JPH0662867A - Arginine deiminase expression vector, transformed microorganism and production of arginine deimidase - Google Patents

Abstract

PURPOSE:To enable the mass-production of arginine deiminase by transforming E.coli with an expression vector containing a DNA sequence coding an arginine deiminase gene and culturing the transformed E.coli under prescribed condition. CONSTITUTION:An expression vector bonded with a fragment of tac promoter, a fragment of the replication starting point (ori) of plasmid puc18 and a DNA fragment coding the amino acid sequence of arginine deiminase or its analog is constructed beforehand. E.coli is transformed by using the expression vector and the transformed E.coli is cultured. The expressed arginine deiminase or its analog is recovered and purified. The obtained arginine deiminase has an activity comparable to that of arginine deiminase originated from mycoplasma and is an antitumor enzyme effectively utilizable as a carcinostatic agent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an expression vector of arginine deiminase or an analog thereof, a transformed microorganism in which the expression vector is incorporated into Escherichia coli, and a large amount of arginine deiminase or an analogue thereof using the transformed microorganism. On how to produce.

[0002]

2. Description of the Related Art Arginine deiminase (enzyme classification numbers 3, 5, 3, and 6) is an enzyme that catalyzes an enzymatic reaction that produces L-citrulline and ammonia from L-arginine and water. It is known to exist.
It has already been clarified that arginine deinase obtained from mycoplasma has a strong cancer cell growth inhibitory activity, and it is expected to utilize it as a novel antitumor agent (JP-A-3-164173).

Conventionally, arginine deiminase was produced from Mycoplasma arginini , which is an arginine deiminase-producing bacterium. However, its low productivity and complicated culture management of mycoplasma make it a large-scale production of this enzyme. However, there are problems that it is difficult to use it as an enzyme source, and that the purification method is complicated.

[0004]

The present invention has been made to solve the above-mentioned drawbacks, and an object of the present invention is high expression of arginine deiminase or its analog, which can be efficiently produced. It is to provide an expression vector and Escherichia coli transduced with this expression vector. Further, an object of the present invention is to provide a method for producing arginine deiminase or its analogs in large quantities at low cost by using Escherichia coli by a genetic engineering method.

[0005]

[Means for Solving the Problems] The present inventors have investigated means for solving the above-mentioned problems, and have found that a fragment of tac promoter and an origin of replication (O) of plasmid pUC18.
ri) fragment, a fragment containing a tetracycline resistance gene, and an arginine deiminase expression vector to which a DNA fragment encoding an amino acid of arginine deiminase or an analog thereof is bound, which is used to transduce E. coli, When this transformed microorganism was cultured, a large amount of arginine deiminase or its analog was produced in the culture, and it was found that this can be easily purified, and the present invention was completed.

That is, the present invention relates to the following arginine deiminase expression vector, a transformed microorganism in which this expression vector is transduced into Escherichia coli, and a method for producing arginine deiminase using this transformed microorganism.

(1) A fragment of the tac promoter, a fragment of the origin of replication (Ori) of the plasmid pUC18, a fragment containing the tetracycline resistance gene, and a DNA fragment encoding the amino acid sequence of arginine deiminase or its analogs are bound together. An expression vector of arginine deiminase or its analog. (2) A transformed microorganism, which is obtained by transducing Escherichia coli with the expression vector of the arginine deiminase or its analog. (3) A method for producing arginine deiminase or an analog thereof, which comprises culturing the transformed microorganism to express arginine deiminase or an analog thereof, and collecting arginine deiminase or an analog thereof from the culture. .

The entire base sequence of arginine deiminase is
Determined by the present inventors (JP-A-3-16417)
3), molecular weight of about 40 thousand amino acids (SDS-PAGE) or about 90,000 (gel filtration HPL)
It is the polypeptide of C). And the origin of the microorganism, for example, M. arginini; M. arthritidis, etc.
The terminal amino acids are slightly different. In the present invention, such N
Arginine deiminase analogs that have different terminal amino acids or have arginine deiminase activity even if they have different amino acids are included as arginine deiminase analogs (hereinafter, these analogs are also referred to as arginine deiminase).

