JPH01144992A - Production of fused protein - Google Patents

Abstract

PURPOSE:To produce a protein composed of a heterogene fused with dihydrofolic acid reductase, in high efficency, by introducing a heterogene into a specific recombinant plasmid. CONSTITUTION:A dihydrofolic acid redcuctase (DHFR) gene originated from E.coli is modified to attain trimethoprim-resistance and ampicillin-resistance. A heterogene is introduced to the 3'-terminal of the modified DHFR gene in a recombinant plasmid pT70-1 containing the modified gene in such a manner as to match the reading frame of the genetic code. A protein composed of a heterogene fused with DHFR can be produced in high efficiency as a protein having a DHFR activity.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to dihydrofolate reductase (hereinafter referred to as
It is abbreviated as DHFR. ) Recombinant plasmid pTP70-1 containing a modified gene (patented in 1983-31)
By introducing a heterologous gene into the 3' end of the modified DHFR gene in 2836) with the open reading frame of the genetic code matching, the heterologous gene product can be transformed into a protein fused with DHFR and a fusion protein having DHFR activity. The present invention relates to a method for producing a fusion protein, which is characterized by efficient production as a fusion protein.

The present invention is suitable for use in the fields of microbial industry, fermentation industry, and pharmaceutical manufacturing.

BACKGROUND OF THE INVENTION DHFR is an enzyme that catalyzes the reaction of reducing dihydrofolate to produce tetrahydrofolate, and is an important enzyme in the monofolate coenzyme biosynthesis system. Trimethoprim, like sulfa drugs, is an inhibitor of the folate coenzyme biosynthesis system, but this drug strongly binds to DHFR and inhibits enzyme activity. Therefore, the presence of trimethoprim in the culture medium makes it impossible for bacteria such as E. coli to grow. However, DHF
By incorporating the R gene into a plasmid, etc., and increasing the number of copies of the gene, it is increased in bacterial cells.

Bacteria acquire resistance to trimethoprim (M,
Iwakura et at, J, Bioche
mistry, vo191゜p, I205 (1982
)). Taking advantage of this property, the DHFR gene is used as a selection marker for plasmids.

Gene cloning is actually being carried out using a plasmid vector incorporating the DHFR gene (patented).
Showa 56-143520. M, Iwakura et a.
t,, J,Biochemistry, vol, 9
2. p. 815 (1982)).

The trimethoprim resistance marker using the DHFR gene has a small gene size of approximately 500 base pairs, has an easily accessible restriction enzyme site in the gene, and is very good in terms of gene expression level and strength of trimethoprim resistance. It is an excellent genetic marker due to the existence of correlations and is expected to be widely used. The present inventors have developed various vectors incorporating the E. coli DHFR gene (Patent Nos. 1369288 and 136929).
No. 1, Tokikai 1977-110999. JP 69-135
889. Time opening 1983-133769゜Time opening 1986-
184388. Japanese Patent Publication No. 1983-199385, Publication No. 62-69990. JP-A-62-126984. patent
(Sho 61-312836, etc.).

A method for expressing and producing a heterologous gene product as a protein fused with DHFR by introducing a heterologous gene into the 3' end of the DHFR gene by aligning the reading frame of the genetic code with the DHFR gene of Bacillus subtilis. There is a method using .

This work has been carried out by the present inventors (Patent No. 61-234
003, patent 1986-249260. Special application 1986-
079377, Patent No. 62-079378,
Patent: 1986-085406゜Patent: 1982-0928
81 etc.).

Problems However, in the method of ligating a heterologous gene with the open reading frame of the genetic code aligned with the 3'' end of the DHFR gene of Bacillus subtilis and attempting to express and produce the heterologous gene product as a fusion protein with DHFR, The efficiency of gene expression is not so high, and the amount of fusion protein produced within the microbial cell is only a few percent of that of the 9-sided protein.

Therefore, increasing the expression efficiency of 2 and increasing the production amount remained a problem that needed to be solved.

