A pH-stable arginine deiminase mutant and its application in tumor treatment

Abstract

The present invention discloses an arginine deiminase mutant with high pH stability and its application in tumor treatment. The arginine deiminase mutant provided by the present invention has improved pH stability, and after PEG modification, still maintains good pH stability, solves the problems of low storage stability of arginine deiminase and low stability under physiological pH 7.4, especially the mutant K208R / K258H / L318F, the enzyme activity is 110% of the parent, the pH stability and pH stability after PEG modification are good.

Description

Technical Field

[0001] The invention relates to an arginine deiminase mutant with high pH stability and application thereof in tumor treatment, belonging to the technical field of genetic engineering. Background Art

[0002] There is an urgent need to find a new method for treating tumors that can specifically treat tumors without damaging normal cells. Tumor metabolism is a target for the treatment of certain malignant tumors and has become a focus of attention in recent years. Amino acid deprivation therapy, which degrades or deprives tumors of amino acids necessary for survival based on metabolic enzymes, is a typical representative and has been successfully applied in clinical trials. Arginine, as one of the conditionally essential amino acids, is derived from citrulline through the catalytic conversion and synthesis of two key enzymes, arginine succinate synthetase (ASS1) and arginine succinate lyase, and plays a key role in cell division and metabolism. The growth and proliferation of some malignant tumor cells must rely on exogenous arginine, and the loss of the key rate-limiting enzyme ASS1 in the synthesis process makes arginine deprivation therapy an effective method for treating ASS1-deficient tumors.

[0003] Arginine deiminase (ADI, EC3.5.3.6) can irreversibly hydrolyze L-arginine into L-citrulline and ammonia, and is a protein drug for treating arginine auxotrophic cancer. Injecting arginine deiminase into the human blood circulation will consume and cut off the supply of arginine necessary for the growth of arginine auxotrophic cancer cells in the blood, inhibit the growth and proliferation of tumor cells, and thus achieve the purpose of killing cancer cells and treating cancer. Studies have shown that arginine deiminase can effectively treat liver cancer, skin cancer, melanoma, and acute myeloid leukemia. However, for clinical applications, the direct application of microbial ADI to the human body will produce neutralizing antibodies, resulting in short half-life, reduced activity, and immune response. In order to overcome this defect, Polaris Pharmaceuticals in the United States has developed polyethylene glycol-modified ADI (ADI-PEG), using SS-PEG with a molecular weight of 20kDa to modify recombinantly expressed ADI. The modified molecule well maintains the activity of the enzyme, while blocking the antigen epitope, and the level of neutralizing antibody production is also significantly reduced. ADI currently used in clinical research mainly comes from mycoplasma, but still has the defect of low activity to arginine under physiological conditions. Researchers are constantly exploring the application of arginine deiminase from other sources in tumor treatment. CN 112094837A discloses a recombinant arginine deiminase, which has an enzyme activity increased by 980% compared with the wild type, significantly reduced Michaelis constant for arginine, significantly improved catalytic efficiency, and has an in vitro anti-tumor effect on melanoma cells, liver cancer cells and prostate cancer cells, with an IC50 of less than 1ng / mL.

[0004] The normal pH value of the human body is usually 7.35-7.45, which requires that the enzymatic activity and stability of arginine deiminase under physiological pH conditions can be improved to better play a catalytic role; however, the degree of PEG shedding is related to the pH of the PEGylated arginine deiminase injection. The higher the pH of the buffer system, the more serious the PEG shedding, and the storage stability is relatively good below pH 6.5. This puts higher requirements on the pH stability of arginine deiminase. Summary of the invention

[0005] In order to solve the above technical problems, the present invention uses arginine deiminase as a starting enzyme and uses genetic engineering to transform the enzymatic properties of arginine deiminase.

[0006] The object of the present invention is to provide an arginine deiminase mutant with high pH stability, wherein the mutant is obtained by mutating the aspartic acid at position 112 of the arginine deiminase with an amino acid sequence as shown in SEQ ID NO.1 into asparagine, and mutating the lysine at position 258 into histidine; or by mutating the lysine at position 208 of the arginine deiminase with an amino acid sequence as shown in SEQ ID NO.1 into arginine, mutating the lysine at position 258 into histidine, and mutating the leucine at position 318 into phenylalanine.

[0007] The second object of the present invention is to provide a nucleotide sequence encoding the arginine deiminase mutant.

[0008] The third object of the present invention is to provide an expression vector carrying the nucleotide sequence.

[0009] Furthermore, the expression vector is a PET series expression vector.