The method for creating the arginine deiminase expression vector of the present invention will be illustrated and described.

The plasmid which constitutes the vector in the present invention can be prepared as follows. tac
Since the base sequence of the promoter fragment is known, this fragment can be easily produced using a DNA synthesizer or the like. This is a commercially available plasmid pU
It may be combined with a fragment of the replication origin (Ori) of C18. However, a combination of the fragment of the tac promoter and the fragment of the replication origin (Ori) of the plasmid pUC18 can be prepared relatively easily by combining the excision and conjugation of a commercially available plasmid. For example, the commercially available plasmid pKK223-3
(Manufactured by Pharmacia) is digested with restriction enzymes PvuI and NruI to obtain a site portion containing a fragment of tac promoter. On the other hand, the plasmid pUC18 was digested with the restriction enzyme P
A site part containing a fragment of the origin of replication (Ori) cut with vuI and NruI is obtained. Then, both sites were joined with T4 DNA ligase to obtain a plasmid pMK2, which was used as a tac promoter and a plasmid pUC1.
It can be used as a plasmid containing 8 replication origins (Ori).

A DNA fragment encoding the amino acid sequence of arginine deiminase is prepared as follows. Preferably, a gene in which all TGA codons contained in the structural gene of arginine deiminase are replaced with TGG codon, which is a tryptophan codon of Escherichia coli, and a gene containing a 240 bp regulatory gene upstream thereof are commercially available plasmid pUC19. SacI-H
It is inserted into the indIII side to prepare the plasmid pAD12 (see Japanese Patent Laid-Open No. 3-164173).

As shown in FIG. 1, this plasmid pAD12 is cleaved with restriction enzymes SacI and HindIII to purify a DNA fragment encoding arginine deiminase.

Furthermore, commercially available phage vector M1
3mp19 is cleaved with restriction enzymes SacI and HindIII, and the DNA fragment encoding the arginine deiminase is ligated to the cleavage site with T4 DNA ligase to obtain a plasmid M13ADME. Escherichia coli JM101 was transformed with this, and double-stranded DNA prepared from the resulting plaque was treated with restriction enzymes EcoRI and Hind.
Cleavage with III yields a DNA fragment encoding the amino acid of arginine deiminase. When there is a DNA sequence in this fragment that should not be cleaved when it is subsequently treated with a restriction enzyme, the nucleotide sequence may be converted beforehand using a mutation-introducing primer or the like.

Then, this DNA fragment and, if this fragment lacks a part of the base sequence of arginine deiminase, the DNA fragment corresponding to the part is used as a restriction enzyme EcoR of the plasmid pMK2.
A plasmid pMKAD containing the tac promoter; a replication origin (Ori) fragment of the plasmid pUC18 and a DNA fragment encoding the amino acid sequence of arginine deiminase was incorporated into the I and HindIII cleavage sites to transform Escherichia coli JM109 strain. Amplify. And, this plasmid is
It is cleaved with stI and HindIII, and the DNA fragment encoding the C-terminal amino acid sequence lacking this is ligated to obtain plasmid pMKΔCAD.

Further, in order to stabilize the plasmid, the plasmid pBR322 was digested with the restriction enzyme EcoR.
Cleavage with I and AvaI to make a blunt end with Klenow enzyme to obtain a tetracycline resistance gene, which was ligated to the restriction enzyme BamHI cleavage site of plasmid pMKΔCAD, and Escherichia coli JM109 strain was transformed with the arginine deiminase gene. An expression plasmid pADTc1 in which the tetracycline gene is linked in the opposite direction is obtained.

The expression plasmid of the present invention may include other genes derived from the plasmids used in addition to these genes without any problem. However, for example, it contains fragments such as rrnB terminator and ampicillin resistance gene. Arginine deiminase can be highly expressed, and screening is simple, which is particularly preferable.