The present inventors have already created a morning change plasmid pTP64-1 that produces a large amount of enzyme protein for the E. coli DHFR gene (62-6
9990). In E. coli having pTP64-1, DHFR is produced as about 15% or more of the bacterial protein.

However, in the DHFR gene of E. coli, when a heterologous gene is introduced into the 3' end of the gene, DHF
It was completely unknown whether it could be expressed as a fusion protein without losing its RHF groove properties.

purpose of invention The purpose of the present invention is to solve the above problems.

The purpose of this project is to establish a method for efficiently producing a fusion protein in which a heterologous gene product is fused to the carboxy terminal side of DHFR.

As a result of intensive research, the present inventors have developed pTP7 for the purpose of easily using the DHFR gene as a genetic marker.
Focusing on the fact that the 0-1 DHFR gene expresses and produces about 20% of the bacterial protein, modified DHFR that has enzymatic activity even if the 3' end sequence of the gene is artificially modified, we solved the above problems. The present invention was completed by clarifying that the problem can be solved.

Structure of the Invention The present invention relates to a method for producing a fusion protein between DHFR and a heterologous gene product using pTP70-1.

FIG. 1 shows the entire base sequence of pTP70-1. FIG. 2 shows the amino acid sequence of DHFR produced by pTP70-1. The only restriction enzyme BamH1 site present in pTP70-1 (from position 532 to position 5 in Figure 1)
The 37th array) is.

The amino acid sequence of DHFR from 2nd to 4th from the carboxy terminus (159th to 161st from the amino terminus, see Figure 2) is encoded. BamHI has a cohesive end (CQhe
Since it is a restriction enzyme that produces sive end),
When heterologous DNA is introduced into the cleavage site using the complementarity of the ends, the DNA is introduced between positions 532 and 533 in Figure 1, and its sequence is as follows.

5'-G (522nd) GATC- (heterologous DNA)-G
They have a common sequence ATCC (537th)-3'. Therefore, the fusion protein 9 created by introducing heterologous DNA is the D
In the amino acid sequence of HFR, the first methionine (N
The amino acid sequence from the 160th isoleucine (11e) to the 160th isoleucine (11e) is completely common, and the heterologous D
A fusion protein having an amino acid sequence derived from NA is produced.

The present invention utilizes this feature to demonstrate a method for creating a fusion protein between DHFR and a heterologous gene product.

Creation of fusion protein involves (■) Creation of fusion gene,
That is, creation of a recombinant plasmid having a gene in which a heterologous gene is linked to the 3' end of the DHFR gene of pTP70-1, (II), introduction of the I-recombined plasmid into E. coli, and (■) fusion expressed in E. coli. Detection of fusion proteins that are gene products.

This can be done by

(1) Creating a fusion gene The method for creating a fusion gene with the DHFR gene using the BarnHI site of pTP70-1 is to use the cohesive end generated by cleavage with ΦBamH1 to align the open reading frame of the DHFR gene and create the fusion gene. Method for making DHFR: After cutting with BamHI, make blunt ends using DNA polymerase or reverse transcriptase.
A method of aligning the open reading frames of genes and joining heterologous DNA by plant end ligation to create a fusion gene; ■ After cutting with BamHI, nuclease S
A method of creating a fusion gene by making blunt ends using a nuclease that specifically cleaves single- or single-stranded DNA, aligning the open reading frame of the DHFR gene, and joining heterologous DNA by plant end ligation, ■BamH
After cleavage with I, exonuclease BAL31
A method such as a method of creating a fusion gene by making the ends blunt with an exonuclease such as and ligating heterologous DNA by plant end ligation can be performed.

These methods can be easily carried out by anyone who is involved in recombinant DNA work without any problems. In the example, we used the cohesive ends generated by cutting with BamHI to convert E. coli chromosomal DNA into 5au3AI.
This shows that fusion proteins with various molecular weights can be created using DNA fragments obtained by cutting with .

