CN104130996A - Arginine deiminase mutant from arthritis-type mycoplasma and application thereof - Google Patents

Abstract

The invention discloses an arginine deiminase mutant from an arthritis-type mycoplasma, a gene, a polyethylene glycol modified substance and an application thereof. The arginine deiminase mutant is a dipolymer which is formed by two protein subunits, wherein amino sequences of the protein subunits are represented as SEQ ID No.2 in a sequence table. The arginine deiminase mutant is increased in expression quantity in escherichia coli, is increased in renaturation efficiency, is not changed in enzyme activity and is suitable for industrial production. The polyethylene glycol modified substance of the arginine deiminase mutant can be used for preparing anti-tumor drugs, such as anti-hepatoma drugs, anti-leukemia drugs and anti-melanoma drugs.

Description

Mycoplasma arthritis arginine deiminase mutant and its application

technical field

The invention belongs to the pharmaceutical field of biologically active proteins, in particular to a mycoplasma arthritis arginine deiminase mutant and its gene, polyethylene glycol modification and application.

Background technique

Normal human cells rely on arginine succinate synthase to convert citrulline into arginine, so arginine is not an essential amino acid in human cells. Liver cancer cells and some other cancer cells lack arginine succinate synthetase, so they cannot synthesize arginine in cells and must rely on extracellular arginine for survival. Therefore, any enzyme that depletes arginine outside these cancer cells, such as arginine deiminase (arginine deiminase), which can break down L-arginine into citrulline and ammonia, can block these cancer cells from obtaining The supply of exogenous arginine will starve the cancer cells to death.

There is no arginine deiminase gene present in the human genome. Arginine deiminase with anti-cancer effects has been isolated from various microorganisms such as Mycoplasma, including Mycoplasma hominis, Mycoplasma arginini and Mycoplasma arthritidis . David Filpula and Wang Maoliang disclosed a kind of arginine deiminase cloned from ATCC14152 strain Mycoplasma arthritis in U.S. Patent US5804183, and different from the Mycoplasma arthritis arginine deiminase found in the past, each subunit The unit contains 2 cysteines, the isoelectric point is 5.35, and the Km value of the enzyme Michaelis constant is as low as 10.4μM, which can effectively decompose the high-concentration arginine in the plasma. The existing data show that this is so far The most suitable arginine deiminase found so far as a therapeutic drug.

The arginine deiminase expressed in Escherichia coli by genetic engineering technology exists in the form of inclusion body, which is insoluble and lacks enzymatic activity. Like other insoluble proteins expressed in E. coli, recovery of native arginine deiminase from inclusion bodies must undergo a polypeptide protein refolding process. Dissolve the arginine deiminase in the form of inclusion bodies with 6M guanidine hydrochloride, dilute the denatured arginine deiminase 100 times in phosphate buffer, and refold at room temperature for 48 hours to become active Arginine deiminase (see Satoru Misaw et al., High-level expression of Mycoplasma arginine deiminase in Escherichia coli and its efficient renaturation as an anti-tumor enzyme, J. of Biotechnology 36(1994) 145-155). However, in the production of arginine deiminase, it is quite difficult to industrialize the high-fold dilution renaturation process.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the defect that the existing arginine deiminase is difficult to produce industrially, and provide a kind of arginine deiminase mutant and its polyethylene Diol modifiers, genes and applications.

The present invention is based on the modeling of the amino acid sequence of arginine deiminase of Mycoplasma arthritis, designs a variety of mutants that may affect the renaturation of inclusion body proteins from The mutants with sexual efficiency were prepared, their polyethylene glycol modifications were prepared, and their application potential was discovered.

One of the technical solutions provided by the present invention is: a protein subunit, the amino acid sequence of which is shown in SEQ ID NO.2.

The second technical solution provided by the present invention is: an isolated protein, which has arginine deiminase activity and is a dimer composed of two protein subunits.

