.
Having E. E.coli C600 strain
Deposited as P-1579. pTP104-
The containing E. coli produces a large amount of modified DHFR. p
E. coli containing TP104- was added to YT + Ap medium (cultured).
5g NaCl, 8g tryptone g, 5 in 1 liter of ground
g yeast extract and 5 mg ampicillin nato
(Medium containing lithium) from the late logarithmic growth phase to the stationary phase
Of DHFR in cell-free extracts obtained from cultured cells
Specific activity is about 9 to 10 units / mg protein
Is. The value of E. coli containing pTP70-1 is about 7
To 9 units / mg protein, pTP104
Of DHFR by using E. coli containing -4
The output can be increased from 1.0 to 1.4 times.
Suitable for production of modified DHFR. Use pTP104-4
By using the fusion tag of DHFR and a heterologous gene product.
The method of creating the protein quality will be described below. Figure 3 shows pT
Amino acid sequence of DHFR produced by DHFR of P104-4
It is the figure which showed. Only present in pTP104-4
Restriction enzyme BamHI site (from 532 to 53 in FIG. 1)
The 7th sequence) is the carboxy terminal of DHFR
2 to 4 (159 to 161 from the amino terminus)
(See Fig. 3, Fig. 3). Bam
HI gives rise to cohesive ends
Since it is a restriction enzyme,
When heterologous DNA is introduced into the cleavage site, the 532nd position in Fig. 1
DNA is introduced between position 533 and 533, and its sequence
Is 5'-G (522nd) GATC- (heterologous DNA)
-Has a common sequence of GATCC (537th) -3 '
Will be connected. Therefore, the introduction of heterologous DNA
The fusion protein produced by
The first methionine (M
et) to the 160th isoleucine (Ile)
Amino acid sequence is completely the same, heterologous DN after 160th
A fusion protein having an amino acid sequence derived from A was produced
Be done. According to the present invention, the pTP104-4 described above is used.
Of DHFR and heterologous gene products by utilizing the characteristics of
Can make proteins. The fusion tongue of the present invention
As described below, the method of creating a quality
Method for producing combined gene, that is, DTP of pTP104-4
A gene in which a heterologous gene is bound to the 3'end of the R gene
For producing a recombinant plasmid having (B) recombinant plasmid
Method for introducing rasmid into E. coli, (C) expressing in E. coli
A method for detecting a fusion protein that is a fusion gene product
Consists of (A) Method for constructing fusion gene DHFR gene using BamHI site of pTP104-4
As a method of creating a fusion gene with a gene, BamHI
Using the sticky ends generated by cleavage with
Those who make fusion genes by matching the open reading frames of genes
DNA Polymer after cutting by BamHI method
Blunt-ended with a protease or reverse transcriptase
Blunt heterologous DNA by aligning the reading frame of R gene
Create a fusion gene by binding by endoligation
Method, Nuclease after digestion with BamHI
Nucleia that specifically cleaves S1 or single-stranded DNA
Read out DHFR gene
Blunt end ligation of heterogeneous DNA
Method for producing a fusion gene by binding with BamHI
After cutting with exonuclease BAL31
Use any exonuclease to make blunt ends and
DNA is bound and fused by blunt end ligation
A method such as a method for creating a gene can be performed.
These methods are not available to the party performing the recombinant DNA.
It can be done easily without any problems. Reference example
In the case of
The chromosomal DNA of Escherichia coli was transformed into Sau3AI using the terminating ends.
Various molecular weights using DNA fragments obtained by digestion with
Shows that a fusion protein with
It (B) Method for introducing recombinant plasmid into Escherichia coli The recombinant plasmid prepared in (A) above to E. coli cells
Is introduced by the so-called transformation method.
It can be carried out. Recombinant plasmid transform
Although various methods are known as solution methods,
Recombination made with pTP104-4 made in Ming
To introduce the plasmid into E. coli, use recombinant DNA
The parties involved can easily do it without any problems.
Yes, it does not depend on the method of transformation. p
A recombinant plasmid prepared using TP104-4 was introduced.
