JPH01144979A - Manifestation vector ptp104-4 - Google Patents

Abstract

PURPOSE:To produce a substance containing a heterogenic product fused to a carboxy-terminal of dihydrofolic acid reductase(DHFR), in high efficiency, by modifying a plasmid pTP70-1 capable of easily utilizing a DHFR gene as a genetic marker. CONSTITUTION:A terminator region of rrnB gene (one of ribosomal RNA genes) known as an efficient terminator is introduced at the downstream side of a DHFR gene of plasmid pTP70-1 to obtain a plasmid pTP104-4 capable of efficiently producing a substance containing heterogenic product fused to the carboxy-terminal of DHFR. The plasmid has a size of 4,466 base pairs and can impart a host E.coli with trimethoprim resistance and ampicillin resistance.

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 DHPR. ) Contains a modified gene,
By introducing a heterologous gene into the 3' end of the modified DHFR gene so that the open reading frame of the genetic code matches, the heterologous gene product is efficiently produced as a protein fused with DHFR and as a protein with DHFR activity. Recombinant plasmid pTP allows
104-4. 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
If the DHFR content in the facepiece is increased by incorporating the R gene into a plasmid, increasing the number of copies of the gene, etc., the bacteria will acquire resistance to trimethoprim (
M-IWakura ej al-yJ, Bioc
hemistry, vo191, P, 1205 (19
B2)) @Using this property, the DHFR gene is used as a selection marker for plasmids, and cloning of genes is actually carried out using plasmid vectors incorporating the DHFR gene (special training 1987-143)
520, M., Iwakura et al.
J. Biochemistry.

vol, 92. p. 615 (1982)). DHFR
Trimethoprim resistance markers using genes have a small gene size of about 500 base pairs, an easily accessible restriction enzyme site in the gene, and a very good correlation between gene expression level and strength of trimethoprim resistance. It is an excellent genetic marker due to the presence of interrelationships, and is expected to be widely used.

The present inventors have developed various vectors incorporating the E. coli DHFR gene (Patent No. 1369288
No. 1369291, Tokikai 1987-110999
．． Hours open: 59-135889° Hours open: 1982-13376
9. Unexamined publication 1984-184388, published in 1982-19
9385. Japanese Patent Publication No. 62-89990. Tokikai Showa 62-1
26984 etc.). The plasmid containing the modified DHFR gene is pTP70-1, which was created by the present inventors.
There is (special training Sho 6l-312836).

A method for expressing and producing a heterologous gene product as a protein fused with DHFR by introducing a heterologous gene in a manner that matches the frame is Bacillus subtilis DH.
A method using the FR gene.

The present inventors have conducted this work, and it has already been clarified that it is meaningful to create a fusion protein with DHFR (Special training 1986-234003. Patent application 1982-249260゜Special training 1982-07937)
7. Special training 1986-079378, Special training 1986
2-085406゜special training 1986-092881, etc.)
.

Problems However, D) By introducing a heterologous gene into the 3' end of the IFR gene of Bacillus subtilis by aligning the open reading frame of the genetic code, attempts are made to express and produce the heterologous gene product as a protein fused with DHFR. In this method, the gene expression efficiency is not so high, and the amount of fusion protein produced within the bacteria is at most a few percent of the bacterial protein, so the solution must be to improve the expression efficiency and increase the production amount. remained as an issue. The present inventors have created a silk recombinant plasmid pTP64-1 that produces large amounts of E. coli DHFR (Tokikai
(Sho 62-69990). In E. coli having pTP64-1, DHFR is produced as about 15% or more of the bacterial protein. However, it was completely unknown whether the DHFR gene of E. coli would be expressed as a fusion protein without losing the DHFR enzyme activity when a heterologous gene was introduced into the 3' end of the gene.

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 found that the DHFR gene of pTP70-1, which was developed for the purpose of using the present invention, has no enzymatic activity even if the 3'-terminal sequence of the gene is artificially modified. Approximately 2 of the bacterial cell proteins contain modified DHFR with
Focusing on the fact that pTP70 is expressed and produced at around 0%
-1 by modifying the new recombinant plasmid pT
By constructing pTP104-4 and utilizing pTP104-4, it was revealed that the above problems could be solved, and the present invention was completed.

Structure of the Invention The present invention provides (1) pTP10 modified from pTP70-1.
4-4. (2) E. coli containing pTP104-, and (3) DHP by utilizing pTP104-4.
Method 3 of creating a fusion protein between R and a heterologous gene product
It consists of two inventions.