However, in the present invention, a fragment of the tac promoter, a fragment of the origin of replication (Ori) of the plasmid pUC18, a fragment containing the tetracycline resistance gene and a DNA fragment encoding the amino acid sequence of arginine deiminase or its analogs are ligated. Arginine deiminase is not expressed in high yield in the absence of one of the fragments.

Next, the arginine deiminase expression plasmid pADTc1 thus obtained was used for Escherichia coli JM.
The 101 strain is transformed, cultured in a medium containing tetracycline, the bacterial cells are collected, and arginine deiminase is collected therefrom.

Any appropriate medium such as 2 × TY medium and LB medium can be used as the medium, and the culture conditions are 37 ° C.
Incubate with aeration and agitation at the surrounding temperature for one night to several days. The bacterial cells may be collected from the culture by centrifugation or the like. In addition, for collecting arginine deiminase from the collected bacterial cells, first, the bacterial cells are washed with physiological saline or the like, suspended in 0.1M potassium phosphate buffer (pH 7.0), and ultrasonicated by ultrasonic waves. Are crushed and centrifuged to obtain an insoluble fraction. This insoluble fraction was treated with Triton X-100 (Triton X-100).
After removing the membrane fraction of E. coli by washing with a detergent such as guanidine hydrochloride, tris-hydrochloric acid buffer and a reducing agent [for example, dithiothreitol (used for cleavage of -SS-bond) The arginine deiminase is extracted with an extract solution consisting of
Refold. The extract is filtered, and the filtrate is subjected to Q sepharose column chromatography or arginine-sepharose column to purify arginine deiminase. In this way, purified arginine deiminase can be obtained.

The activity of arginine deiminase was determined by the method of Fenske et al. [J. Bacteriol., 126 , 501-510 (1976)] to produce citrulline, and the amount thereof was determined by the method of Archibald [J. Biol. Chem., 156 , 121-142 (1944)].

That is, a 10 mM arginine solution [0.
Dissolved in 1M phosphate buffer (pH 7.0)] 1.0 ml
After adding 20 μl of the arginine deiminase sample solution to the mixture and performing the enzyme reaction at 37 ° C. for 5 to 30 minutes, 1 ml of the acid mixture (H ₂ SO ₄ : H ₃ PO ₄ = 1: 3) was added to the enzyme reaction. To stop. To the reaction solution, 25 μl of a 3% aqueous solution of diacetyl monooxime was added, and the mixture was heated at 100 ° C. for 20 minutes in the dark, then allowed to cool at room temperature for 30 minutes, and 490 n
The citrulline produced is quantified by measuring the absorbance at m. The arginine deiminase activity that produces 1 μmol of citrulline per minute is defined as 1 unit (U).

The present invention will be specifically described below with reference to examples.

[0023]

Example 1 Construction of arginine deiminase expression vector pADTc1
Production ( see FIG . 1 ) As shown in FIG. 1, the arginine deiminase expression vector pAD12 (JP-A-3-164173) was cleaved with restriction enzymes SacI and HindIII to purify a DNA fragment encoding arginine deiminase.
This fragment and the fragment of the plasmid M13mp19RF cleaved with the restriction enzymes SacI and HindIII were ligated with T4 DNA ligase, and J. Messing [Meth
ods in Enzymology, 101, 21-78 (1983)], Escherichia coli JM101 was transformed. A single-stranded DNA was prepared from the obtained plaque, and E present in the nucleotide sequence at positions 251-252 of the amino acid sequence of arginine deiminase was prepared.
Kukel's method for crushing the coRI site [Proc. Nat
l. Acad. Sci. USA, 82, 488 (1985)], a mutation was introduced using a mutation-introducing primer such as GAATTC → GAATTT. The double-stranded DNA containing the mutant arginine deiminase gene thus obtained was cleaved with restriction enzymes EcoRI and HindIII to purify a fragment lacking the nucleotide sequence encoding the N-terminal 10 amino acids of arginine deiminase. did. Further, the E. coli expression vector pMK2 was cleaved with the restriction enzyme EcoRI, blunt-ended with Mung Bean nuclease, and a vector fragment cleaved with the restriction enzyme HindIII was prepared. The above two fragments were ligated and the linker DNA encoding the N-terminus shown below was synthesized in order to prepare an expression vector.

