JP3004788B2 - Arginine deiminase expression vector, transformed microorganism and method for producing arginine deiminase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION This invention is, write sets the expression vector of arginine deiminase or its analogs in E. coli
The present invention relates to a method for producing arginine deiminase or an analog thereof in a large amount using the transformed microorganism.

[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 to produce L-citrulline and ammonia from L-arginine and water, and is useful for animals, bacteria, yeasts Its existence is known.
It has already been revealed that arginine deinase obtained from mycoplasma has a strong cancer cell growth inhibitory activity, and it is expected that this arginine deinase will be used as a novel anticancer agent (JP-A-3-164173).

[0003] Arginine deiminase has been conventionally produced from Mycoplasma arginini , which is an arginine deiminase-producing bacterium. However, there are problems in that the method is difficult to use as an enzyme source and that the purification method is complicated.

[0004]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned drawbacks, and an object of the present invention is to express arginine deiminase or its analogs at a high level and to produce them efficiently. the expression vector in E. coli
An object of the present invention is to provide a method for producing arginine deiminase or an analog thereof in large quantities at low cost by genetic engineering using the transduced Escherichia coli.

[0005]

Means for Solving the Problems The present inventors have studied means for solving the above-mentioned problems, and have found that a fragment of the tac promoter, an origin of replication of plasmid pUC18 (O
ri), an arginine deiminase expression vector to which a fragment containing a tetracycline resistance gene and a DNA fragment encoding an amino acid of arginine deiminase or an analog thereof are linked, and Escherichia coli is transduced using the arginine deiminase expression vector; When this transformed microorganism was cultured, a large amount of arginine deiminase or an analog thereof was produced in the culture, and it was found that the arginine deiminase could be easily purified, thereby completing the present invention.

Namely, the present invention relates to a method of incorporating the arginine deiminase expression vector as follows: E. coli, to produce the arginine deiminase using a transformant microorganism obtained by the <br/> transduction.

(1) A fragment of the tac promoter, a fragment of the replication origin (Ori) of the plasmid pUC18, a fragment containing a tetracycline resistance gene, and a DNA fragment encoding the amino acid sequence of arginine deiminase or an analog thereof are combined. Arginine deiminase or an analog thereof. (2) A transformed microorganism obtained by transducing Escherichia coli with the expression vector for the arginine deiminase or an analog thereof. (3) culturing the transformed microorganism to express arginine deiminase or an analog thereof, collecting arginine deiminase or an analog thereof from the culture ,
Soluble with a solution containing guanidine hydrochloride and a reducing agent
A method for producing arginine deiminase or an analog thereof, comprising refolding with a potassium phosphate buffer after the conversion .

[0008] The entire nucleotide sequence of arginine deiminase is
Determined by the present inventors (JP-A-3-16417)
3), a molecular weight consisting of all 409 amino acids of about 45,000 (SDS-PAGE) or about 90,000 (gel filtration HPL
The polypeptide of C). The origin of the microorganism, for example, M. arginini; by M. arthritidis like N
Terminal amino acids are slightly different. In the present invention, such N
An arginine deiminase analog includes an amino acid having an arginine deiminase action even if the terminal amino acid is different or the amino acid is different (hereinafter, these analogs are also referred to as arginine deiminase).

The arginine deiminase expression vector of the present invention will be described by way of an example of the method for its creation.

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

A DNA fragment encoding the amino acid sequence of arginine deiminase is prepared as follows. Preferably, a gene obtained by substituting all TGA codons contained in the structural gene of arginine deiminase with TGG codon, which is a tryptophan codon of Escherichia coli, and a gene containing a 240 bp regulatory gene upstream of this gene is used as a commercially available plasmid pUC19. SacI-H
Plasmid pAD12 is prepared by inserting it into the indIII side (see JP-A-3-164173).

The plasmid pAD12 is digested with restriction enzymes SacI and HindIII as shown in FIG. 1 to purify a DNA fragment encoding arginine deiminase.

Furthermore, a commercially available phage vector M1
3mp19 is cleaved with restriction enzymes SacI and HindIII, and the DNA fragment encoding arginine deiminase is ligated to the cleavage site with T4 DNA ligase to obtain plasmid M13ADME. Using this, Escherichia coli JM101 was transformed, and the double-stranded DNA prepared from the resulting plaque was digested with restriction enzymes EcoRI and Hind.
Cleavage with III yields a DNA fragment encoding the amino acid of arginine deiminase. When a DNA sequence which must not be cleaved after treatment with a restriction enzyme is included in this fragment, the nucleotide sequence may be previously converted using a mutagenic primer or the like.

When this DNA fragment and this fragment lack a part of the base sequence of arginine deiminase, the DNA fragment corresponding to the part is replaced with the restriction enzyme EcoR of plasmid pMK2.
Plasmid pMKAD containing the tac promoter, a fragment of the replication origin (Ori) of plasmid pUC18 and a DNA fragment encoding the amino acid sequence of arginine deiminase, and transformed into E. coli JM109 strain. Amplify. Then, this plasmid is replaced with the restriction enzyme P.
The plasmid is digested with stI and HindIII, and a DNA fragment encoding the deleted C-terminal amino acid sequence is ligated to obtain plasmid pMKΔCAD.