[0010] The fourth object of the present invention is to provide a recombinant bacterium expressing the arginine deiminase mutant.

[0011] Furthermore, the recombinant bacteria uses Escherichia coli as a host.

[0012] Furthermore, the Escherichia coli is E. coli BL21 (DE3).

[0013] The fifth object of the present invention is to provide the use of the arginine deiminase mutant in the preparation of tumor therapeutic drugs.

[0014] Furthermore, the tumor is liver cancer or melanoma.

[0015] Beneficial Effects

[0016] The arginine deiminase mutant provided by the present invention has improved pH stability, and after PEG modification, still maintains good pH stability, thereby solving the problems of low storage stability of arginine deiminase and low stability under physiological pH 7.4. In particular, the mutant K208R / K258H / L318F has an enzyme activity of 110% of the parent, and has good pH stability and pH stability after PEG modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the pH stability of the parent arginine deiminase and the mutant;

[0018] Figure 2 It is the pH stability of the mutant modified by PEG of the present invention. DETAILED DESCRIPTION

[0019] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0020] Example 1: Construction of recombinant arginine deiminase ADI genetically engineered bacteria

[0021] (1) Target gene synthesis

[0022] The gene of arginine deiminase with the amino acid sequence shown in SEQ ID NO.1 was synthesized by Shanghai Bioengineering Co., Ltd.

[0023] (2) Construction of recombinant expression vector pET28a(+)-ADI

[0024] The pET28a(+) plasmid and the gene synthesized in step (1) were double-digested with BamH I and Xho I, respectively, and the digestion products were recovered. The target gene after double digestion was ligated and recombined with the linearized pET-28a vector using Solution I ligase to construct a recombinant expression vector pET28a-ADI. The recombinant expression vector was transformed into E. coli BL21(DE3) cells, and the cells were plated on LB plates containing 50 μg / mL kanamycin to screen positive clones, and E. coli BL21(DE3) / pET28a(+)-ADI was sequenced and stored.

[0025] (3) Site-directed mutagenesis of arginine deiminase, construction of recombinant expression plasmid and recombinant bacteria

[0026] Through computer software simulation calculation, and based on conservative sequence analysis and structural characteristics, four mutation sites were selected: D112N, K208R, K258H, and L318F. Single mutations and further combined mutations were performed for a total of 10 groups of mutation sequences. The sequences were sent to Shanghai Bioengineering Co., Ltd. for synthesis.

[0027] According to the method in step (2), the synthesized sequence was connected to the expression vector, and then transformed into E. coli BL21 (DE3), and the positive clones were screened on LB plates containing 50 μg / mL kanamycin, and the mutants were sequenced and preserved.

[0028] Example 2: Expression and purification of parent arginine deiminase and mutants

[0029] The strain E. coli BL21 (DE3) / pET28a (+) -ADI of Example 1 and the mutant strain obtained by mutation were inoculated into LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37 ° C, 180 rpm for 12 h. The inoculum was inoculated into a fresh LB liquid culture medium containing a final concentration of 50 μg / mL kanamycin at a volume concentration of 2.0% (v / v), and cultured at 37 ° C, 180 rpm for 2 h. Then, IPTG with a final concentration of 0.20 mM was added to the culture solution. After culturing at 28 ° C for 12 h, the culture solution was centrifuged at 4 ° C, 8000 rpm for 10 min to obtain the corresponding wet bacterial cells.

[0030] The obtained wet cells were centrifuged at 10000rpm, 4℃ for 10min and the cells were collected and washed twice with 0.9% (w / v) saline. According to the total amount of 25g / L of the cells, they were added to pH 7.0, 100mM PBS buffer and resuspended, and ultrasonically disrupted on an ice-water mixture for 6min. The ultrasonic disruption conditions were: power of 400W, disruption for 1s, and pause for 1s to obtain the parent arginine deiminase and the mutant crude enzyme solution. The supernatant was collected by centrifugation at 10000rpm, 4℃ for 10min, and after microfiltration through a 0.45μm membrane, the mutant protein was purified using Super-Q anion exchange resin, and desalted with PD-10 to obtain the parent arginine deiminase and the mutant protein.

[0031] Example 3: Determination of the enzyme activity of the parent arginine deiminase and mutants

[0032] Arginine deiminase can catalyze the hydrolysis of substrate arginine to generate product citrulline. Citrulline reacts with a color developing solution and absorbs at a wavelength of 530 nm after color development. The enzymatic activity of arginine deiminase is calculated by detecting the content of product citrulline generated under specific reaction temperature, specific reaction pH and specific reaction time.