[0024]

[Chemical 1]

The above three fragments were ligated with T4 DNA ligase and Escherichia coli JM109 strain was transformed to obtain arginine deiminase expression plasmid pMKAD. Next, in order to delete the 3'-untranslated region of the arginine deiminase gene, the plasmid pMKAD was digested with restriction enzymes PstI and HindIII, and the vector fragment was purified.

Further, D encoding the C-terminal amino acid sequence
NA was divided into four types of DNA as shown below and synthesized and constructed.

[0027]

[Chemical 2]

After purifying 4 kinds of DNA, 2 kinds of DNA except the 5'-end DNA were phosphorylated and then annealed. Ligation with T4 DNA ligase gave a double-stranded DNA. This was ligated to the above vector and the plasmid pM
I got KΔCAD. Furthermore, considering the stabilization of the plasmid, we decided to introduce a tetracycline resistance gene. Plasmid pBR322 was digested with restriction enzymes EcoRI and A
After cutting with vaI and purifying the 1.4 Kbp tetracycline resistance gene fragment, it was blunt-ended with Klenow enzyme. In addition, after the plasmid pMKΔCAD was cleaved with the restriction enzyme BamHI, a vector fragment blunt-ended with Klenow enzyme was prepared, and both fragments were ligated with T4 DNA ligase.
The 109 strain was transformed to obtain an expression plasmid pADTc1 in which the tetracycline gene was linked in the opposite direction to the arginine deiminase gene.

[0029]

Example 2 Expression of Arginine Deiminase Escherichia coli JM101 strain transformed with the above-mentioned expression plasmid pADTc1 [Mikokukenjojo 3617 (FER
M BP-3617)] in 2 × TY medium containing 16 μg / ml of tetracycline (16 g of bactotryptone /
l, Bacto yeast extract 10g / l, NaC
5 g / l) at 37 ° C. overnight. After culturing, 1
The cells were collected in a volume of 100 ml, suspended in 100 μl of a Laemli sample buffer, dissolved by heating, and 3 μl was subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, it was stained with Coomassie Brilliant Blue and decolorized.
Expression of arginine deiminase was observed at position 5,000 (see FIG. 2).

[0030]

Example 3 Fermentation of Arginine Deiminase-Producing Bacteria in a 5 L Fermenter In a 5 L fermentor , E. coli JM101 / pADTc1 in 2 × TY medium containing 2 L of 2% glycerol.
When the cells of (Microtechnology Research Institute No. 3617) were cultured at 37 ° C. for 24 hours with aeration and agitation, the expression level of arginine deiminase reached 15% of the total bacterial protein. Moreover, the growth of the bacterial cells was A660 = 25, and about 150 m / l of the culture solution.
The expression level of g was obtained.

[0031]

Example 4 Arginine deiminase from an arginine deiminase-producing bacterium
Purification method of nuclease (solubilization and activity regeneration method ) After fermentation, 2 l of culture solution was centrifuged (6,000 xg, 1
The cells were collected for 0 minutes). Wet cells were suspended in 200 ml of 10 mM phosphate buffer pH 7.0 and sonicated to sterilize the cells. This suspension was centrifuged to obtain a precipitate which was an insoluble fraction. The insoluble fraction was precipitated with 4% Trito.
nX-100, 10 mM phosphate buffer, 1 mM EDT
A The membrane fraction was solubilized by washing with 200 ml of a pH 7.0 solution. The precipitate was washed with 10 mM phosphate buffer pH 7.0, and then 6M guanidine hydrochloride, 50 mM Tris-H.
Cl (pH 8.5), 2 mM 10 mM dithiothreitol
50 ml was added and solubilized at room temperature. This one
It was diluted 100-fold with 0 mM potassium phosphate buffer (pH 7.0), stirred at 10 to 20 ° C. overnight, and refolded. After refolding, an activity of 4900 U was obtained.