(n)! Introduction of the recombinant plasmid into E. coli The recombinant plasmid produced in (I) can be introduced into E. coli cells by a so-called transformation method. Various methods are known for the transformation of Mi recombinant plasmids, but the p
The recombinant plasmid produced using TP70-1 can be easily introduced into E. coli by anyone who has performed recombinant DNA without any problems.

It does not depend on the method of transformation.

E. coli into which the morning change plasmid created using pTP70-1 has been introduced can be easily detected using an agar medium. pTP70-1 is.

It has ampicillin resistance and trimethoprim resistance as genetic markers. Since ampicillin resistance is not manipulated at all when creating a fusion gene with the DHFR gene, the ampicillin resistance is inherited as is in the two recombinant plasmids. Also,
The DHPR gene, which confers trimethoprim resistance, loses its DHF and R enzyme activities even if a foreign peptide or protein binds to the carboxy-terminal side of DHFR, which is created by the fusion gene creation procedure described in Section 2 (1). It has the ability to confer resistance to Trimedobrum. Therefore, E. coli into which the recombinant plasmid created using pTP70-1 has been introduced exhibits resistance to ampicillin and trimethoprim. When an agar medium prepared by adding ampicillin and trimethoprim to a medium in which E. coli used for transformation can grow, the transformed strain that grows on this medium is pTP70-
This is an E. coli strain into which a recombinant plasmid made using 1 was introduced.

(I [I) Detection of fusion protein pTP70-1, which is a fusion gene product expressed in E. coli, can produce a large amount of modified DHFR. E. coli containing pTP70-1 was transformed into YT+A
Hedrons cultured in p medium from the late logarithmic growth phase to the stationary phase were subjected to SDS polyacrylamide gel electrophoresis (hereinafter referred to as 5
It is abbreviated as DS-PAGE. ) was suspended and lysed in a sample preparation solution (see Example 3), and separated by 5DS-PAGE.

When a protein is stained with Coomassie brilliant blue, the most obvious band (main band) is shown at a molecular weight of approximately 20,000.

E. coli containing a recombinant plasmid created using pTp 104-4 can produce a large amount of DHFR fusion protein.2 The produced fusion protein is pTP
It can be detected using 5DS-PAGE in the same manner as the modified DHFR produced by E. coli containing 70-1. The 5DS-PAGE method can be easily performed according to Laemmli's method. In 5DS-PAGE, proteins with a small molecular weight have a high electrophoretic mobility, and proteins with a large molecular weight have a small electrophoretic mobility.

Furthermore, it is known that there is a very good correlation between electrophoretic mobility and molecular weight. From this.

The molecular weight of the fusion protein can be estimated.

Considering the sequence of the introduced gene, it is possible to determine whether the fusion protein is the desired fusion protein.

Next, examples and reference examples of the present invention will be shown.

Example 1 Preparation of fusion protein Approximately 1 μg of pTP70-1 was cleaved with BamHI and then treated with alkaline phosphatase. The alkaline phosphatase-treated DNA was treated with phenol to denature and remove coexisting enzyme proteins, and then the DNA was precipitated with ethanol. 70% of precipitated DNA
After washing with ethanol, the ethanol was removed and the precipitate was dried under reduced pressure.

Approximately 5 μg of E. coli chromosomal DNA was added to 5au3A! After cutting, the DNA was precipitated with ethanol.

After washing the precipitated DNA with 70% ethanol.

The ethanol was removed and the precipitate was dried under reduced pressure.

The dried DNA was added to a 50 μm ligase reaction solution (10 mM Tris-HCI, pH 7, 4, 5).
mM MgCl2゜10mM dithiothreitol, 5
After dissolving in mM ATP), combine the two, add 5 units of T4-DNA ligase, and incubate at 25'C for 40 minutes.
The DNA ligation reaction was allowed to take place for several hours. This reaction product was subjected to a transformation method (trans-formation method).
d, as described in document l above).

Incorporated into E. coli. A facepiece that has undergone this treatment.