Among them, the arginine deiminase derived from Mycoplasma arthritis has a subunit amino acid sequence as shown in SEQ ID No.1, and the cysteine at position 251 of the enzyme subunit is replaced by other amino acids to form the For the mutant of the enzyme, the amino acid sequence of the mutant protein subunit is shown in SEQID No.2. The mutant protein subunit is serine, threonine, glycine, alanine, leucine, isoleucine, tyrosine, phenylalanine, proline or valine at position 251, preferably serine and threonine.

Wherein, the preparation method of the protein subunit or protein is a conventional preparation method in the art, such as isolating from an expressing transformant expressing the protein recombinantly, or obtaining it by artificial synthesis, or isolating it from a naturally occurring protein in nature get.

The third technical solution provided by the present invention is: an isolated nucleic acid, the nucleic acid encoding the protein subunit.

Wherein, the nucleic acid is any nucleic acid encoding a protein whose amino acid sequence is as shown in SEQ ID NO.2, as long as the amino acid sequence of the protein encoded by it is as shown in SEQ ID NO.2 due to codon degeneracy. Preferably, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO.3. The preparation method of the nucleic acid is a conventional preparation method in the art, such as obtaining the nucleic acid encoding the above-mentioned protein subunit by gene cloning technology, or obtaining the nucleic acid encoding the above-mentioned protein subunit by artificial full-sequence synthesis, or extracting the nucleic acid from nature A naturally occurring nucleic acid encoding a subunit of the above-mentioned protein.

The fourth technical solution provided by the present invention is: a recombinant expression vector comprising the nucleic acid according to claim 3 .

Wherein the recombinant expression vector can be obtained by conventional methods in the art, such as connecting nucleic acid molecules encoding the above protein subunits to various expression vectors, and the expression vectors can be various conventional vectors in the art . The vector preferably includes: various plasmids, cosmids, phage or virus vectors, etc., and the vector of the present invention is preferably the plasmid pET-27b(+).

The fifth technical solution provided by the present invention is: a recombinant expression transformant comprising the recombinant expression vector.

The preparation method of the recombinant expression transformant is conventional in the art, such as transforming the above recombinant expression vector into host microorganisms. The host microorganisms can be various conventional host microorganisms in the field, as long as the above-mentioned recombinant expression vector can stably replicate itself and the gene encoding the protein subunit can be effectively expressed. Wherein the host microorganism is preferably Escherichia coli (E.coli) or Bacillus subtilis, more preferably Escherichia coli BL21. Transform the aforementioned recombinant expression plasmid into E.coli BL21 to obtain the preferred genetic engineering strain of the present invention. Wherein the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electroporation method.

The sixth technical solution provided by the present invention is: a preparation method of recombinant arginine deiminase, which includes the following steps: cultivating the recombinant expression transformant, and obtaining recombinant arginine deiminase from the culture enzyme.

Wherein, the culture method is a conventional method.

Preferably, said obtaining recombinant arginine deiminase from culture comprises the following steps:

1) After disrupting the bacterial cells, isolate the recombinant arginine deiminase inclusion body;

2) Denature the inclusion bodies with guanidine hydrochloride, then dilute them at a ratio of 1:10-1:40, preferably 1:20, and refold in phosphate buffer for 32-60 hours, preferably 48 hours.

Wherein, the method for disrupting bacterial cells in step 1) is conventional, preferably ultrasonic disruption. The method for isolating inclusion bodies of recombinant arginine deiminase is conventional, preferably using 1-10% TritonX-100 at 0-10°C for 20-60 minutes, centrifuging to remove the precipitate Instantly. The centrifugation is preferably performed at 10000-15000g for 15-60 minutes, more preferably at 13000g for 30 minutes.

The method of denaturation with guanidine hydrochloride described in step 2) is a conventional method, preferably, the recombinant arginine deiminase inclusion body is suspended in a Tris solution containing guanidine hydrochloride and dimercaptothreitol (DTT) undergo a sex change. Preferably, the Tris solution is a 50mM Tris (pH8.5) solution containing 6M guanidine hydrochloride and 10mM dimercaptothreitol. The sodium phosphate buffer of described sodium phosphate buffer is preferably 10mM pH7.0 sodium phosphate buffer.

