JPH02142481A - Novel recombinant plasmid pddm1 - Google Patents

Abstract

NEW MATERIAL:A recombinant plasmid pDDM1 having a DNA sequence expressed by the formula. USE:For producing dihydrofolate reductase. PREPARATION:For example, a plasmid pTP64-1 is cleaved with restriction enzymes BclI and BglII and then subjected to treatment with an alkaline phosphatase and phenol to denature and remove coexisting enzymic proteins. The resultant DNA is subsequently precipitated with ethanol and linked to a plasmid pTP70-1 cleaved with the restriction enzyme BglII using a T4-DNA ligase, inserted into Escherichia coli and the obtained transformant is then cultured to select a strain capable of producing dimer of dihydrofolate reductase(DHFR) from the formed colony. Thereby, the objective recombinant plasmid pDDM1 is obtained.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a protein (hereinafter referred to as DHFR) in which two molecules of dihydrofolate reductase (hereinafter referred to as DHFR) are linked together.
pDDM1) and a novel recombinant plasmid that enables its production.

The novel recombinant plasmid pDDM1 of the present invention is.

It has the DNA sequence shown in FIG. DHFR
The dimer has the amino acid sequence shown in FIG. pDDMl, Escherichia coli containing pDDMl, and DHFR dimer are suitable for fields such as fermentation industry and pharmaceutical industry.

[Prior Art and Problems] DHPR produced by Escherichia coli is a monomeric enzyme having one enzymatic activity unit in a single polypeptide chain consisting of 159 amino acids. Already 9 the inventors have 9
We have established a method for mass expression and production of DHFR in Escherichia coli using recombinant DNA technology (patented in 1982-
135889. JP-A-Sho (32-69990, patent Sho 62-302152). The DHPR dimer of the present invention is
Unlike the above-mentioned monomeric HPR, it is a completely new protein having two enzymatically active units in one polypeptide chain, and is a substance that was unknown until the completion of the present invention. Furthermore, plasmid pDDM1 encoding DHFR dimer is also a novel Mi-transforming plasmid.

As the basic technology for developing the DHFR dimer of the present invention, there are nine methods for producing fusion proteins invented by the present inventor (Japanese Patent Application No. 302153/1982).

[Objective of the Invention] The DHF R dimer of the present invention is a protein having two enzymatic activity units expressing DHFR enzyme activity in its polypeptide chain, and is a completely unknown substance until the present invention. Met.

As a result of intensive research aimed at creating a novel protein, DHFR dimer, the present inventors utilized the fusion protein production method developed by the present inventors to create two molecules of DHFR by linking the DHPR genes. A recombinant plasmid p D D based on the idea of combining
By constructing M1 and expressing pDDMI in Escherichia coli, we successfully isolated and purified the DHFR dimer from Escherichia coli containing pDDMI, leading to the completion of the present invention.

[Configuration of the Invention] The present invention provides (1) a novel recombinant plasmid pDDMl, (
It consists of 2) E. coli containing pDDMI, (3) DHPR dimer, which is a novel enzyme protein encoded by pDDMI, and (4) a method for separating and purifying DHFR dimer from E. coli containing pDDMI.

(1) New recombinant plasmid pr'lDM] Figure 1 shows 9
The entire base sequence of pDDM1 of the present invention is shown. The diagram is
One of the DNA strand sequences of the double-stranded circular DNA is cut at the cleavage recognition site that is closer to the restriction enzyme H1ndm site among the two restriction enzyme CIaI sites present in the plasmid,
5'-ATCGAT-3°, counting the first "A"' as number 1 and writing in the direction from the 5' end to the 3' end. pDDMI of the present invention:,t, is a novel recombinant plasmid. pI)DMI has a size of 5779 base pairs and can confer trimethoprim and ampicillin resistance to the host E. coli. pDDMl.

As shown in the examples, between the restriction enzyme BglIr and Bc11 sites of pTP64-1 (described in Tokikai 62-69990) prepared by the present inventors, It can be produced by inserting a DNA fragment of about 500 base pairs obtained by cutting pTP70-1 (Japanese Patent Application No. 61-312836) developed by et al. with the restriction enzyme Bgln. However, two different methods are possible for producing a recombinant plasmid whose sequence has been clarified, and therefore the present invention is not limited to the two methods for producing a recombinant plasmid.

The sequence encoding the DHFR dimer is the sequence from 57th to 1013th @ in FIG.

Upstream of the sequence encoding the DHF R dimer.