50mg/I of Ann and Silin Sodium and 10mg
Nutrient agar medium containing /I trimethoprim (medium 1l
Inside, 2g glucose, 1g dipotassium phosphate, 5
g yeast extract, 58 polypeptone.

Contains 158 agars. ) and cultured at 37°C for 24 hours, approximately 500 colonies could be obtained. Randomly select 20 colonies from these colonies and
．． 5 ml of YT+Ap medium (5 g of Na in medium 11)
C1.5g yeast extract.

Contains 8g tryptone, 50mg ampicillin sodium. ), the bacterial cells were cultured overnight at 37°C. Transfer each culture solution to an Eppendorf centrifuge tube.

The mixture was centrifuged at 12,000 rpm for 10 minutes, and the bacterial cells were collected as a precipitate. To this, add 0.1 ml of electrophoresis sample preparation solution (0,0625 M Tris-HCI, p
H6,8,2% Sodium Lauryl Sulfate (SDS).

loz glycerin, 5x 2-mercaptoethanol.

Contains 001% bromophenol blue. ) and the bacterial cells! ! ! , keep it cloudy for 5 minutes in boiling water,
The bacterial cells were lysed. This treated sample was subjected to 5DS-polyacrylamide gel electrophoresis (lJ, lamm).
l i ; Nature, vol, 227. p, 6
80 (1970)).

Escherichia coli containing pTP70-1 was treated in the same manner as a standard sample, and lactalbumin (molecular weight 14,200) was used as a molecular weight marker.

Trypsin inhibitor (molecule ffi 20,10
0),) Lipsinoken (molecule ff124,000)
, carbonic anhydrase (molecule ff129,00
0), glyceraldehyde 3-phosphate dehydrogenase (molecular weight 36,000).

Samples containing ovalbumin (molecular weight [45,000) and bovine serum albumin (molecular weight 66,000) were run on polyacrylamide gradient gels ranging from lO to 20%. As a result, out of the 40 colonies investigated,
In 32 cells, the DHFR band of pTP70-1 disappeared and a new protein with a clearly larger molecular weight was produced (the position of the band of the newly appeared protein in each bacterial cell was different). . By comparing the mobility of molecular weight marker proteins, the molecular weight of those proteins ranges from about 21.000 to about 35.
The value was between 000 and 000. DHFR of pTP70-1 (
Under these conditions, the molecular weight 18.379) is 20,
000 protein. Therefore, by fusing the 5au3AI cut fragment of E. coli chromosomal DNA, one molecular weight of approximately 1.
000 to about 15,000 peptides or proteins were produced. Since all E. coli strains that produce the fusion protein are trimethoprim resistant, the fusion protein is
It is clear that it has FR activity.

Also, from the state of staining of newly appeared protein bands with Kumamaji Brilliant Blue.

The amount of protein produced is estimated, but both pTP7
It is about the same level as or more than the 0-1 DHFR band,
It was demonstrated that the fusion protein was stably produced.

Furthermore, in this example, the chromosomal DNA of E. coli was
DNA fragments obtained by cutting in humans are used to create fusion genes, and the resulting fusion protein happens to have the same open reading frame of the gene and the sequence up to the appearance of the translation stop code on that open reading frame. It is thought that this was obtained by fusion of the two.

Therefore, it is suggested that the method of the present invention is not restricted by the DNA sequence introduced.