Preferably, the product obtained by renaturation is further purified with Q Sepharose FF ion exchange chromatography column.

The seventh technical solution provided by the present invention is: a polyethylene glycol modification of the protein.

Wherein, preferably, the lysine of the protein is modified by polyethylene glycol. Preferably, the polyethylene glycol is polyethylene glycol 5000-20000. Preferably, one molecule of the protein is modified with 1-64 polyethylene glycol molecules.

The eighth technical solution provided by the present invention is: the application of the polyethylene glycol modification of the protein in the preparation of antitumor drugs.

Wherein, said tumor is conventional, preferably liver cancer, malignant melanoma or leukemia.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The present invention replaces a cysteine group, while retaining the original enzyme activity of arginine deiminase, increases the expression level of arginine deiminase in Escherichia coli, and improves arginine deiminase The renaturation efficiency of the deiminase is suitable for the industrialization of the recombinant arginine deiminase.

Description of drawings

Figure 1 is a three-dimensional structure diagram of arginine deiminase of Mycoplasma argininoformis, in which the 251st cysteine is replaced by other amino acids (serine, threonine, glycine, alanine, leucine, isoleucine amino acid, tyrosine, phenylalanine, proline, or valine) substitution.

Detailed ways

The present invention mutates the 251st cysteine of mycoplasma arthritis arginine deiminase, the mutation does not affect the enzyme activity, but improves the expression efficiency of the recombinant arginine deiminase in Escherichia coli The output is improved, the renaturation efficiency is improved, and it is suitable for industrial production.

1. Serine group replaces cysteine group C251

M. arthritis arginine deiminase is a dimer composed of the same two subunits. The gene or DNA molecule encoding arginine deiminase mentioned in the present invention, and the protein molecule of arginine deiminase both refer to one of the two subunits. Each subunit polypeptide contains two cysteine groups, C251 and C398. The catalytic center of M. arthritis arginine deiminase is a triangular structure consisting of cysteine group C398, glutamic acid group E213 and histidine group H269. The present invention is based on the coded amino acid sequence of arginine deiminase cloned from ATCC14152 mycoplasma arthritis disclosed in U.S. Patent No. 5,804,183, and uses computer software to simulate the function of arginine deiminase of mycoplasma arthritis. Three-dimensional structure, replacing the C251 or C398 cysteine group with a serine group, respectively. When the cysteine group C251 is replaced, the three-dimensional structure of arginine deiminase does not change significantly; while the cysteine group C398 is replaced by a serine group, it will destroy arginine deiminization Enzyme Catalytic Center.

Further studies have shown that other amino acids replacing the cysteine group C251 of arginine deiminase have similar effects to serine, such as threonine, glycine, alanine, leucine, isoleucine , tyrosine, phenylalanine, proline and valine. Serine and threonine are preferred, most preferred is serine, see Examples below.

2. Recombinant expression of arginine deiminase gene with directed mutation

The nucleotide sequence shown in SEQ ID NO.3 is generated by directed gene mutation technology on the arginine deiminase gene of Mycoplasma arthritis. The generated arginine deiminase-directed mutant gene can be expressed in various prokaryotic gene expression systems. All extrachromosomal vectors used for expression in Escherichia coli, such as pET and pBAD, can be used to express the arginine deiminase gene of the present invention. In order to controllably and efficiently express arginine deiminase, the extrachromosomal vector should contain promoter, operator, ribosome binding site, transcription terminus, replication origin, antibiotic marker and regulatable suppressor gene. Promoters include, but are not limited to, T7, araBAD, phoA, trc, and the like. T7 promoter is preferred. Methods for inducing expression include, but are not limited to, changing the temperature of the culture medium, adding arabinose, or adding isopropyl-β-dextranthiogalactoside (IPTG), etc. The method of inducing the lactose operon with IPTG is preferred. For the Escherichia coli containing the arginine deiminase gene, the selected antibiotic markers include, but are not limited to, ampicillin, kanamycin or tetracycline. The plasmid containing the mutated gene of arginine deiminase was transformed into competent Escherichia coli BL21 (Escherichia coli B series 834 strain, Escherichiacoli834strain) for expression. During the fermentation process, the composition, temperature, pH, concentration of inducing factors and induction time of E. coli culture solution can be adjusted. When the optical density OD ₆₀₀ of the Escherichia coli culture medium reaches 5-50, the expression inducing factor IPTG is added, and after an induction process of 0.5 to 50 hours, the Escherichia coli cells are harvested from the culture medium.