(1) pTP104 pTP104-4 contains rrn8 (known as an efficient terminator) downstream of the DHFR gene of pTP70-1 in order to increase the production efficiency of DI-IFR.
This is a recombinant plasmid obtained by introducing the terminator region of one of the ribosomal RNA genes, and is a new recombinant plasmid. FIG. 1 shows the entire base sequence of pTP104-4 of the present invention. pTP104-4 has a size of 4466 base pairs and can confer trimethoprim resistance and ampicillin resistance to host E. coli. pTP10
4-4 is restriction enzyme Aat II, BamHI
.

BclI, BglII, C1aI, EcoRI.

HindI TI, HpaI, Pstl, PvulI,
1 (431B-4
323), 1(532-537), 1(58-63
), 2(13-18,538-543), 2(1-
6,545-550), 1(471-478), 2
(19-24, 4481-4466), 2 (7-12
, 4434-4439), 1 (3641-3646)
, 1 (1822-1827), and 1 (825
-830) (The cleavage recognition site of each restriction enzyme in FIG. 1 is shown in parentheses.

).

Terminator-D introduced into pTP104'7-'4
NA is the terminator region of the rrrIB gene. A plasmid containing the terminator region of the rrnB gene was described by Brosius et al.
t, at,, J, Mo1Bio1. vol, 14
8. p. 107 (1981)).
A plasmid containing the terminator region of the rrnB gene can be purchased from Pharmacia.

For the construction of pTPl 04-4, plasmid pKK17
5-6 is used. The sequence from 1600th to 1824th in FIG. 1 is the sequence containing the terminator region of the rrnB gene.

By introducing this sequence, we were able to increase the production amount of modified DHFR in E. coli by 1.0 to 1.4 times.
(See section on bacteria).

(2) Large intestine surface containing pTP104-4.

The above pTP104-4 can confer trimethoprim resistance and ampicillin resistance to host E. coli;

It was introduced into E. coli strain C600 and kept stable.

ε, coli C800 containing pTP104-4
The stock is.

It has been deposited with the Institute of Fine Technology as FERM8P-1579.

E. coli containing pTP104- produces large amounts of modified DHFR. E. coli containing pTp 104-
YT+Ap medium (+58 NaCl 1,8 g in medium 11
tryptone 8.5g yeast extract, and 5mg
The specific activity of DHFR in the cell-free extract obtained from bacterial cells cultured from the late logarithmic growth phase to the stationary phase in a medium containing ampicillin sodium is approximately 9 to 10 units)/
The value is mg protein. The value of E. coli containing pTP70-1 is about 7 to 9 units/mg protein, and the use of E. coli containing pTP104-4 reduces the production of DHFR to 1. It can be increased from 0 to 1.4 times and is suitable for producing modified DHFR.

(3) DHF by using pTP 104-4
Method for creating a fusion protein between R and a heterologous gene product Figure 2 shows the amino acid sequence of DHFR produced by DHFR of Kawa p'rp 104-4. pTP 1
04-4, the only restriction enzyme Bam) (1 site (
The array (532nd to 537th array in Figure 1) 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 cohesive ends (Cohe
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, DNA is introduced between the 532nd and 533rd positions in Figure 1, and the sequence is δ'-G (522nd)G.
ATC-(heterogeneous DNA)-GATCC(537th)-
They will have a common array called 3'. Therefore, heterogeneous D
Two fusion proteins are created by introducing NA.

Among the amino acid sequences of DHFR shown in FIG.

The amino acid sequence from the first methionine (Net) to the 160th isoleucine (Ile) is completely common, and 1
A fusion protein is produced that has an amino acid sequence derived from the foreign DNA starting from the 60th position.

The present invention utilizes this feature to demonstrate a method for creating a fusion protein between DHFR and a heterologous gene product. The production of the fusion protein consists of (A) production of a fusion gene, that is, production of a recombinant plasmid having a gene to which a heterologous gene is linked to the 3' end of the DHFR gene of pTP104-4; (B) production of a recombinant plasmid. Introduction to E. coli,
(C) It can be performed by detecting a fusion protein, which is a fusion gene product expressed in E. coli.

(A) Creation of fusion gene A method for creating a fusion gene with the DHFR gene using the BamH1 site of pTP104-4.