Effects of the Invention According to the present invention, it is easy to fuse a useful peptide or protein to the carboxy terminal side of DHFR. It is expected that peptides or proteins that are unstable in E. coli cells will be produced as fusion proteins, and the present invention aims at mass production of useful peptides or proteins by producing fusion proteins with DHFR. It is important to contribute.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the entire base sequence of pTP70-1, and the DNA strand sequence of one of the double-stranded DNA is
It is written in the direction from the 'end to the 3' end. The symbols in the figure represent nucleic acid bases, A is adenine, C is cytosine,
G represents guanine and T represents thymine. The number in the figure indicates the 15' ATCGAT of the C1al cleavage recognition site that is closer to the restriction enzyme HindlII cleavage recognition site among the two restriction enzyme CIaI cleavage recognition sites that exist in pTP70-i.
-3', indicates the number counted with the first "A" as number 1. FIG. 2 is a diagram showing the base sequence of the portion that exists in pTP70-1 and encodes HFR, and the amino acid sequence of the protein. The symbols in the figure represent nucleobases and amino acids, and A represents adenine. C stands for cytosine, G stands for guanine, and T stands for thymine. Ala is alanine, Arg is arginine, Asn is asparagine, Asp is aspartic acid, Cys is cysteine, Gln is glutamine, Glu is glutamic acid, Gly is glycine, I (is is histidine, Ile is Isoleucine, Leu for leucine, Lys for lysine, Met for methionine, Phe for phenylalanine,
Pro stands for proline and Ser stands for serine. Thr is threonine, Trp is tryptophan, T
yr represents tyrosine, and Val represents valine. The numbers in the figure indicate the numbers counted starting from "A" of the ATG codon that encodes the first amino acid, methionine. ATCGATGTTA ACAGATCTAA GCT
TAACTAAAACTTCCATGATCAGTCT
GAT TGCGGCGTTAllo 1
20 130CATGGAAAACGCCA
TGCCGT GGAACCTGCCAAACGCAAC
ACCTTAAATAAACCCGTGATTATC
AATCGGTCGTCCGTTGCCAGGACGC
AAAACCGGGTACG GACGATCGCG
TAACGTGGGTTCGCGGCGTGTGGT
GACGTA CCAGAAATCAGTTTATGA
ACAGTTCTTGCCAAAAGCGCAACTA
ACTCCGG AAAAGGAGGAGCGGTAG
ATCGCGTTATCGGTGCCGATCTCGC
CTGGTTTATGGGCCGCCA TACCTG
GGAAAATATTATATCCTCAGCAGTCAG
AAGTCGGTGGATGAAGCCATGGTG
ATTGG CGGCGGTCGCAAAACTGTAT
CTGACGCATATTGCAGGAGTCGCAT
AAGGGAAAACCCAGTCAGCTCCTTC