Entered E. coli can be easily detected using agar medium.
You can pTP104-4 is a genetic marker
It has ampicillin resistance and trimethoprim resistance.
Ampicillin resistance creates a fusion gene with the DHFR gene.
Since it does not undergo any manipulation when
Is inherited as is. Also resistant to trimethoprim
The DHFR gene, which is a gene that imparts
Of DHFR produced by the manipulation of the fusion gene
A heterologous peptide or protein is attached to the
When combined, the DHFR enzyme activity is not lost,
It has the ability to confer topram resistance. Therefore, p
A recombinant plasmid prepared using TP104-4 was introduced.
Entered E. coli is ampicillin and trimethoprim
Show resistance to. Used for transformation as agar medium
Ampicillin and trime
When a medium supplemented with toprim is used, it grows on this medium
The transformant is a recombinant produced using pTP104-4.
E. coli in which the plasmid has been introduced. (C) A fusion gene that is a fusion gene product expressed in Escherichia coli
E. coli containing pTP104-4 contained modified DHFR
Make a lot. E. coli containing pTP104-4 was transformed into Y
Cultured in T + Ap medium from late logarithmic growth phase to stationary phase
SDS polyacrylamide gel electrophoresis (hereinafter
Below, it is abbreviated as SDS-PAGE. ) For sample preparation
Suspended and lysed (see Examples), and used this for SDS-PAG
Separation with E, protein Coomassie-Brilliant Blue
When dyed with
A clear band (main band) is also shown. On the other hand, p
Includes recombinant plasmids created using TP104-4
Escherichia coli has a large amount of DHFR fusion protein.
It This fusion protein was modified from pTP104-4.
Heterologous polypeptide chain on the carboxyl terminal side of modified DHFR
Are bound and the molecular weight is high. Therefore,
The obtained fusion protein should be used for SDS-PAGE.
And the molecular weight became larger than pTP104-4.
As a clear band at the protein position, pTP104
-1 can be detected in the same manner as the detection of the modified DHFR.
You can The method of SDS-PAGE is Laemml.
It can be easily performed according to the method i. SDS-P
In AGE, mobility of proteins with small molecular weight
Is large, and the mobility of proteins with large molecular weight is small
is there. Also, there is a very good correlation between mobility and molecular weight.
Known to be related. From this,
The molecular weight of the protein can be estimated and
Whether or not it is the target fusion protein considering the child sequence
It is possible to determine whether or not. Next, an example of the present invention
And a reference example. (Example 1) Preparation of fusion protein About 1 µg of pTP104-4 was cleaved with BamHI.
After that, it was treated with alkaline phosphatase. Alkaline phos
Phenase-treated DNA with fatase treatment
Denaturing and removing the coexisting enzyme protein,
The DNA was precipitated with Tanol. 70% of the precipitated DNA
After washing with% ethanol, remove ethanol and
The precipitate was dried. About 5 μg of E. coli chromosomal DNA,
After digestion with Sau3AI, the DNA was precipitated with ethanol.
Let The precipitated DNA was washed with 70% ethanol
After that, ethanol was removed and the precipitate was dried under reduced pressure. Dry
Add 50 μl of dried DNA to each ligase solution.
Reaction solution (10 mM Tris-HCl, pH 7.4,
5 mM MgCl _Two , 10 mM dithiothreitol,
After dissolving in 5 mM ATP), combine both and add 5
Add Knit T4-DNA ligase at 25 ° C,
The ligation reaction of DNA was performed for 4 hours. This reactant is shaped
Quality conversion method
od, described in reference 1)).
It was The treated cells were treated with 50 mg / l ampicillin
Sodium and 10 mg / l trimethoprim
Nutrient agar medium (2g glucose, 1
g potassium diphosphate, 5 g yeast extract, 5 g
Contains polypeptone, 15 g of agar. ) Apply 3 on
By culturing at 7 ° C for 24 hours, about 500
I got a knee. From these colonies
Twenty selected, 1.5 ml YT + Ap medium (in 1 liter of medium)
5g NaCl, 5g yeast extract, 8g
Lipton, containing 50 mg sodium ampicillin
Mu. ), The cells were cultured overnight at 37 ° C. Culture fluid
Take each in an Eppendorf centrifuge tube and perform 12,000 times.