■A method of creating a fusion gene by aligning the open reading frame of the DHFR gene using the cohesive ends generated by cutting with BamHI, ■After cutting with BamHI, D
The ends are made blunt using NA polymerase or reverse transcriptase, the open reading frame of the DHFR gene is aligned, and the heterologous DNA is
A method for creating a fusion gene by ligating A by plant end ligation, ■ After cutting with BamHI,
Make blunt ends using a nuclease that cleaves DHF.
A method of aligning the open reading frames of the R gene and joining the heterologous DNA by plant end ligation to create a fusion gene; ■ After cutting with BamHI, the ends are made blunt using an exonuclease such as exonuclease BAL31, and the heterologous DNA is Methods such as a method of linking by plant end ligation to create a fusion gene can be performed. These methods can be easily performed by anyone who is involved in DNA recombination without any problems. Example 3 shows that it is possible to create fusion proteins with various molecular weights by utilizing the sticky ends generated by cleavage with BamH and the DNA fragments obtained by cleavage with Escherichia coli chromosomal DNA 2Sau3AI. ing.

(B) Introduction of recombinant plasmid into E. coli The recombinant plasmid produced in (A) can be introduced into E. coli cells by a so-called transformation method. Various methods are known for the transformation of alternative plasmids, but the p'
rpi.

The recombinant plasmid produced using 4-4 can be easily introduced into Escherichia coli by those involved in the 2 recombinant DNA process without any problems.

It does not depend on the method of transformation.

E. coli into which the recombinant plasmid created using pTPI 04-4 has been introduced can be easily detected using an agar medium. p'rp 104-4 has ampicillin resistance and trimethoprim resistance as genetic markers. Ampicillin resistance.

Since no manipulation is performed when creating the fusion gene with the DHFR gene, the 2M recombinant plasmid can also carry over the gene as it is. In addition, the DHFR gene, which is a gene that confers trimethoprim resistance, loses DHFR enzyme activity 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 (A) above. Because it is not, it has the ability to confer resistance to Trimedobrum. Therefore, E. coli into which the recombinant plasmid created using pTP104-4 was introduced,
Resistant to ampicillin and trimethoprim. If a medium containing ampicillin and trimethoprim is used as an agar medium for E. coli used for transformation, the transformed strain growing on this medium will be pT.
This is E. coli into which a recombinant plasmid made using P104-4 has been introduced.

(C) Detection of fusion protein, which is a fusion gene product expressed in E. coli pTP104-4 can be produced in modified DHFR Odo. E. coli containing pTP104-4-4,
Hedrons cultured in YT+Ap medium from the late logarithmic phase to the stationary phase were subjected to SDS polyacrylamide gel electrophoresis (
Hereinafter, it will be abbreviated as 5DS-PAGE. ) in the sample preparation solution (see Example 3).
When the protein is stained with Coomassie brilliant blue, the most obvious band (main band) is shown at a molecular weight of about 20,000.

E. coli containing the recombinant plasmid created using pTP104-4 can produce a large amount of DHFR fusion protein.2 The produced fusion protein is pTP1.
It can be detected using 5DS-PAGE in the same manner as the modified DHFR produced by E. coli containing 04-4. The 5DS-PAGE method can be easily performed according to Laemmli's method. In 5DS-PAGE, two proteins with a small molecular weight have a high electrophoresis, and two proteins with a large molecular weight have a small electrophoresis.

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.

Approximately 1 μg of pTP70-1 (Special training Sho 6l-312
836) was digested with 5alI and PvuII, 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 the precipitated DNA
After washing with % ethanol, the ethanol was removed and the precipitate was dried under reduced pressure. The operations of DNA cleavage with restriction enzymes, alkaline phosphatase treatment, phenol treatment, and ethanol precipitation are all carried out using ``Molecul.
arCloning A Laboratory M
annual” (T, Maniatis.

E., F., Fr1tsch, J., Sambrook, ed.
s, Cold Spring Harbor Labo
(1982), hereinafter referred to as Reference 2.

) according to the method described. pK of approximately 1μ8
K175-6 (purchased from Pharmacia).

After cutting with 5all and PvuII, the DNA was precipitated with ethanol. After washing the precipitated DNA with 70% ethanol, remove the ethanol.

The precipitate was dried under reduced pressure.