There are sequences that allow efficient gene expression (Tokikai 1986-69990). That is, the sequence from position 43 to position 50 is called the SD sequence and contributes to efficient translation, and sequence from position 5737@ to position 5765e is a consensus transcription promoter and contributes to efficient transcription. From this, when pDD41 is introduced into E. coli, it is possible to make a large amount of DHFR dimer. The produced DHFR dimer accumulates in a soluble state within the bacterial body, accounting for about 20% of the bacterial protein.

This makes E. coli containing pDDMl resistant to trimethoprim.

Furthermore, pDDM1 has an ampicillin resistance gene derived from J) TP64-1. From this, pDD
E. coli into which Ml has been introduced also exhibits resistance to ampicillin. pDDMl was introduced into E. coli and kept in a stable state, and E. coli containing I) DD11 was transferred to FER to FER.
It has been deposited as M BP-2150.

(2) E. coli containing 9DDM1 E. coli containing pDDM1 exhibits resistance to trimethoprim and ampicillin. As a result of efficient expression of the DHPR dimer gene, E. coli containing pDDM1 accumulates a large amount of DHPR dimer in a soluble state within the bacterial body. E. coli containing pDD41 was transformed into YT+A
DHFR dimer accumulates when cultured at 37°C to stationary phase using p medium (liquid medium containing 5 g of NaCl, 8 g of tryptone, 5 g of yeast extract, and 50 mg of ampicillin sodium in medium 11)! i
, reaching about 20% of M-body proteins.

When cultured bacterial cells are suspended in an appropriate buffer such as phosphate buffer, disrupted by the French press method or sonication method, and separated into supernatant and precipitate by centrifugation, all DHFR dimers are removed. recovered in the supernatant. Escherichia coli containing pDDMl was supplied to the Microtech Institute as FERM BP-215.
It has been deposited as 0.

(3) DHFR dimer Figure 2 shows the part D that encodes the DHFR dimer.
Shows the NA sequence and the amino acid sequence of the protein made from it. DHFR dimer.

It is a novel protein consisting of 319 amino acids.

The molecular weights of the DHFR dimers are 36 and 116, respectively. DHFR dimer is a completely new protein. DHFR dimer is derived from pTP70-1
HFR (amino acid sequence from 1st to 162nd in Figure 2) and a protein obtained by removing the N-terminal methionine from pTP64-1-derived DHFR (163 in Figure 2).
It has a structure in which two molecules (amino acid sequence from position 319 to position 319) are bonded in series. Despite this structure, the DHFR dimer has DHFR enzymatic activity. The enzyme activity per molecule of DHPR dimer is DHF
This is twice the activity per molecule of R. Also, dimer 1
DHFR dimer is completely inhibited when two molecules of mesotrixate, an inhibitor, are combined per molecule. Thus, the DHFR dimer of the present invention is a protein having two enzymatic activity units expressing DHFR enzymatic activity in one polypeptide chain.

In addition, when E. coli produces large amounts of DHFR dimer, D
They become resistant to trimethoprim, an HFR inhibitor and antibacterial agent.

(4) Separation and purification of Dli, PR dimer The method for separating and purifying the fusion protein of the present invention includes: (1) Cultivation of bacterial cells, (2) Crushing of bacterial cells, (2) DEAE-) Yopar force column chromatography 9 (2) Toyopearl HW55 column chromatography. It consists of the process of chromatography, and DEAE-) Jobar force chromatography.

■Culture of bacterial cells For culturing E. coli containing pDDMl, use YT+Ap medium (5 g of NaCl, 8 g of tryptone in medium 11,
It can be cultured in a liquid medium containing 5 g of yeast extract and 50 mg of ampicillin sodium).

Other examples of the medium include ST+Ap medium (a liquid medium containing 2 g of glucose, 1 g of dipotassium phosphate, 5 g of polypeptone, 15 g of yeast extract, and 50 mg of ampicillin sodium in medium 11).

Any medium can be used as long as it allows bacterial cells to grow, but as far as we have investigated, YT+Ap medium is optimal.

E. coli containing pDDM) was inoculated into a medium.

Culture at 37°C until after the logarithmic growth phase or until the stationary phase. The cultured bacterial cells are collected by centrifugation at 5,000 rpm. From the medium 11, bacterial cells with a wet weight of 2 to 5 g are obtained.

Bacterial collection and subsequent operations are performed at low temperatures (between 0 and 10°C, preferably 4°C) unless otherwise specified.