[0032]

Example 5 Purification Method of Activated Arginine Deiminase The diluted solution obtained in Example 4 was filtered through a 3.2 μm filter (manufactured by Pall). The obtained filtrate is 10 mM
Q equilibrated with potassium phosphate buffer (pH 7.0)
-Sepharose FF (Pharmacia) column (4.
4 × 2 cm). After application, washing with equilibration buffer and stepwise elution of active AD with a buffer containing 0.2 M NaCl resulted in the recovery of 4506 U of activity.
The active fraction was treated with 10 mM potassium phosphate buffer (pH 7.
It was applied to an arginine-Sepharose 4B (Pharmacia) column equilibrated with 0). After washing with an equilibration buffer containing 0.2 M NaCl, elution was performed under the following conditions.

Column size: 2.2 × 20 cm Flow rate: 76 ml / hr Fraction amount: 7.6 ml Eluent: 10 mM phosphate buffer (pH 7.0) (0.2-1.0 M sodium chloride concentration gradient) )

The absorbance at 280 nm and the arginine deiminase activity of each of the obtained fractions were measured. The results are shown in FIGS. 3 and 4. As you can see in this result,
Arginine deiminase was found to be contained in fractions 30-52. Finally, an activity of 4160 U was obtained. The above active fraction was subjected to the method of Remuri et al. [Nature,
727, 680-685 (1970)].
-When polyacrylamide gel electrophoresis was performed, it was detected as a single band at the position of molecular weight 45,000,
It was confirmed that a completely purified recombinant arginine deiminase was obtained (FIG. 5). As a result of measuring the enzyme activity of the purified sample, it was found that the specific activity was 32 U / mg, which was equivalent to the arginine deiminase derived from Mycoplasma. The degree of purification in each step is shown in Table 1.

[0035]

[Table 1] ──────────────────────────────────── Purification step Total protein Total activity Total activity Specific activity yield (mg) (U) (U / mg) (%) ───────────────────────────────────── Refolding ND 4900 ─── 100 Q-Sepharose FF 140.8 4506 32.0 92 Arginine-Sepharose 4B 128.2 4160 32.4 85 ────────────────────────── ──────────

[0036]

Example 6 Physical Properties of Recombinant Arginine Deiminase The optimum pH range is 6.0 to 7.0 as shown in FIG.
The optimum reaction temperature was 45 to 50 ° C as shown in Fig. 7. As a result of measurement by SDS-polyacrylamide gel electrophoresis, the molecular weight was about 45,000. In addition, as a result of measurement by a gel filtration HPLC method using a TSK G3000SWXL (manufactured by Tosoh Corporation) column, the molecular weight was about 90,000, and it was considered that a dimer was formed. As a result of measuring the isoelectric point by the isoelectric focusing method, the isoelectric point was about 4.7, which was in agreement with mycoplasma-derived arginine deiminase. When the amino acid composition and the N-terminal amino acid sequence were examined, the amino acid composition values are shown in Table 2.
Shows almost the same value as mycoplasma-derived arginine deiminase, the N-terminal sequence is Ser-Val-Phe as shown in Table 3, and it was confirmed that Met derived from the start codon was completely removed. Was done.