Dried DNA (pTP70-1 and pKK17
5-6 cut with restriction enzymes), 50
Reaction solution for μ+ glue gauze (10mM Tris-HCI
, p)I 7.4.5mM MgCl2.10mM
After dissolving in dithiothreitol, 5mM ATP)
Both were combined, 10 units of T4-DNA ligase was added thereto, and the DNA ligation reaction was carried out at 25°C for 4 hours. This reaction product was transformed using the transformation method.
tion method (described in Document 1 above).

The treated cells were grown in a nutrient universal medium containing 50 mg/l ampicillin sodium and 10 mg/l trimethoprim (y 2 g of glucose in medium 11, 1
g dipotassium phosphate t 5 g yeast extract +
Contains 5 g polypeptone, 15 g agar. ) and cultured at 37°C for 24 hours, approximately 50 colonies could be obtained. Select 8 colonies from these colonies and add 1.5 ml of YT+Ap medium (medium 11
y5g of NaCl and 5g of yeast were cultured in it. Transfer the culture solution to each Etzpendorf centrifuge tube and incubate at 12,000
The mixture was centrifuged at 0 rpm for 10 minutes, and the bacterial cells were collected as a precipitate. to this.

0.1ml electrophoresis sample preparation solution (0,0625
M Tris-HCI, pH 6, 8, 2% sodium lauryl sulfate (SDS), 10% (7) glycerin, 5x 2-mercaptoethanol, 0.001%
Contains bromophenol blue. ) was added to suspend the dihedron, and this was kept in boiling water for 5 minutes to dissolve the bacterial cells.

This treated sample was subjected to 5DS-polyacrylamide gel electrophoresis (U, Lamm1i; Natur
e, vol, 227. p., 680 (1970)). As a standard sample, E. coli containing pTP70-1 was treated in the same way, and as a molecular weight marker, lactalbumin (molecular weight 14,200
), ) ribsin inhibitor (molecule ffi 2
0,100),) Ribsinogen (molecule fi24,00
0), carbonic anhydrase (molecular weight 29.0
00), glyceraldehyde 3-phosphate dehydrogenase (molecular weight 36,000), egg albumin (molecular weight
f145,000) and bovine serum albumin (molecular weight 66,000) were run on a 10 to 20% polyacrylamide gradient gel. As a result, among the eight colonies examined, the colonies were pTP
It was revealed that the protein produced is approximately the same size as DHFR 70-1.

These five bacterial cells were cultured in YT+Ap medium, and Tana
ka and Weisblum's method (T, Tanaka,
B, Weisblum; J.

Bacteriology, vol, 121. p, 3
54 (1975)).

A plasmid was prepared. When each of the obtained plasmids was attempted to be cleaved with the restriction enzyme AvaI, none of the plasmids was cleaved with AvaI. Also,
When the plasmid was cut with EcoRI and analyzed by agarose gel electrophoresis, only one site was cut in each case, and the molecular size of the plasmid was approximately 4.5 kilobase pairs, which was slightly smaller than pTP70-1. It became clear.

Select one appropriately from the five plasmids obtained and transform it into pTP.
It was named 104-4.

The larger of the two DNA fragments obtained by 5ail and PvulI digestion of pTP70-1 and pKK
It should have a structure in which the smaller of the two DNA fragments obtained by cutting 175-6 with Sal I and Pvu II are joined together. pTP104-4 5a
When cleaved with IL and PvuII, two DNA fragments were obtained as expected, and the larger DNA fragment was

The larger and smaller DNA fragments of the two DNA fragments obtained by 5alI and pvull digestion of pTP70-1 are pKKl 75-6.
It completely matched the smaller of the two DNA fragments obtained by digestion with 5ail and PvulI.

Since the complete nucleotide sequences of pTP70-1 and pKK175-6 have already been determined, the entire nucleotide sequence of pTP104-4-4 was determined using the results as shown in FIG.

Example 2 DHFRHF type of E. coli containing pTP104-4 E. coli C600 containing pTP104-4
E. coli C600 containing pTP70-1 and pTP70-1.
and after overnight culture in 50 ml of YT+Ap medium, respectively.