Further, in order to stabilize the plasmid, plasmid pBR322 was replaced with a restriction enzyme EcoR.
I and AvaI, and blunt-ended with Klenow enzyme to obtain a tetracycline resistance gene, which was ligated to the restriction enzyme BamHI cleavage site of plasmid pMK 、 CAD, transformed into Escherichia coli JM109 strain, and transformed with the arginine deiminase gene. An expression plasmid pADTc1 in which the tetracycline gene is linked in the reverse direction is obtained.

The expression plasmid of the present invention may contain any other gene derived from the plasmid used besides these genes, without any problem. However, for example, it may contain a fragment such as rrnB terminator or ampicillin resistance gene. Arginine deiminase is particularly preferable because it can highly express arginine deiminase and facilitates screening.

However, in the present invention, a fragment of the tac promoter, a fragment of the replication origin (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 an analog thereof are ligated. Arginine deiminase is not expressed in high yields even if one of these fragments is missing.

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

An appropriate medium such as 2 × TY medium or LB medium can be used as the medium.
Incubate with aeration and agitation at ambient temperatures for one to several days. Bacterial cells may be collected from the culture by centrifugation or the like. Arginine deiminase is collected from the collected cells by first washing the cells with physiological saline, suspending the cells in a 0.1 M potassium phosphate buffer (pH 7.0), and sonicating the cells by ultrasonication. Is crushed and centrifuged to obtain an insoluble fraction. This insoluble fraction was subjected to Triton X-100.
After removing the membrane fraction of Escherichia coli by washing with a surfactant or the like, guanidine hydrochloride, Tris-HCl buffer and a reducing agent [for example, dithiothreitol (used for cleavage of -SS- bond) Arginine deiminase is extracted with the extract comprising the following steps, and diluted with the above potassium phosphate buffer to refold. The extract is filtered, and the filtrate is applied to a Q Sepharose column chromatography or an arginine-Sepharose column to purify arginine deiminase. Thus, a purified arginine deiminase can be obtained.

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

That is, a 10 mM arginine solution [0.
Dissolved in 1 M phosphate buffer (pH 7.0)] 1.0 ml
, After the addition of arginine deiminase sample solution 20 [mu] l, was carried out for 5 to 30 minutes the enzyme reaction at 37 ° C., an acid mixture _{(H 2 SO 4: H 3} PO 4 = 1: 3) was added 1ml enzyme reaction To stop. To the reaction solution, 25 μl of a 3% aqueous solution of diacetylmonooxime was added, and the mixture was heated at 100 ° C. for 20 minutes under light shielding, and then allowed to cool at room temperature for 30 minutes, followed by 490 n
The resulting citrulline 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).

Hereinafter, the present invention will be described specifically with reference to examples.

[0023]

Example 1 Construction of Arginine Deiminase Expression Vector pADTc1
(See FIG . 1 ) As shown in FIG. 1, the arginine deiminase expression vector pAD12 (JP-A-3-164173) was digested with restriction enzymes SacI and HindIII to purify a DNA fragment encoding arginine deiminase.
This fragment and the fragment obtained by cutting the plasmid M13mp19RF with the restriction enzymes SacI and HindIII were ligated with T4 DNA ligase, and J. Messing [Meth
ods in Enzymology, 101, 21-78 (1983)]. A single-stranded DNA was prepared from the obtained plaque, and E was present in the nucleotide sequence at positions 251-252 of the amino acid sequence of arginine deiminase.
Kukel's method [Proc. Nat.
In accordance with l. Acad. Sci. USA, 82, 488 (1985)], a mutation was introduced as in the case of GAATTC → GAATTT using a mutagenesis primer. The thus obtained double-stranded DNA containing the mutant arginine deiminase gene is 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. After the E. coli expression vector pMK2 was cleaved with the restriction enzyme EcoRI, the ends were blunt-ended with Mung Bean nuclease, and a vector fragment cut with the restriction enzyme HindIII was prepared. The above two fragments were ligated, and a linker DNA encoding the N-terminus shown below was synthesized to prepare an expression vector.

[0024]

Embedded image

The above three fragments were ligated with T4 DNA ligase to transform Escherichia coli strain JM109 to obtain an arginine deiminase expression plasmid pMKAD. Next, 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 and synthesized and constructed as shown below.