Centrifuge at 10 rpm for 10 minutes to collect bacterial cells as a precipitate
It was Add 0.1 ml of the sample preparation solution for electrophoresis.
(0.0625M Tris-HCl, PH6.8,2
% Sodium lauryl sulfate (SDS), 10% green
Serine, 5% 2-mercaptoethanol, 0.001
% Bromphenol blue. ) Is added,
Suspend and keep it in boiling water for 5 minutes to dissolve the cells
It was A sample subjected to this treatment was used for SDS-polyacrylic acid.
Mid-gel electrophoresis (UK Lammli; Natu
re, vol. 227, p. 680 (1970))
Was analyzed. PTP104-4 as a standard sample
E. coli containing the same treatment, and molecular weight
Lactalbumin (molecular weight 14,20 as a marker
0), trypsin inhibitor (molecular weight 20,10
0), trypsinogen (molecular weight 24,000), cal
Bonic anhydrase (molecular weight 29,000), green
Ceraldehyde 3-phosphate dehydrogenase (molecular weight 3
6,000), ovalbumin (molecular weight 45,000),
And bovine serum albumin (molecular weight 66,000)
Samples should be concentrated at 10 to 20% polyacrylamide
Run on a gradient gel. As a result, 20 rollers that I examined
Of the knees, 18 had a DHFR version of pTP104-4.
Disappeared, and the molecular weight became apparently higher than that
Protein (Protein newly appeared in each bacterial cell
The band positions were different. ) Is newly produced
It was Comparing the mobility of molecular weight marker proteins
Therefore, the molecular weight of these proteins is about 23.00.
It was a value between 0 and about 35,000. pTP104
-4 DHFR (molecular weight 18,379)
It migrated as a protein having a molecular weight of about 20,000. Servant
Therefore, the Sau3AI digestion fragment of E. coli chromosomal DNA
By fusing, the carboxy terminal side of DHFR
A peptide having a molecular weight of about 3,000 to about 15,000
Or a fusion protein in which the proteins are fused produces
It was shown that E. coli producing the fusion protein
Are fused because they are resistant to trimethoprim.
The protein appears to have DHFR activity
It In addition, the appearance of new protein bands
From the state of staining with brilliant blue, the protein
The production amount of pTP104-4 is estimated.
A band that is at least as good as the DHFR band,
It was shown that the quality of the pak is produced stably. Furthermore, the book
In the examples, E. coli chromosomal DNA was cut with Sau3AI.
DNA fragments obtained by cutting were used to create the fusion gene
And the resulting fusion protein happens to read the gene.
When the reading frames match, the translation stop cipher appears on the reading frame.
Sequence until appearance was obtained by fusion
It is considered to be a thing. Therefore, the method of the present invention has been introduced.
It is suggested that the DNA sequence is not regulated. Reference Example 1 Preparation of pTP104-4 About 1 μg of pTP70-1 (JP-A-63-46193)
Gazette) was cut with SalI and PvuIl,
Lucari phosphatase treatment was performed. Alkaline phosphata
By treating the treated DNA with phenol,
Denature and remove coexisting enzyme proteins, and then ethanol
The DNA to precipitate. 70% of the precipitated DNA
After washing with nol, remove ethanol and precipitate under reduced pressure.
Dried. Cleavage of DNA by restriction enzymes, alkaline
Sphatase treatment, phenol treatment, and ethanol
Each of the precipitation operations was performed using "Molecular C
longing A Laboratory Manual
l "(T. Maniatis, EF Fritsc.
h, J. Sambrook, eds. Cold Spr
ing Harbor Laboratory (198
2), hereinafter referred to as Reference 1. )
I went. About 1 μg of pKK175-6 (pharmacy
(Purchased from A.) was cut with SalI and PvuII.