Bacterial cells were collected by centrifugation. The facepiece was prepared with 1 containing 0.1 mM disodium ethylenediaminetetraacetate (EDTA).
After washing with 0 mM phosphate buffer pH 7.0 (hereinafter referred to as buffer 1), the suspension was suspended in 2 ml of buffer 1 and sonicated. The sonicated bacterial cell fluid was centrifuged at 20,000 rpm for 1 hour to obtain a supernatant. The DHFR enzyme activity and protein amount were measured for the obtained supernatant. From the measured enzyme activity and protein amount, 1 mg of supernatant protein was obtained.
DHFR enzyme activity (specific activity (units/Bpr)
otein)) was calculated. This value is an amount proportional to the amount of DHFR produced within the bacterial body. As a result, pTP 1
In E. coli C600 containing 04-4, 9
The values of ゜02.9.85 and 9.90 (tested 3 times) are
pTPj7. E. coli containing cy, -1
In C600.

Values of 7.05, 8.20, and 8.95 were obtained.

In both cases, the bacterial cells containing pTP104-4 had a higher DH
FR production exceeded that amount.

Example 3 Creation of fusion protein Approximately 1 μg of pTP104-4-4 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. After washing the precipitated DNA with 70% ethanol, the ethanol was removed and the precipitate was dried under reduced pressure.

Approximately 5 μg of E. coli chromosomal DNA was cut with 5au3AI, and then 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 mixed with a 50 μm ligase reaction solution (10 mM Tris-HCI, pi (7, 4,
5mM MgCl2゜10mM dithiothreitol, 5
After dissolving in mM ATP), the two were combined, 5 units of T4-DNA ligase was added thereto, and the mixture was incubated at 25°C.
The DNA ligation reaction was allowed to proceed for 4 hours. This reactant is
Transformation method
od, described in the above-mentioned document 1).

Incorporated into E. coli. Bacterial cells that have undergone this treatment.

50 mg/I ampicillin sodium and 10 mg
Nutrient agar medium containing /l trimethoprim (medium 11
Inside + 2g glucose + Ig dipotassium phosphate + 5g yeast extract + 5g polypeptone, 1
Contains 5g of agar. ) and cultured at 37"C for 24 hours, approximately 500 colonies were obtained. Approximately 20 colonies were selected from these colonies and 1
．． 5 ml of YT+Ap medium (5 g of Na in medium 11)
C1, 5g yeast extract, 8g tryptone, 50
Contains mg of ampicillin sodium. ) at 37°C
, the bacterial cells were cultured overnight.

Transfer the culture solution to each Eppendorf centrifuge tube and incubate at 12.0
Centrifuge for 10 minutes at 00 rpm and 1M sodium lauryl sulfate (SDS), 8.2x of sodium lauryl sulfate (SDS), 5x of glycerin, 5z of 2-mercaptoethanol.

o, ootx bromophenol blue. ) was added to suspend the dihedron, and this was kept in boiling water for 5 minutes to dissolve the bacterial cells. This treated sample was subjected to 5DS-polyacrylamide gel electrophoresis (U, Lamm1i;
Nature, vol, 227. p, 680 (1
970)). pT as a standard sample
Escherichia coli containing P104-4 was treated in the same way, and the molecular weight markers were lactalbumin (molecular fi 14.200), ) ribsin inhibitor (molecular weight 20.100), and ) ribsinogen (molecular f 124).
,000), carbonic anhydrase (molecule!2
9,000), glyceraldehyde 3-phosphate dehydrogenase (molecular weight 36,000), egg albumin (
Samples containing bovine serum albumin (molecular weight 45,000) and bovine serum albumin (molecular weight 66,000) were run on a 10 to 20% polyacrylamide gradient gel. As a result, out of 20 colonies examined, 18 had pT
The DHFR band of P104-4 disappeared, and a protein with a clearly larger molecular weight was newly produced (the position of the new protein band in each bacterial cell was different). By comparison with the migration density of molecular weight marker proteins, the molecular weight of those proteins was between about 23,000 and about 35,000. Under these conditions, the DHPR of p'rp 104-4 (molecular weight 18,379) was
0 protein. Therefore.

By fusing the S au 3 A I cut fragment of E. coli chromosomal DNA, a fusion protein is produced in which two peptides or proteins with a molecular weight of about 3,000 to about 15,000 are fused to the carboxy terminal side of FHFR. It was shown that it was done. Since all E. coli strains that produce the fusion protein are trimethoprim resistant, it is clear that the fusion protein has DHFR activity.

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

The estimated amount of protein d produced was comparable to or greater than the DHFR band of pUni1 TP 104-4, indicating that the fusion protein was stably produced.