[0037]

[Table 2] Amino acid composition analysis (0.5 nmol = 22.5 μg) ───────────────────────────────────── Amino Acids Mycoplasma AD Recombinant AD Theoretical ──────────────────────────────────── Asx 43.6 42.6 47 Thr 17.0 17.1 18 Ser 20.0 20.5 23 Glx 43.2 43.0 44 Gly 23.0 23.0 23 Ala 25.1 25.0 25 Cys 1.0 1.2 2 Val 29.0 29.9 34 Met 9.4 9.6 10 Ile 24.7 25.3 28 Leu 42.4 43.0 43 Tyr 13.5 14.0 14 Phe 17.5 18.0 18 His 10.2 10.5 11 Lys 27.8 28.0 28 Arg 16.3 16.7 17 Pro 18.9 19.5 19 Trp nd nd 5 ──────────────────────────────────── ─

[0038]

[Table 3] N-terminal amino acid sequence analysis (1 nmol = 45 μg) ──────────────────────────────────── ─ Cycle amino acid content (pmol) ──────────────────────────────────── 1 Ser 203 2 Val 916 3 Phe 756 4 Asp 299 5 Ser 186 6 Lys 801 7 Phe 705 8 Lys 333 9 Gly 580 10 Ile 618 ─────────────────────────── ──────────

[0039]

According to the present invention, arginine deiminase can be produced in large quantities by genetic engineering. The obtained arginine deiminase exhibits an activity equivalent to that of arginine deiminase derived from mycoplasma, and can be effectively used as an antitumor enzyme for a carcinostatic agent.

[Brief description of drawings]

FIG. 1 shows a conceptual diagram of a method for constructing an arginine deiminase expression vector of the present invention.

FIG. 2 shows an electropherogram showing the expression of arginine deiminase in E. coli.

[Explanation of symbols]

a: Mycoplasma-derived arginine deiminase b: Escherichia coli c: Recombinant arginine deiminase-producing bacterium (flask culture) d: Recombinant arginine deiminase-producing bacterium (jar culture) e: d soluble fraction f: d insoluble fraction g: f 6M guanidine hydrochloride soluble fraction

FIG. 3 shows the activity of arginine deiminase in each fraction by affinity chromatography.

FIG. 4 shows an electropherogram showing the expression of arginine deiminase in each fraction of FIG.

FIG. 5 shows a purity test by electrophoresis of purified preparations of arginine deiminase derived from Mycoplasma and the recombinant arginine deiminase of the present invention.

FIG. 6 shows the relationship between the arginine deiminase activity of the present invention and pH.

FIG. 7 shows the relationship between arginine deiminase activity of the present invention and temperature.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] August 24, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 shows a conceptual diagram of a method for constructing an arginine deiminase expression vector of the present invention.

FIG. 2 shows an electropherogram showing the expression of arginine deiminase in E. coli.

FIG. 3 shows the activity of arginine deiminase in each fraction by affinity chromatography.

FIG. 4 shows an electropherogram showing the expression of arginine deiminase in each fraction of FIG.

FIG. 5 shows a purity test by electrophoresis of purified preparations of arginine deiminase derived from Mycoplasma and the recombinant arginine deiminase of the present invention.

FIG. 6 shows the relationship between the arginine deiminase activity of the present invention and pH.

FIG. 7 shows the relationship between arginine deiminase activity of the present invention and temperature.

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Correction target item name] All drawings

[Correction method] Change

[Correction content]

[Figure 4]

[Figure 5]

[Figure 1]

[Fig. 2]

[Figure 3]

[Figure 6]

[Figure 7]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display area // (C12N 1/21 C12R 1:19) (C12N 9/78 C12R 1:19)

Claims (4)

[Claims] 1. A fragment of the tac promoter, a fragment of the origin of replication (ori) of the plasmid pUC18, a fragment containing the tetracycline resistance gene, and a DNA fragment encoding the amino acid sequence of arginine deiminase or its analogs. An expression vector of arginine deiminase or an analog thereof, which is characterized in that: 2. A transformed microorganism, which is obtained by transducing Escherichia coli with the expression vector according to claim 1. 3. The Micro Works Research Article No. 3617 (FERM B
The transformed microorganism according to claim 2, which is P-3617). 4. Culturing the transformed microorganism of claim 2,
A method for producing arginine deiminase or an analog thereof, which comprises expressing arginine deiminase or an analog thereof and collecting arginine deiminase or an analog thereof from a culture.