3. PEG Modification of Directed Mutagenesis of Arginine Deiminase

The directed mutation arginine deiminase of the present invention is preferably modified with polyethylene glycol. Among them, polyethylene glycol can modify the mutated arginine deiminase through a variety of active groups, and the amino group (nucleophilic group) of lysine is the most optimal. After being modified by polyethylene glycol, the molecular volume of arginine deiminase becomes larger, the molecular weight increases, the plasma half-life is prolonged, and more importantly, it is more stable in the body and is not easily decomposed by proteases. Although the enzymatic activity per milligram of protein is weaker than that before modification, as long as the same amount of enzymatic activity of polyethylene glycol-modified arginine deiminase is given, the weakened activity per milligram of protein can be compensated.

The polyethylene glycol used for modification in the present invention can be linear, branched or multi-chain. The modification of the polyethylene glycol of the present invention may be permanent or transient. Each targeted mutagenized arginine deiminase dimer is preferably modified with 1-64 polyethylene glycol molecules. The molecular weight of polyethylene glycol preferably varies from 1,000 to 40,000. The molecular weight of mycoplasma arthritis arginine deiminase is 46309 Daltons, and in the reaction process of polyethylene glycol modified arginine deiminase, the mole of polyethylene glycol and arginine deiminase The ratio is preferably from 10:1 to 200:1.

4. Application of polyethylene glycol-modified optimized arginine deiminase

The polyethylene glycol-modified mutated arginine deiminase of the present invention can be used to prepare anti-tumor drugs, and the anti-tumor drugs include, but not limited to, anti-liver cancer drugs, anti-malignant melanoma drugs, anti-tumor drugs, Prostate cancer drugs, etc. The polyethylene glycol-modified product solution of arginine deiminase can be injected subcutaneously, intramuscularly, or intravenously in an appropriate dose for treatment.

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

The design of the directed mutation of the arginine deiminase gene of embodiment 1 mycoplasma arthritis

Based on the coding nucleotide sequence of the arginine deiminase of ATCC14152 strain Mycoplasma arthritidis (Mycoplasma arthritidis) in U.S. Patent US5804183, simulate the arginine deiminase in Mycoplasma arthritidis with PyMOL0.99rc6 software The three-dimensional structure (see Figure 1), it can be seen that the cysteine group C251 is far away from the active center of arginine deiminase, and other amino acids (serine, threonine, glycine, alanine, leucine, isoleucine, tyrosine, phenylalanine, proline or valine) to replace the cysteine group C251, and 10 mutants were designed. Three-dimensional structure analysis showed that these mutations did not significantly change the three-dimensional structure of arginine deiminase.

Example 2 Directed Mutation and Recombinant Expression of Mutant Arginine Deiminase Gene

Based on the nucleotide sequence encoding arginine deiminase from ATCC14152 strain Mycoplasma arthritis described in U.S. Patent No. 5,804,183, according to Escherichia coli encoding serine, threonine, glycine, alanine, leucine, Different codons for isoleucine, tyrosine, phenylalanine, proline and valine, design mutated nucleotide sequences, and entrust the gene company to synthesize nucleotides with the original enzyme sequence and mutant sequence .

The proenzyme gene and the arginine deiminase gene produced by directed mutation were inserted behind the T7 promoter of the plasmid pET-27b(+). Then transform into competent Escherichia coli BL21 cells (Escherichia coli B series 834 strains, Escherichia coli834strain). The DNA sequence of the targeted mutation gene was confirmed by nucleotide sequence analysis as shown in SEQ ID NO.3.