Furthermore, in this example, the chromosomal DNA of E. coli was
The DNA fragment obtained by cutting with h I is used to create a fusion gene, 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. In the production of unstable peptides or proteins in E. coli cells, it is expected that they will be produced as '#11 conjugated proteins.2 The present invention aims at producing useful peptides or proteins by producing fusion proteins with DHFR. It is important to contribute to mass production.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the entire base sequence of pTP104-4, and the DNA strand sequence of one of the double-stranded DNA is
It is written in the direction from the 5' end to the 3' end. The symbols in the figure represent nucleic acid bases: A is adenine, C is cytosine, and G is guanine. T indicates thymine. The numbers in the figure are pTP104-
Of the two restriction enzyme C1al cleavage recognition sites present in 4, C1aI is the one closer to the restriction enzyme HindIII cleavage site.
The numbers are shown starting from the first "A" of the cleavage recognition site, 5'-ATCGAT-3', as number 1. FIG. 2 is a diagram showing the base sequence of the portion encoding DHFR and the amino acid sequence of the protein present in pTP104-4. The code in the figure is. Represents a nucleobase and an amino acid; A represents adenine. C stands for cytosine, G stands for guanine, and T stands for thymine. sn stands for asvagine, Asp stands for aspartic acid, C
ys is cysteine, Gin is glutamine, Glu is glutamic acid, cty is glycine,) (is is histidine, Ile is isoleucine, Leu is leucine, Lys is lysine, Met is methionine, Phe is phenylalanine) of,
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. ]0 20 ATCGATGTTA ACAGATCTAAAACTT
CCATGA TCAGTCTGATllo
120 CATGGAAAACGCCATGCCGTAACGC
AACACCTTAAATAAATCAAATCGGTC
GTCCGTTGCCACCGGGTACG GACG
ATCGCGTCGCGGCGTGTGGTGACG
TAGTTTATGAACAGTTCTTGCC7i1
n d2n GCTTAACTAA CTAACTCCGG AAA
AGGAGGATGCGGCGTTA GCGGTAG
ATCGCGTTATCGGGGAACCTGCCTG
CCGATCTCGCCTGGTTTACCCGTGA
TTA TGGGCCGCCA TACCTGGGAA
AGGACGCAAAAAATATTATCCTCAGC
AGTCATAACGTGGGTGAAGTCGGT
G GATGAAGCCACCAGAAATCA T
GGTGATTGG CGGCGGTCGCAAAA
GCGCAA AAAACTGTATCTGACGCAT
ATdT) din
d', (1CGACGCAGAA GTGGAAGGC
GACTGGGAATCGGTATTCAGCCACA
GCTATG AGTTCGAAATGATCCTCT
ACGCCGGACGCACGGTTGCTGG CG
CCTATATCCGCCACTTCGGGCTCA
TGAGCCCGTGGCCGGGGGACTGTT
TGCGGCGGCG GTGCTCAAACG No.
] ACACC, CATTT CCCGGATTACGAG
CCGGATGGAATTCCACG ATGCTGA
TGCGCAGAACTCTTCTGGAGCGG C
GGATCCAGA TCTAATCGATTCGTG
GCCGG CATCACCGGCGCCACAGGT
GGCCGACATCA CCGATGGGGA AG
ATCGGGCTCGCTTGTTTCGGCGTGG