■ Bacterial cells obtained by crushing and culturing the bacterial cells are buffered with twice the wet weight of 1121
(10mM potassium phosphate buffered TFF containing 0.1mM sodium ethylenediaminetetraacetate (EDTA)
, pH 7.0) and disrupt the bacterial cells using a French press. The cell suspension was ultracentrifuged at 35°,000 rpm for 1 hour to obtain a supernatant (cell-free extract).

■DEAE-) Jobal Lamb chromatography Transfer the cell-free extract to buffer 1 containing 50mM KCI in advance.
Wash the column with 1 buffer solution containing 50 mM KCI in the same volume as the column volume. Enzyme elution was performed using buffer 1 at 0. IM
to 0.3M KCI using a linear concentration gradient, and the eluate is fractionated in fixed amounts using a fraction collector. D) Measure the FR activity of the fractionated eluate and collect both fractions containing enzyme activity.

■Toyopearl HW55 column chromatography The enzyme solution obtained by the above procedure was filtered using an ultrafiltration membrane.
Concentrate to 4 ml, apply to Toyopearl HW 55 column equilibrated with buffer 1, and elute the enzyme using the same buffer. Fractionate a certain amount of the eluate using a fraction collector. DH for fractionated eluate
Measure PR activity and collect fractions containing enzyme activity.

(2) DEAE-) Jobal Lamb Chromatography The enzyme solution obtained by the above procedure is adsorbed on a DEAE-) Jobal Lamb that has been equilibrated with preconditioning buffer 1. After adsorption, a linear concentration gradient from 50mM to 0.3M KCl containing 50mM KCl is used, and the eluate is fractionated in fixed amounts using a fraction collector. The fractionated eluate is measured for absorbance at 280 nm and on DHFRHF. Enzyme activity/absorbance value at 280 nm.

Collect a certain amount of money.

By the above operations, the fusion protein can be highly purified and homogenized with good reproducibility.

According to the present invention, purification of the fusion protein can be carried out within one week, including culturing the bacterial cells.

A uniform enzyme preparation can be obtained with a recovery rate of 35% or more.

DHFR enzyme activity was determined by 9 reactions i & (0,05mM dihydrofol, 0,06mM NADPH, 12mM 2
- Take 1ml of mercaptoethanol, 50mM phosphorus (pH 7,0) into a cuhette, and use this -
This is done by adding an enzyme solution to the sample and measuring the change in absorbance at 340 nm over time. One unit of enzyme is defined as the amount of enzyme required for one minute under the above reaction conditions. This i
Il determination can be easily carried out using a 9 spectrophotometer.

Next, examples of the present invention will be shown.

Example 1 Construction of pDDMl In order to create a DNA sequence encoding a protein in which two molecules of HFR are linked in series, p'rp(34-1(
Described in Jikai 1982-69990. ) restriction enzyme Bgl
n (sequence corresponding to the 188th from 12@giant in Figure 1)
and the Bc11 site (sequence corresponding to positions 5388 to 5438 in Figure 1), pTP70-1 (patented in 1983)
We considered inserting a DNA fragment of approximately 500 base pairs (corresponding to the sequence between positions 14 and 538 in FIG. 1) obtained by digestion of restriction enzyme BglII of 1-312836).

About 1 μg of pTP64-1 was treated with the restriction enzyme Be11tase. 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, remove the ethanol.

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 performed using "tlol".
Erural CloningA Loborator
y Manual” (T, Maniatis, E, F
, Frltsch, J., Sambrook, eds.

Co1d Spring Harbor Labora
tory (1982) + below.

This was carried out according to the method described in Reference 1). By cutting approximately 1 μg of pTP70-1 with the restriction enzyme Bgl■ and then treating it with phenol.

Coexisting enzyme proteins were denatured and removed, and then 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 two types of DNA treated in this way were treated with 25 μm of ligase ff1M dithiothreitol,
5mM ATP), mix the two together, add 1 unit of 74-DNA ligase,
The DNA ligation reaction was carried out at 'C for 12 hours. This reaction product was subjected to a transformation method (trans-formation).
method, described in the above-mentioned document l), the large intestine r
jIJH8101 strain.

A nutrient agar medium containing the treated bacterial cells, 50 mg/ml ampicillin sodium and 10 mg/ml trimetobri 1 (in medium 11, 2 g glucose,
1g dipotassium phosphate + 5g yeast extract, 5g
of polypeptone, 15 g of agar. ) on top,
By culturing at 37@C for 24 hours, more than 100 colonies could be obtained. Select 40 colonies from these colonies and add 1.5 ml of YT+Ap to each.
medium (containing 5 g NaCI, 5 g yeast extract, 8 g tryptone, 50 mg ampicillin sodium in medium 11) at 37°C.