The wild-type proenzyme or BL21 Escherichia coli containing the arginine deiminase gene plasmid produced by directed mutation was fermented at 37°C in a 10-liter fermenter (5 liters of medium). Add 10 mg of kanamycin per liter of bacterial culture (LB medium). When the optical density OD ₆₀₀ reading value reached 20, 1 mM isopropyl-β-dextrothiogalactoside (IPTG) was added to induce expression for 16 hours, and the E. coli cells were harvested. The inclusion bodies of the wild-type proenzyme and different mutants are shown in Table 1 below, and the expression of the mutants increased.

Table 1. The amount of inclusion body after expression of different mutants of wild-type proenzyme (g/5L)

Refolding and purification of embodiment 3 directed mutation arginine deiminase

The Escherichia coli cells harvested in Example 2 were centrifuged to obtain a bacterial cell precipitate. Suspend 10 g of bacterial sediment in 100 ml of 10 mM sodium phosphate buffer (pH 7.0). After sonicating the cells, 4% TritonX-100 was added and stirred at 4°C for 30 min. Inclusion bodies were obtained by centrifugation at 13000g for 30 minutes. Then the proenzyme inclusion body was suspended in 10 ml of 50 mM Tris (pH 8.5) solution containing 6M guanidine hydrochloride and 10 mM dimercaptothreitol (DTT), and stirred at 4°C for 15 minutes. Finally, the solution was added to 1 liter of 10 mM sodium phosphate buffer (pH 7.0), stirred at 15°C for 90 hours, and 70 mg of purified arginine deiminase was obtained per liter of culture solution.

Suspend the mutated arginine deiminase inclusion body in 10 ml of 50 mM Tris (pH 8.5) solution containing 6M guanidine hydrochloride and 10 mM dimercaptothreitol (DTT), and stir at 4°C for 15 min . Finally, add the solution to 300 ml of 10mM sodium phosphate buffer solution (pH7.0) and stir at 15°C for 90 hours. The amount of purified arginine deiminase per liter of culture solution can be seen in Table 2. The renaturation efficiency of the mutants was improved, not only the ratio of renaturation solution was reduced to 1:30, but also the amount of active protein was increased.

Table 2. Concentration of inclusion body refolding protein of proenzyme and different mutants (mg/L)

The expressed directional mutant arginine deiminase and wild-type proenzyme were purified by 5 ml QSepharose FF ion-exchange chromatography column (GE Healthcare) to obtain purified refolded mutant arginine deiminase, The homogeneous protein was confirmed by SDS-polyacrylamide gel electrophoresis.

The activity measurement of embodiment 4 arginine deiminase

The activity of arginine deiminase is measured by the amount of citrulline produced by catalyzing arginine (see: Evelyn L. Oginsky: Methods Enzymology 3 (1957): 639-643). One unit of arginine deiminase activity is the amount of enzyme activity required to convert 1 micromole molecule (μmol) of arginine to citrulline in one minute at 37°C.

Arginine deiminase activity was assayed in 96-well microplates. Mix 5 microliters of citrulline samples with a standard concentration of 1-10mM and 5 microliters of samples to be tested with 5 microliters of arginine substrate, react at 37°C for 15 minutes, add 200 microliters of acidified 3% The biacetyl monoxime solution was incubated at 80°C for 30 minutes, and the absorbance value at a wavelength of 490nm was read. According to the curve equation of the citrulline standard sample, the enzyme activity of the sample to be tested is calculated. The activity of arginine deiminase did not change after directed mutation, as shown in Table 3.