GTA TGGTGGCAGGGGGCGCCATCT
CCTTGCATG CACCATTCCTGCCTC
AACCTACTACTGGGCTGCTTCCTA
AL Figure l TGCAGGAGTCGCATAAGGGA GAGC
GTCGACAACCCAGTCAGCTCCTTC
CG GTGGGCGCGGACTTATGACT G
TCTTCTTTA TCATGCAACTCGCTC
TGGGT CATTTTCGGCGAGGACCGC
Tlolo 1020 1030A
TCGGCCTGT CGCTTGCGGT ATTC
GGAATCCTTCGTCACT GGTCCCGC
CA CCAA, A, CGTTTllo
1120 1130TCGCCGGCAT G
GCGGCCGACGCGCTGGGCTACGCGA
GGCT GGATGGCCTT CCCCATTAT
GCGATGCCCTT GAGAGCCTTCGGC
ATGACTA TCGTCGCCGCCGTAGGA
CAG GTGCCGGCAGTTCGCTGGAG
CGCGACGATGTTGCACGCCCTCGCT
CAAGCCGGCGAGAAG CAGGCCATT
AACGTCTTGCT GGCGTTCGCGATT
CTTCTCCGCTTCCGGCGGCATCGGG
ATG CCCGCGTTGCAGGCCATGCTA
CCATCAGGG ACAGCTTCAA GGAT
CGCTCGTCGATCACTG GACCGCTG
AT CGTCACGGGCGCACATGGAACGG
GTTGGCAT GGATTGTAGGGCCTC
CCGCGTTGCGTCGCGGTGCATGGAA
TGGAAGCCG GCGGCACCTCGCTA
ACGGATGCCAATCAATTCTTGCGG
AG AACTGTGAATFigure GTCCAGGCAG GTAGATGACGCGGC
TCTTACCAGCCTAACTATTTATGCC
G CCTCGGCGAGCGCCGCCTA TA
CCTTGTCTGCCGGGCCACCTCGACC
TGATCACCACTCCAAGAATTGGAGC
GCAAACCA ACCCTTGGCA 2 CGGCTGTTTT GGCGGATGAGCAGA
ACGCAG AAGCGGTCTGGCGGTGGT
CCCACCTGACCCCCGCCGATGGT AG
TGTGGGGTCATCAAATAAAACGAA
AGGC1860187O CTGTTGTTTG TCGGTGAACGCGGA
TTTGAA CGTTGCGAAGCCCGCCATA
AA CTGCCAGGCAsn1n
Qnsu^ AGAAGATTTT CAGCCTGATA CAG
ATTAAATATAAAAACAGA ATTTGCC
TGG CGGCAGTAGCCATGCCGAACT
CAGAAGGTGA AACGCCGTAGCTCCC
CATGCGAGAGTAGGG AACTGCCAG
GTCAGCTGAAA GACTGGGCCT TT
CGTTTTATCTCTCCTGAG TAGGAC
AAAT CCGCCGGGAGCAACGGCCCG
GAGGGTGGCG GGCAGGACGCTCA
AATTAAG CAGAAGGCCA TCCTGA
CGGArin'>n/)nAn
/) ncn1 = 2, υ heat υ 4 = 2 1% JLυ TGGCCTTTTT GCGTTTCTACATCC
CCCCACAGATACGGTACTGCCTCGC
G CGTTTCGGTGTCCCGGAGACGGT
CACAGCTGCCCGTCAGGGCGCGTC
AGCGACCCAGTCA CGTAGCGATAA
TCAGAGCAG ATTGTACTGA 1st LUJU Lυ1v
LυJυAAAACTCTTCCTGT
CGTCATA TCTACAAGCCAACTAG
CTCGTTTTGCATCAGGAAAGCAGAT
GACGGTGA AAACCTCTGA CACAT
GCAGCTGTCTGTAAG CGGATGCCG
G GAGCAGACAAGGGGTGTTGGCGGGG
TGTCGGG GCGCAGCCATGCGGAG
GTA TACTGGCTTA ACTATGCGGC
GAGTGCACCA TATGCGGTGT GAA
ATACCGC diagram 3 CTCACTGACT CGCTGCGCTCTCAC
TCAAAGGCGGTAATACGAAAGAAC
AT GTGAGCAAAAGCCGCGTTGCTG
GCGTTTTTCAAAAAATCGA CGCTCA
AGTCGATACCAGGCGTTTCCCCTA
CCCTGCCGCTTTACCGGATAGGCGCT
TTCT CAATGCTCACGGTCGTTCGG
CTGCGGCGAG CGGTATCAGCGGT
TATCCACAGAATCAGGG GATAACG
CAGGGCCAGCAAA AGGCCAGGAA
CCGTAAAAAAGCCATAGGCTCCGCC
CCCTG ACGAGCATCAAGAGGTGGC
G AAACCCGACA GGACTATAAAAGG
AAGCTCCCTCGTGCGCTCTCCTGTT
CCGCCTGTCCGCCTTTTCTCCCTTC
GGGAAGCGTGCTGTAGGTATCTCA
GTTCG GTGTAGGTCGdo81L)
1!8doυTTCGCTCCAA GCTG
GGCTGTTGCGCCTTAT CCGGTAA
CTACTTATCGCCA CTGGCAGCAG
ATGTAGGCGG TGCTACAGAGACT
AGAAGGA CAGTATTTGGCGGAAA
AAGA GTTGGTAGCTGCGGTGGTT
T TTTTGTTTGC No. l Do8jU Doδ4U Dobb
uGTGCACGAACCCCCCGTTCA GCC
CGACCGCTCGTCTTGAGTCCAACC
CGG TAAGACACGACCACTGGTAA