The bacterial cells were cultured overnight. Each culture solution was centrifuged for 0 minutes using Elapen and collected as a bacterial precipitate. To this, 0.1
ml electrophoresis sample preparation solution (0,0625M T
ris-HcI, p)! 6.8.2'X Sodium Lauryl Sulfate (Sr)S), 10% (7) Glycerin, 5X (7) 2-Mercaptoethanol, 0.0
Contains bromophenol blue of 01χ. ) was added to suspend the bacterial cells, 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 (tJ, Laemmli; N
ature, vol, 227°p, 680 (+970)
). pTP64- as a standard sample
Escherichia coli containing 1 and pTP70-1 were treated in the same way, and the molecular weight markers were lactalbumin (molecular weight ff 114,200), ) ribsin inhibitor (molecular weight 20.100), and ) ribsinogen (molecular weight 24,000). , carbonic anhydrase (molecular weight 29,000), glyceraldehyde 3-phosphate dehydrogenase (molecular weight 36,
000), egg albumin (molecular weight 45,000).

and bovine serum albumin (molecular weight 66,000). As a result, it was revealed that two of the 40 colonies produced a large amount of protein estimated to have a molecular weight of about 37,000. Appropriately select one plant from the two colonies obtained,
This was cultured in YT+Ap medium, and Tanaka and We
isblum method (T, Tanaka, B, Wei
s-blum: J, Bacteriology, v
ol, I21. Plasmids were prepared according to P. p., 354 (1975)). The obtained plasmid was pDDMl
It was named.

Since 41 to pDD should have a structure in which a sequence derived from pTP70-1 is inserted into pTP64-1, in order to confirm this, from 49548th to 49th in Figure 1,
9515 in Figure 1 from the Pst1 site located at position 59
It reaches the EcoR1 site located at position 1 and 9568. The dideoxy method using M13 phage (J, Messing; Meht
ods in Enzymology.

vol, +01. p, 20(+983)),
The base sequence was determined. As a result, 4 of the arrays shown in Figure 1.
The sequence up to 954th and 6th was confirmed. Also, all.

The entire nucleotide sequence of pTP64-1 is clear (described in Jikai 1986-69990), and the remaining part was determined by cutting pTP64-1 with restriction enzymes AatII, PvulI, and Taql. Approximately 4 kilobase pairs of D obtained by EcoRI and PstI cleavage
It was shown to be completely identical to the NA fragment.

From the above results, it was determined that the entire base sequence of pDDMl was the sequence shown in FIG.

Example 2 DHFR dimer produced by E. coli containing pDDMl The dino acid sequence of a DHFR fusion protein produced by E. coli containing pDDMl can be predicted from the base sequence of the gene. Since the sequence from 57th to 1013th in Figure 1 encodes the fusion protein, the amino acid sequence was deduced using the triblet code table.
An amino acid sequence was obtained showing the DHFR dimer.

It consists of 319 amino acids and has a molecular weight of 36. It was 116 Daltons.

DHFR dimer was isolated and purified from E. coli containing pDD11, and the properties of the protein were investigated.

[Purification of DHFR dimer] A. Amount of bacterial cells used: Wet weight ffi 10 gB, Enzyme purification table The purification process in the table is ■Cell-free extract, ■DEAE-)
Jobal Column Chromatography ■Toyopearl) (W55 Column Chromatography and ■DEAE
-) represents Yopal force chromatography.

Purification Enzyme solution Recovered protein Recovered enzyme Yield process Member (ml) Lithium (mg) Activity (]nit) (2) ■
33 661 8.841
+00■ 30 129 6,
777 76.7■ 28 85
3,190 35.7 The obtained enzyme protein was analyzed by SDS electrophoresis (method described in the above example).

A single protein bundle of approximately 37,000 molecules was demonstrated, indicating the homogeneity of the resulting enzyme preparation.

[Properties of the separated and purified DHF R dimer The activity of the purified DHFR dimer and the protein concentration of the enzyme used at that time were measured, and the activity (specific activity) per 1 mg of protein was determined to be 35.2 It was one stone per unit/mg protein. In the same manner, the specific activity of wild-type DHFR (DHFR produced by E. coli containing pTP64-1) was determined to be 34.8 units/mg protein, which is the difference between the enzyme activity of DHFR dimer and wild-type DHFR. The specific activities showed the same values. D.H.
The molecular weight of the FR dimer is approximately twice that of wild-type DHFR, indicating that it is twice that of wild-type DHFH.