Table 3. Enzyme specific activity of the original enzyme and different mutants (U/mg)

Example 5 Preparation of SC-PEG Modified Objects of Arginine Deiminase Before and After Targeted Mutation and Its Enzyme Activity

In 10 ml of 0.1M sodium phosphate buffer (pH 8.0), 50 mg of arginine deiminase (active enzyme protein non-inclusion body, obtained by renaturation in Example 3) was mixed with 1.0 g of Dalton's SC-PEG (purchased from Korea ID Biochemical Company) was reacted at 4°C with electromagnetic stirring for 30 minutes. The reaction product was then added to a 3 ml Superdex200 chromatography column (purchased from GE Healthcare), and eluted with 50 mM sodium phosphate buffer (pH 6.5) containing 150 mM sodium chloride to obtain purified SC-PEG-modified arginine Deiminase. Enzyme activity was measured according to the method of Example 4. After SC-PEG modification, the activity of arginine deiminase was 20 units per milligram, and the activity of arginine deiminase after targeted mutation was shown in Table 4. Compared with directed mutation, arginine deiminase can retain more enzymatic activity after being modified by SC-PEG (5KD, 12KD and 20KD).

Table 4. Enzyme specific activity of the original enzyme and different mutants after PEG modification (U/mg)

Example 6 Anti-tumor effect of arginine deiminase before and after being modified by SC-PEG

Dilute the sample with PBS to obtain diluted samples with concentrations of 1000, 100, 10, 1, 0.1 and 0.01 μg/ml respectively; add the diluted samples to a flat-bottomed 96-well plate, 10 μl per well, and make two parallels at each point test. The DMSO phase was serially diluted and added to the plate as a control; the cells in the logarithmic growth phase were taken and the density of the cell suspension was adjusted to 2×10 ⁵ /ml. In a flat-bottom 96-well plate, add 90 μl of cell suspension to each well, and incubate for 48 hours in a 37° C., 5% CO ₂ cell incubator. Add 10 μl of MTT solution with a concentration of 5 mg/ml to each well, continue to incubate in the incubator for 3 to 4 hours, add 100 μl of DMSO to each well, and continue to incubate overnight in the incubator to fully dissolve the formed formazan crystals. The absorbance at 580 nm was measured. The data were analyzed with XLfit Wizard software, and the half inhibitory concentration (median inhibitory concentration, IC50) of each group of drugs on cell growth inhibition was calculated. The results are shown in Table 5 and Table 6. Table 5 shows that the arginine deiminase proenzyme involved in the present invention and 251 cysteine are replaced by different amino acids to form mutants that have the activity of inhibiting melanoma, liver cancer cells, and leukemia cells. Table 6 shows that the proenzyme and the corresponding mutants after PEG modification also have the activity of inhibiting three kinds of tumor cells, indicating that it can be used to develop drugs for treating three kinds of tumors (anti-liver cancer, leukemia, melanoma, etc.).

Table 5. In vitro anti-tumor effects of different mutants before PEG modification

Table 6. In vitro anti-tumor effects of different mutants modified with PEG

Claims (10)

1. A protein subunit, characterized in that the amino acid sequence of the protein subunit is as shown in SEQ ID NO.2. 2. An isolated protein, characterized in that the protein has arginine deiminase activity, and the protein is a dimer composed of two protein subunits as claimed in claim 1. 3. An isolated nucleic acid, characterized in that, the nucleic acid is a nucleic acid encoding a protein subunit as claimed in claim 1. 4. A recombinant expression vector comprising the nucleic acid according to claim 3. 5. A recombinant expression transformant comprising the recombinant expression vector according to claim 4. 6. A preparation method for recombinant arginine deiminase, characterized in that it comprises the steps of: cultivating the recombinant expression transformant as claimed in claim 5, obtaining recombinant arginine deiminase from the culture enzyme. 7. A polyethylene glycol modification of protein as claimed in claim 2. 8. The polyethylene glycol modification of protein as claimed in claim 7, characterized in that, the lysine of the protein is modified by polyethylene glycol. 9. The polyethylene glycol modification of protein according to claim 7, wherein said polyethylene glycol is polyethylene glycol 5000-20000. 10. The use of the polyethylene glycol modification of protein as claimed in claim 7 in the preparation of antitumor drugs.