CAGGATTAGCAGAGCGAGGTTTCTT
GAAGT GGTGGCCTAA CTACGGCT
ACTATCTGCGCTCTGCTGAAGCCA
GTTACCTTCTTGATCCGG CAAACA
AACCACCGCTGGTAAAGCAGCAGA
TTACGCGCAG AAAAAAAGGAFigure 4 CGAAAACTCA CGTTAAGGGATCAC
CTAGAT
GT AAAACTTGGTCACCTATCTCAG
CGATCTGTCCCGTCGTA GATAA
CTACGGCTGCAATGA TACCGCGAG
AAATAAAACCAG CCAGCCGGAATAT
CCGCCTCCATCCAGTCT'''2r:%1n
'282nTTTTGGTCAT G
AGATTATCA AAAAGGATCTTAAAA
ATGAA GTTTTAAATCAAATCTAAAG
TTGACAGTTACCAATGCTTAATCA
GTGAGGCTATTTCGTTCATCCATAG
TT GCCTGACTCCATACGGGAGG G
CTTACCATCTGGCCCCAGTCCCACCG
CTCA CCGGCTCCAG ATTTATCAG
CGGGCCGAGCG CAGAAGTGGT CC
TGCAAACTTATTAATTGTTGCCGGG
AAGCTAGAGTAAGTAGTTCGCCAG
TTAATAGTTTCGTGGTGTCA CGCT
CGTCGTAACGATCAAGGCGAGTTA
CAAGCTCCTTCG GTCCTCCGATAT
CACTCATGGTTATGGCAGCCGTAA
GATG CTTTTCTGTGGAATAGTGTA
TGCGGCGACC 1st GCGCAACGTT GTTGCCATTG CTG
CAGGCATTTGGTATGGCTTCATTCA
GCTCCGGTTCCCTGATCCCCCA TG
TTGTGCAA AAAAGCGGTTCGTTGT
CAGA AGTAAGTTGG CCGCAGGTGT
TCACTGCATAA TTCTCTTACT GT
CATGCCATACTGGDGINGGAGTACTCA
ACCAA GTCATTCTGAGAGTTGCT
CT TGCCCGGCGT CAACACGGGAFigure 5 GTTCTTCGGG GCGAAAACTC-TCG
ATGTAACCCACTCGTGCノCACCAGC
GTT TCTGGGTGAG (AGGGATAA
G GGCGACACGG) CAATATTATTG
AAGCATTTA”ATTTGAATGT ATTT
AGAAAAANOCCCGAAAAGGT GCCACCT
GAC・(ACCTATAAAAATAGGCGTA
T (TGTTATCCGCTCACAATTAA 1
rCAAGGATCT TACCGCTGTT GAG
ATCCAGT\CCCAACTGA TCTTCAG
CATCTTTACTTT:AAAAAACAGG
AAGGCAAAAT GCCGCAAAAA\AAT
GTTGAA TACTCATACT CTTCCTT
TTTrCAGGGTTA TGTCTCATGA
GCGGATACAT\TAAACAAAT AGGG
GTTCCG CGCACATTTC;TCTAAGA
AA CCATTATTAT CATGACATTA:
ACGAGGCCCTTTCGTCTTCAAGAAT
TAATrTCTTGACAA TTAGTTAACT
ATTTGTTATA'JrL+OU')')
00 Figure l 6

Claims (1)

[Scope of Claims] 1. It is stably replicated in E. coli and can impart trimethoprim resistance and ampicillin resistance to the host large intestine. By introducing a heterologous gene with matching reading frames, the heterologous gene product can be efficiently expressed as a protein fused with dihydrofolate reductase, which has a size of 4466 base pairs and is shown in FIG. Expression vector pTP104-4 with the DNA sequence. 2. Expression vector pTP10 according to claim 1
E. 4-4. coli strain C600. 3. Using pTP104-4 described in claim 1, a heterologous gene is ligated to the 3' end of the dihydrofolate reductase gene that confers trimethoprim resistance by aligning the open reading frame of the genetic code to create a recombinant plasmid. A method for producing a fusion protein, which comprises introducing the produced recombinant plasmid into Escherichia coli, and causing the E. coli to produce a protein in which a heterologous gene product is fused to the carboxy terminal side of dihydrofolate reductase.