Additionally, 0.74 μM of DHFR dimer and wild-type DH
When various concentrations of mesotrixate, an inhibitor of DHFH, were added to 1.02 μM of FR enzyme and the effect on enzyme activity was investigated, it was found that 0.
．． 145 μM mesotrixate and wild type DHFR
It was revealed that the effect was completely inhibited by 1.19 μN1 of mesotrixate. This result shows that two molecules of mesotrixate bind to one molecule of DHFR dimer, and one molecule of mesotrixate binds to one molecule of wild-type DHFR. Based on the above results.

DHFR dimer consists of -ζ DH in the polypeptide chain
It was shown that the protein has two enzyme activity units expressing FR enzyme activity.

[Effects of the invention] As mentioned above, the novel recombinant Blasmi F' I) D D
Ml is the j7i enzyme protein DHFR
It encodes the dimer and has pDDM1. Furthermore, the produced DHFR dimer has two enzymatically active units in the bonopeptite chain and is a novel HFR enzyme protein. like this.

This substance did not exist until the invention of this invention, and it is expected that it will be used in a variety of applications in the future.

In addition, DHFR is an important enzyme for the synthesis of 9-folate coenzyme, and by using this enzyme activity, it is also possible to quantify dihydrofolate and tetrahydrofolate, and at least it is useful in this field. It is clear that it contributes to

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the entire base sequence of pDDMl,
The DNA strand sequence of one of the double-stranded DNA is written in the direction from the 5' end to the 3' end. The symbols in the figure represent nucleic acid bases; A represents adenine, C represents cytosine, G represents guanine, and T represents thymine. The numbers in the figure are p
The number shown is the 5' cleavage recognition site of the restriction enzyme C1al, which is the only one present in DDM1. FIG. 2 is a diagram showing the base sequence of the portion encoding the DHFR dimer present in pDDM1 and the amino acid sequence of the protein. 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. Ala is alanine, Arg is arginine, Asn is asparagine, Asp is aspartic acid, Cys is cysteine, Gln is glutamine, GIIJ is glutamic acid, cry is glycine, His is histidine, and Ile is isoleucine. , Leu is leucine, L
ys is lysine. Met is methionine, Phe is phenylalanine,
Pro stands for proline and Set 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. 1 in Figure 1 ACTGCTGCTG CAAAACGTCT GCG
ACCTGAG CAACAACATG AATGGT
CTTC2960297029802990300GG
GTTrCCGTG TTTCGTAAAG TCTG
GAAACG CGGAAGTCAG CGCCCTG
CAC 2 in Figure 1 3 in Figure 1 αχKiAGCAGA CAAGCCCGTCAGGG
CGCGTCAGCGGGTGrT Gfχ℃α';T
GTCGGGGCGCAGCCATGACCCAG T
CACGTAGCG ATAGCGGAGT GTAT
ACTα℃nmuACTATGCGGCATCAGAG
CAGATTGTACTGAGAGTGCA CCAT
ATGCGGTGTGAAADINGACCGCACAGAT
G CGTAAGGAGA AAATACCGCA
TCAGGCGCTCrrccαgoTCCTCGCTC
ACTG ACTCGCTGCG CrCIATCGT
T CGGCTGCGGC0AGCGGTATCAGC
rCACTCA AAGGCGGTAA TACGGT
TATCCACAGAATCA3810 38
20 3830 3840,
3850GGGGATAACG CAGGAAAGAA
CATGTGAGCA Al11Aαχ:CAGCA
AAAαffAGGAACCGTAAA AAAGGCC
GCGT TGCTαmrigoCCATAGG CTC
CGCCCCCCTGACGAGCA TCACAAA
AAT CGACGCTCAA GTCAGAGGTG
GCGAAAACCCGACAGGACTATAAA
GATACCA GGCGrTTCCCCCrGGAA
GCr CAχgoCGTα℃ACrAmGTr ATA
ATGTATr CATAAGCTT 6 in Figure 1

Claims (1)

[Claims] 1. A novel recombinant plasmid pDDM1 having the DNA sequence shown in FIG. 2. Novel recombinant plasmid p described in claim 1
Large intestine surface containing DDM1. 3. Dihydrofolate reductase having the amino acid sequence shown in FIG. 4. A method for separating and purifying dihydrofolate reductase according to claim 3.