CGACTTATGACT GTCTTCTTTACG
CTCTGGGT CATTTTCGGClolo
1020 ATCGGCCTGT CGCTTGCGGTCTTC
GTCACT GGTCCCGCCAllo
1120 TCGCCGGCAT GGCGGCCGACACGC
GAGGCT GGATGGCCTTGAGCGTCG
ACCGATGCCCTT GAGAGCCTTCGT
GGGCGCGG GGCATGACTA TCGTC
GCCGCTCATGCAACT CGTAGGACA
G GTGCCGGCAGGAGGACCGCT TT
CGCTGGAG CGCGACGATGATTCGG
AATCTTGCACGCCCTCGCTCAAGCC
CAAACGTTT CGGCGAGAAG CAGG
CCATTAGCGCTGGGCT ACGTCTTG
CT GGCGTTCGCGCCCCATTATG A
TTCTTCTCCG CTTCCGGCGGldoHJ
ldodoU CATCGGGATG CCCGCGTTGC. ACCATCAGGG ACAGCTTCAA 1TC
GATCACTG GACCGCTGAT1CACAT
GGAACGGGTTGGCAT 1GCCTCCCC
GCGTTGCGTCGC1ATGGAAGCCG G
CGGCACCTC1GCCAATCAATTCTT
GCGGAG. No.l I and j AGGCCATGCT GTCCAGGCAG GTA
GATGACGGGATCGCTCCGCGCTCT
TACCAGCCTAACTCGTCACGGCG A
TTTATGCCG CCTCGGCGAGGGATT
GTAGG CGCCGCCCTA TACCTTGT
CTGGTGCATGGAGCCGGGCCACCT
CGACCTGAGCTAACGGATTCACCA
CTCCAAGAATTGGAAAACTGTGAAT
GCGCAAACCAACCCTTGGCA diagram
2 CGGGCAGCGT TGGGTCCTGGTTGA
GGACCCGGCTAGGCTGATCACCGAT
A CGCGAGCGAACGACCTGAGCAAC
AACATGACTGGAAACGCGGAAGTCA
GCCGCAGGATGCTGCTGGCTACAGC
GCTGGCA TTGACCCTGAACCGCCA
GTT GTTTACCCTCATCAGTAACCC
GTATCGTGACCACGGGTGCGCATGA
TCGT GCTCCTGTCGGCGGGGTTGC
CTTACTGGTT AGCAGAATGACGTG
AAGCGA CTGCTGCTGCAAACGTC
TGATGGTCTTCG GTTTCCGTGT T
TCGTAAAGTGCCCTGCACCATTATATG
TTCCGGATCTGCATCCTGTGGAACA
CCTACATCTGTATTAACGAGTGAT
TTTTCTCTGGTCCCG CCGCATCCA
TACAACGTTCCAGTAACCGGG CAT
GTTCATCGCATCCTCTCTCGTTTCA
TCGGTATCATTACCCCCATGAA CA
GAAAATTCCAGGAAAAAAACCGCCCTT
AACACGCTTCTGAGAAAACTCAAC
TGTGAATCGCTTCACGACCCGCGGT
TTCGG TGATGACGGTACGGTCACA
G CTTGTCTGTA 1st CCCTTACACG GAGGCATCAA GTG
ACCAAACATGGCCCGCT TTATCAG
AAG CCAGACATTACGAGCTGGACG
CGGATGAACAGGCAGACATACGCTG
ATGA GCTTTACCGCAGCTGCCTCG
GAAAACCTCT GACACATGCA GCT
CCCGGAGAGCGGATGCCGGGAGCAG
ACAAGCCCGTCAGCGGGTGTCG GG
GCGCAGCCATGACCCAGT diagram
3 --S A- CACGTAGCGA TAGCGGAGTG"AGA
TTGTACT GAGAGTGCAC1GTAAGG
AGAA AATACCGCAT +CTCGCTGC
GCTCGGTCGTTC1AGGCGGTAAT A
CGGTTATCCrATGTGAGCAAAAGG
CCAGCAΔGCTGGCGTTTTTCCATA
GGC'GACGCTCAAG TCAGAGGTGG
17R1n 7R7n DING ATACTGGCT TAACTATGCG G
CATCAGAGCCATATGCGGT GTGAA
ATACCGCACAGATGCCAGGCGCTCT
TCCGCTTCCT CGCTCACTGA:l;
GCTGCGGCG AGCGGTATCA GCTC
CAGAATCAG GGGATAA to ACTCAA
CGCAGGAAAGAAC HAGGCCAGGG AA
CCGTAAAAAGGCCGCGTTDingCCGCC
CCCCTGACGAGCAT CACAAAAAT
C: GAAACCCGA CAGGACTATA AA
GATACCAGGCGTTTCCCCCTGGAAG
CTC1GCTTACCGGA TACCTGTCCG
(CTCAATGCTCACGCTGTAGG“AA
GCTGGGCTGTGTGCACGA)ATCCG
GTAACTATCGTCTTGノCACTGGCAG
CAGCCACTGGTノGGTGCTACAG AG
TTCTTGAA (No. 1 CTCGTGCGCTCTCCTGTTCCGACC
CTGCC:CTTTCTCCCTTCGGGAAGC
GTGGCGCTTTTrATCTCAGTT CGGT