[0033] Arginine deiminase activity assay: L-arginine hydrochloride was used as substrate, pH 6.5, 0.05 mol·L -1 PBS was prepared with 0.2 mol·L -1Substrate solution. Take 100 μL of appropriately diluted enzyme solution and add it to 1 mL of substrate solution. React at 37°C for 30 minutes. After the reaction is completed, add 1 mL of trichloroacetic acid to the test tube and cool it on ice. Then boil it in a 100°C boiling water bath for 5 minutes to inactivate the enzyme and terminate the reaction. After the product L-citrulline undergoes a color reaction, measure its absorbance at 530 nm.

[0034] Definition of ADI enzyme activity: At 37°C, the amount of enzyme required to convert 1 μmol of L-arginine hydrochloride into L-citrulline per minute is defined as 1 U.

[0035] Drawing of L-citrulline standard curve: Preparation concentration is 0.1 g·L -1 The L-citrulline standard solution was diluted with deionized water to 0.002, 0.005, 0.01, 0.015, and 0.02 g·L -1 The color reaction was carried out according to the method reported in the literature, and the absorbance at 530 nm was measured. A standard curve was drawn with OD530 as the abscissa and different concentrations of L-citrulline solution as the ordinate.

[0036] The parent arginine deiminase and mutants of Example 1 were tested for enzyme activity using the above method. The results are shown in Table 1:

[0037] Table 1 Relative enzyme activities of the parent arginine deiminase and mutants

[0038] Arginine deiminase Relative enzyme activity (%) WT 100％ D112N 94％ K208R 78％ K258H 103％ L318F 124％ D112N / L318F 135％ K208R / L318F 108％ D112N / K258H 95％ K208R / K258H 65％ D112N / K258H / L318F 138％ K208R / K258H / L318F 110％

[0039] Example 4: pH stability determination of parent arginine deiminase and mutants

[0040] The parent arginine deiminase and the mutant in Example 1 were placed in PBS buffer at pH 5.0-8.0 (interval 0.5), and the residual enzyme activity at different times at 37°C was measured, with the highest enzyme activity set as 100%. The results are shown in FIG. Figure 1 The results showed that compared with the parent, the mutants of the present invention, K208R, K258H, D112N / L318F, D112N / K258H, K208R / K258H and K208R / K258H / L318F all had improved pH stability, especially better pH stability in a neutral environment.

[0041] Embodiment 5:

[0042] The mutants of K208R, K258H, D112N / L318F, D112N / K258H, K208R / K258H and K208R / K258H / L318F were added with PEG modifier with molecular weight of 20k at a mass ratio of 1:20 and stirred at room temperature for 2h. The reaction solution was added to a 100kDa ultrafiltration tube and centrifuged at 4000r / min. -1 , add 20mmol·L every 10min -1 PBS, pH 7.0 was used for filtration and washing, and this was repeated 3-5 times to remove free PEG molecules and obtain PEG-ADI.

[0043] The prepared PEG-ADI was placed in PBS buffer at pH 5.0-8.0 (interval 0.5), and the residual enzyme activity at different times at 37°C was measured, and the highest enzyme activity of the corresponding unmodified mutant was set as 100%. Figure 2 shown.

[0044] The results showed that although the above mutants had improved stability without modification, the stability of some mutants decreased after PEG modification. D112N / K258H and K208R / K258H / L318F maintained good stability.

[0045] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An arginine deiminase mutant with high pH stability, characterized in that The mutant is a mutant in which the aspartic acid at position 112 of the arginine deiminase in the amino acid sequence as shown in SEQ ID NO.1 is mutated to asparagine, and the lysine at position 258 is mutated to histidine; or a mutant in which the lysine at position 208 of the arginine deiminase in the amino acid sequence as shown in SEQ ID NO.1 is mutated to arginine, the lysine at position 258 is mutated to histidine, and the leucine at position 318 is mutated to phenylalanine.

2. A nucleotide encoding the arginine deiminase mutant according to claim 1.

3. An expression vector carrying the nucleotide sequence of claim 2.

4. The expression vector according to claim 3, characterized in that The expression vector is a PET series expression vector.

5. A recombinant bacterium expressing the arginine deiminase mutant according to claim 1.

6. The recombinant bacterium according to claim 5, characterized in that The recombinant bacteria uses Escherichia coli as a host.

7. The recombinant bacterium according to claim 6, characterized in that: The Escherichia coli is E. coli BL21 DE3.

8. Use of the arginine deiminase mutant according to claim 1 in the preparation of a drug for treating tumors, wherein the tumor is liver cancer or melanoma.

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