Then, the DNA was precipitated with ethanol. Precipitated DNA
After washing with 70% ethanol, remove ethanol and depressurize
The precipitate was dried below. Dried DNA (pTP70
-1 and pKK175-6 were digested with restriction enzymes
50 μl of the reaction solution for ligase (10
mM Tris-HCl, pH 7.4, 5 mM MgC
l _Two , 10 mM dithiothreitol, 5 mM AT
After dissolving in P), combine both and add 10 units of T
4-DNA ligase is added, and DNA is added at 25 ° C for 4 hours.
Was ligated. This reaction product was transformed (t
transformation method, above document
E. coli) according to the method described in 1.). This process
50 mg / l of ampicillin sodium
And nutrient agar containing 10 mg / l trimethoprim
Ground (2g glucose, 1g phosphoric acid 2 in 1 liter of medium)
Potassium, 5g yeast extract, 5g polypept
It contains 15g of agar. ) Apply it on and apply it at 37 ° C for 24
About 50 colonies can be obtained by culturing for a long time.
I was able to. Choose 8 from these colonies, and
ml YT + Ap medium (5 g of NaC in 1 l of medium)
1, 5g yeast extract, 8g tryptone, 50m
g of ampicillin sodium. ) At 37 ° C,
The cells were cultured overnight. Eppendorf cultures
Transfer to a centrifuge tube and centrifuge at 12,000 rpm for 10 minutes.
The cells were separated and the cells were collected as a precipitate. Add 0.1 ml of this
Sample preparation for electrophoresis (0.0625M Tris-
HCl, pH 6.8, 2% sodium lauryl sulfate
(SDS), 10% glycerin, 5% 2-merca
Ptoethanol, 0.001% bromphenol blue
Including ) Is added to suspend the cells, and this is placed in boiling water.
The cells were kept for 5 minutes to dissolve the cells. Samples that have this processing
SDS-polyacrylamide gel electrophoresis (U.
K. Lammli; Nature, vol. 227,
p. 680 (1970)). Standard sun
Similar treatment to E. coli containing pTP70-1 as a pull
And lactal as a molecular weight marker
Bumin (molecular weight 14,200), trypsin inhibitor
-(Molecular weight 20,100), Trypsinogen (Molecular weight
24,000), carbonic anhydrase (molecular weight
29,000), glyceraldehyde 3-phosphate dehydrate
Logenase (molecular weight 36,000), egg albumin (min
Bovine serum albumin (molecular weight 45,000)
66,000) containing a sample containing polyacrylamide
Run on a 10 to 20% gradient gel. That conclusion
As a result, 5 of the 8 colonies examined were p
A protein having almost the same size as DH70 of TP70-1
It became clear that it will produce. These five fungus bodies are Y
Cultivated in T + Ap medium, Tanaka and Weisblue
m. method (T. Tanaka, B. weisblum;
J. Bacteriology, vol. 121, p.
354 (1975)).
It was Restriction enzyme AvaI was added to each of the obtained plasmids.
Tried to cleave with
It was not cut with aI. Also, cut with EcoRI
When analyzed by agarose gel electrophoresis,
Is also cut at only one location, and the molecular size of the plasmid
Is about 4.5 kilobase pairs, and is pTP70-1
5 pluses that became a little smaller
An appropriate one was selected from the amides and named pTP104-4.
PTP104-4 obtained by the above operation is pTP7.
Obtained by Sal1 and PvuII cleavage of 0-1
The larger of the two DNA fragments, pKK175-
2 obtained by digestion of 6 with SalI and PvuII
The structure in which the smaller one of the DNA fragments of the book is bound
It should be doing. pTP104-4 with SalI and
When cut with PvuII, two
A DNA fragment was obtained, and the larger DNA fragment was pTP7.
Obtained by SalI and PvuII digestion of 0-1
The larger of the two DNA fragments
The other DNA fragment was SalI and pKK175-6.