[0027]

Embedded image

After purifying the four DNAs, the two DNAs except for the 5'-end DNA were phosphorylated and annealed. Ligation was performed with T4 DNA ligase to obtain a double-stranded DNA. This was ligated into the above vector and the plasmid pM
K △ CAD was obtained. Furthermore, in consideration of the stabilization of the plasmid, it was decided to introduce a tetracycline resistance gene. Plasmid pBR322 was replaced with restriction enzymes EcoRI and A
After cleavage with vaI, a 1.4 Kbp tetracycline resistance gene fragment was purified and then blunt-ended with Klenow enzyme. Further, after the plasmid pMK @ CAD was digested with the restriction enzyme BamHI, a vector fragment having blunt ends with Klenow enzyme was prepared, and both fragments were ligated with T4 DNA ligase.
109 strains were transformed to obtain an expression plasmid pADTc1 in which a tetracycline gene was ligated in the reverse 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 [Microtechnical Laboratories No. 3617 (FER)
MBP-3617)] in 2 × TY medium containing 15 μg / ml tetracycline (16 g of bactotryptone /
1, Bacto yeast extract 10g / l, NaC
(5 g / l) overnight at 37 ° C. After culture, 1
The cells were collected, suspended in 100 µl of Laemmli's sample buffer, dissolved by heating, and then subjected to 3 µl of SDS-polyacrylamide gel electrophoresis. After electrophoresis, the cells were stained with Coomassie brilliant blue and decolorized to a molecular weight of 4
Arginine deiminase expression was observed at 5,000 positions (see FIG. 2).

[0030]

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

[0031]

Example 4 Arginine Deimi from Arginine Deiminase Producing Bacteria
Purification of Nase (Solubilization and Activity Regeneration ) After fermentation, 2 l of the culture was centrifuged (6,000 xg, 1
(0 min) to collect the cells. The wet cells were suspended in 200 ml of 10 mM phosphate buffer, pH 7.0, and sonicated to kill the bacteria. This suspension was centrifuged to obtain a precipitate which was an insoluble fraction. Precipitate the insoluble fraction with 4% Trito
nX-100, 10 mM phosphate buffer, 1 mM EDT
A Washed with 200 ml of pH 7.0 solution to solubilize the membrane fraction. The precipitate was washed with 10 mM phosphate buffer pH 7.0, and then washed with 6 M guanidine hydrochloride, 50 mM Tris-H.
Cl (pH 8.5), 10 mM dithiothreitol
It was added to 50 ml and solubilized at room temperature. This one
The mixture was diluted 100-fold with 0 mM potassium phosphate buffer (pH 7.0), stirred at 10 to 20 ° C. overnight, and refolded. After refolding, 4900 U of activity was obtained.

[0032]

Example 5 Purification of Activated Arginine Deiminase The diluent obtained in Example 4 was filtered through a 3.2 μm filter (manufactured by Pall Corporation). 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, the plate was washed with an equilibration buffer, and the active AD was eluted stepwise with a buffer containing 0.2 M NaCl. As a result, 4506 U of the activity was recovered.
The active fraction was treated with a 10 mM potassium phosphate buffer (pH 7.
The mixture was applied to an arginine-Sepharose 4B (Pharmacia) column equilibrated in 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) )

For each of the obtained fractions, the absorbance at 280 nm and the arginine deiminase activity were measured. The results are shown in FIGS. As you can see from this result,
Arginine deiminase was found to be contained in fractions 30-52. Finally, an activity of 4160 U was obtained. The active fraction was subjected to the method of Remuri et al. [Nature,
727, 680-685 (1970)].
When subjected to polyacrylamide gel electrophoresis, a single band was detected at a position of a molecular weight of 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, the specific activity was found to be 32 U / mg, which was equivalent to that of mycoplasma-derived arginine deiminase. Table 1 shows the degree of purification in each step.

[0035]

[Table 1] 精製 Purification step Total protein 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 The optimum pH range of the physical properties of the recombinant arginine deiminase was 6.0 to 7.0 as shown in FIG.
The optimum reaction temperature was 45 to 50 ° C. as shown in FIG. As a result of measurement by SDS-polyacrylamide gel electrophoresis, the molecular weight was about 45,000. Further, 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. The isoelectric point was measured by isoelectric focusing, and the isoelectric point was about 4.7, which coincided with mycoplasma-derived arginine deiminase. When the amino acid composition and the N-terminal amino acid sequence were examined, the amino acid composition values were as 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 initiation 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 value ──────────────────────────────────── 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)サ イ ク ル 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 shows the same activity as arginine deiminase derived from mycoplasma, and can be effectively used as an antitumor enzyme as an antitumor agent.

[Brief description of the 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 electrophoretogram 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 a purified sample of arginine deiminase derived from mycoplasma and a 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.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 9/78 C12R 1:19) (56) References JP-A-3-76579 (JP, A) JP-A-3-133383 (JP, A) JP-A-3-133381 (JP, A) JP-A-3-164173 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15 / 900 C12N 9/00-9/99 C12N 1/00-1/38 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A fragment of a tac promoter, a fragment of an origin of replication (ori) of a plasmid pUC18, a fragment containing a tetracycline resistance gene, and a DNA fragment encoding an amino acid sequence of arginine deiminase or an analog thereof. E. coli transformed with expression vector
Cultivating the organism to produce arginine deiminase or
Expression of analogs, from culture, arginine deminer
And its analogs are collected and
And a reducing agent, solubilize
Characterized by refolding with buffer
A method for producing active arginine deiminase. 2. The method according to claim 2, wherein said transformed microorganism is
No. (FERM BP-3617)
2. The active arginine deminer according to claim 1, which is Escherichia coli.
Method for producing ze.