GTAGGT CGTTCGCTCC~CCCCCCCG
TT CAGCCCGACCGCTGCGCCTT~G
TCCAAACCCGGTAAGACACGACTTAT
CGC~ACAGGATTA GCAGAGCGAG
GTATGTAGGC3TGGTGGCCT AACT
ACGGCT ACACTAGAAG 4 GAGTTGGTAG CTCTTGATCCTTT
TTGTTT GCAAGCAGCAAGATCCT
TG ATCTTTTTCTACACGTTAAGG G
ATTTTGGTCATCCTTTTAA ATTA
AAAA DING GGTAAAACTTGG TCTGACAG
TTCAGCGATCTG TCTATTTCGTTA
GATAACTA CGATACGGA'IF, 1n
”(67nGGCAAACAAA
CCACCGC DINGGG TAGCGGTGGTGAT
TACGCGCAGAAAAAAAG GATCTCA
AGACGGGGTCTGA CGCTCAGTG
AACGAAAAACTATGAGATTAT CAAA
AAGGAT CTTCACCTAGAAGTTTTTA
AA TCAATCTAAA GTATATATGAA
CCAATGCTTT AATCAGTGAG GCAC
CTATCTTCATCCATAG TTGCCTGA
CT CCCCGTCGTGGGGCTTACCA T
CTGGCCCCCA GTGCTGCAAT"(6'(
n”(64(1F65(1GATACC)
GCGA GACCCACGCTAGCCAGCCGG
AAGGGCCGAGTCCATCCAGT CTA
TTAATTGAGTTAATAGT TTGCGCA
ACGCACGCTCGTCGTTTGGTATGAG
GCGAGTTA CATGATCCCCCGGTCC
TCCG ATCGTTGTCA 1st CACCGGCTCCAGATTTATCA GCAA
TAAACCCGCAGAAGTG GTCCTGCA
ACTTTATCCGCCTTGCCGGGAA GC
TAGAGTAA GTAGTTCGCCGAAGTA
AGTT GGCCGCAGTG TTATCACTC
Figure A 5 TGCTTTTCTG TGACTGGTGA GTA
CTCAAACCTATGCGGCGACCGAGTT
GCT CTTGCCCGGCCGCCATAG
CAGAACTTTTA AAAGTGCTCAGGGC
GAAAACTCTCAAGGAT CTTACCGC
TGACCCACTCGT GCACCCAACTG
ATCTTCAGCTTTCTGGGTG AGCAA
AAACA GGAAGGCAAAAGGGCGACA
CGGAAATGTTG AATACTCATATTG
AAGCATT TATCAGGGTT ATTGTC
TCAT7141 (1lL4.) (144'tnAAG
TCATTCT GAGAATAGTGGTCAACA
CGG GATAATAACCGTCATTGGAAA
ACGTTCTTCGTTGAGATCCA GTTC
GATGTAATCTTTTACT TTCACCAG
CGATGCCGCAAAAAAGGGAATACT
CTTCCTTTTTCAATATTAGAGCGGG
ATACATATTTGAATGTATTTAGAA
AAATAAAACAA ATAGGGGTTCGTGC
CACCTG ACGTCTAAGA AACCATT
ATTAAAATAGGCGT ATCACGAGGCC
CTTTCGTCTGCTCACAATTAATTC
TTGACAATTAGTTAAATAAGCTT CGCGCACATT TCCCCGAAAAATCA in Figure 1
TGACATTAACCTATAATCAAGAAT
TA ATTGTTATCCCCTATTGTTA T
AATGTATTCGGCGACACCCAnTCCC
GGATTACGG1yAspThrHisPhePr
oAspTyrGCAGATCTAA 1n11e Figure 2 Procedural Amendment November 30, 1988 1°.

Claims (1)

[Claims] 1. Utilizing the recombinant plasmid pTP70-1, which is stably replicated in E. coli and can confer trimethoprim resistance and ampicillin resistance to the host E. coli, the 3' end of the dihydrofolate reductase gene conferring trimethoprim resistance was A heterologous gene is introduced by matching the open reading frame of the code, a recombinant plasmid is created, the created recombinant plasmid is introduced into E. coli, and a protein in which the heterologous gene product is fused to the carboxy-terminal side of dihydrofolate reductase is produced in E. coli. A method for producing a fusion protein, characterized by producing a fusion protein.