Of the two DNA fragments obtained by digestion with PvuII
It was a perfect match with the smaller fragment. Already pTP7
0-1 and pKK175-6 reveal complete nucleotide sequences
Therefore, using the result, pTP104
The entire nucleotide sequence of -4 was determined as shown in FIG. (Reference Example 2) E. coli containing pTP104-4 and DHFR production amount of E. coli containing pTP104-4. coli C600
E. coli containing pTP70-1. coli C600
Each was cultured overnight in 50 ml of YT + Ap medium
After that, the bacterial cells were collected by centrifugation. 0.1 mM of cells
Contains ethylenediaminetetraacetic acid disodium (EDTA)
Mu 10 mM phosphate buffer pH 7.0 (hereinafter, buffer 1)
After washing with water, suspend in 2 ml of buffer solution 1 and
Wave crushed. 20,000 revolutions of sonicated cell fluid
The mixture was centrifuged at 1 / min for 1 hour to obtain a supernatant. In the obtained supernatant
Then, the DHFR enzyme activity and the amount of protein were measured.
Supernatant protein 1 from the measured enzyme activity and protein amount
DHFR enzyme activity per mg (specific activity (units / m
g protein)) was calculated. This value is DHFR
The amount is proportional to the amount produced in the cells of. As a result, pT
E. coli containing P104-4. With the coIi C600,
Values of 9.02, 9.85, 9.90 (done three times).
Of E. coli containing pTP70-1. coli C600
Then, the values of 7.05, 8.20, and 8.95 were obtained.
In all cases, the microbial cells containing pTP104-4 were DH
FR production was higher. (Effects of the Invention) According to the present invention, according to the present invention,
Fusion of useful peptide or protein to the terminal end
It is easy to do. Some peptides are unstable in E. coli
Or as a fusion protein for protein production.
Expected to be produced, the present invention
If a useful peptide by making a fusion protein
For the most part, it contributes to the mass production of proteins.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the entire base sequence of pTP104-4, in which only one DNA strand sequence of double-stranded DNA is
The description is from the 5 ′ end to the 3 ′ end. Symbols in the figure represent nucleic acid bases, A is adenine, C is cytosine, G is guanine, and T is thymine. The numbers in the figure are the restriction enzymes Cl present at two sites in pTP104-4.
5'-ATCG of the ClaI cleavage recognition site closer to the restriction enzyme HindIII cleavage site among the aI cleavage recognition sites
The number counted from the first "A" of AT-3 'is 1. FIG. 2 is a diagram showing a restriction enzyme digestion map of pTP104-4. In the figure, the arrow in the white frame indicates the position and expression direction of the DHFR gene, and the black frame indicates the portion containing the terminator region of the rrnB gene. FIG. 3 is a diagram showing the nucleotide sequence of the portion encoding DHFR existing in pTP104-4 and the amino acid sequence of protein. Symbols in the figure represent nucleobases and amino acids, A is adenine, C is cytosine, G is guanine, T is thymine, Ala is alanine, Arg is arginine, Asn is asparagine, and Asp is aspartic acid. , Cys is cystine, Gln is glutamine, Glu is glutamic acid, Gly is glycine,
His for histidine, Ile for isoleucine, Le
u is leucine, Lys is lysine, Met is methionine, Phe is phenylalanine, Pro is proline, Ser is serine, Thr is threonine, and Trp is Trp.
Indicates tryptophan, Tyr indicates tyrosine, and Val indicates valine. The numbers in the figure indicate the numbers counted from "A" of the ATG codon that encodes the first amino acid, methionine, as number 1.

Claims (1)

What is claimed is: 1. A stable copy of Escherichia coli, which is capable of conferring trimethoprim resistance and ampicillin resistance to E. coli as a host. By introducing a heterologous gene at the same time, the heterologous gene product can be efficiently expressed as a protein fused with dihydrofolate reductase, has a size of 4466 base pairs, and has an expression vector pTP having the following DNA sequence:
104-4 was used to bind a heterologous gene by aligning the reading frame of the genetic code with the 3'end side of the dihydrofolate reductase gene to prepare a recombinant plasmid, which was introduced into E. coli. A method for producing a fusion protein, which comprises producing a protein in which a heterologous gene product is fused to the carboxy terminal side of dihydrofolate reductase.