Genetically engineered arginine deiminase modified by site-directed mutagenesis

Abstract

A genetically engineered arginine deiminase reconstructed by site-directed mutagenesis belongs to the technical field of genetic engineering technology. Its amino acid sequence is shown as SEQ ID No. 1. In the amino acid sequence of the arginine deiminase reconstructed by site-directed mutagenesis, glycine at position 264 is mutated to proline, compared to an amino acid sequence of native arginine deiminase. Compared with wild type enzyme, the effective pH range effect of the mutated arginine deiminase according to the present invention is broadened to a certain extent, and especially a good enzyme activity is achieved at physiological pH 7.4. With the broadening of the effective pH effect range, the mutant enzyme still has higher stability under the condition of pH 5.5-7.5. Therefore, the problem that the arginine deiminase generally is low in enzymatic activity and short in half-life in vivo under physiological conditions in clinical application for tumor therapy is solved, and a good condition for using the enzyme and an encoding gene thereof for clinical treatment is created.

Claims

1. An arginine deiminase mutant, comprising the amino acid sequence of SEQ ID NO: 1, wherein the mutant possesses improved enzyme activity and stability relative to a wild type arginine deiminase of Enterococcus faecalis.

2. The arginine deiminase mutant of claim 1, wherein the mutant is encoded by the nucleotide sequence of SEQ ID NO: 2.

3. A host cell comprising the nucleotide sequence of SEQ ID NO: 2 encoding the arginine deiminase mutant of claim 1.

4. The host cell of claim 3, wherein the host cell is an Escherichia coli cell.

5. The host cell of claim 4, wherein the host cell is an E. coli BL21(DE3) cell.

6. A method of treating a subject who has an arginine-deficient tumor, comprising: administering a pharmaceutically effective amount of a composition comprising the arginine deiminase mutant of claim 1 to the subject.

7. A method of treating leukemia in a subject, comprising: administering a pharmaceutically effective amount of a composition comprising the arginine deiminase mutant of claim the subject.

8. A method of enzymatically producing citruline, comprising adding the arginine deiminase mutant of claim 1 to a solution comprising arginine under conditions in which the mutant catalyzes the conversion of arginine to citruline, thereby producing citruline.

9. The method of claim 6, wherein the arginine-deficient tumor is a breast cancer tumor.

10. The method of claim 6, wherein the arginine-deficient tumor is a liver cancer.

11. The arginine deiminase mutant of claim 1, wherein the stability of the mutant is increased by at least 2 pH units at 45 C. as compared to a wild type arginine deiminase enzyme from Enterococcus faecalis.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1: Construction map of recombinant plasmid pET-28a-ADI.

(2) FIG. 2: A graph showing optimal pH changes of wild enzyme and mutant enzyme.

(3) FIG. 3: A graph showing pH stability changes of wild enzyme and mutant enzyme.

DETAILED DESCRIPTION

(4) The present invention is further clarified below by examples, and the following examples are intended to illustrate and not to limit the scope of the present invention.

(5) Materials and reagents: Restriction enzymes, Solution I ligase, PCR reagents and the like were all purchased from TaKaRa Biotechnology Co., Ltd.; plasmid extraction kit, genome extraction kit, agarose purification kit, E. coli DH5 , BL21(DE3) strains and primers were all purchased from Sangon Biotech (Shanghai) Co., Ltd.; other reagents were all analytically pure reagents purchased domestically or abroad.

EXAMPLE 1: CONSTRUCTION OF RECOMBINANT PLASMID

(6) Enterococcus faecalis SK32.001 was cultured to the mid-exponential growth phase. 2 mL of a bacteria solution was centrifuged at 10000 r/min for 10 min. Supernatant was discarded. After lysozyme treatment for 30 min, genomic DNA was extracted according to a kit instruction.

(7) The following primers were designed for amplification of arcA:

(8) TABLE-US-00001 FADI-2: (SEQIDNO:5) 5-CGCGGATCCATGAGTCATCCAATTAATGT-3 (containingBamHIrestrictionsite), and RADI-2: (SEQIDNO:6) 5-CCGCTCGAGTTAAAGATCTTCACGGT-3 (containingXhoIrestrictionsite).

(9) PCR amplification conditions: denaturation at 95 C. for 3 min, 30 cycles (95 C. for 30 s, 55 C. for 30 s, and 72 C. for 210 s) and finally extension at 72 C. for 2 min.

(10) After purification of an amplified product, the PCR product and a vector pET-28a-c(+) were double-digested with BamH I and Xho I, and enzyme-digested products were separately recovered. The products were ligated with Solution I ligase at 16 C. for 2 h and transformed into DH5 cells by heat shock. After transformants were grown on a plate, single colonies were picked into an LB medium, and the plasmid was extracted. The recombinant plasmid pET-28a-ADI was verified by enzyme digestion. The plasmid was transformed into BL21(DE3) cells to obtain BL21(DE3)/pET-28a-ADI engineered bacteria.

EXAMPLE 2: SITE-DIRECTED MUTAGENESIS

(11) Primer design was performed based on the encoding gene encoding arcA in Enterococcus faecalis SK23.001.

(12) TABLE-US-00002 G264P-forwardprimer: (SEQIDNO:7) 5-CTTGGCTTTTGATATCCCTGAACATCGTAAATTC-3, and G264P-reverseprimer: (SEQIDNO:8) 5-GATATCAAAAGCCAAGATATTTTTGAATCCTA-3.

(13) The underlined portion represents a codon corresponding to glycine at position 264 encoded by the mutant gene. The PCR amplification system is:

(14) TABLE-US-00003 10 X Reaction Buffer 5 dNTP mix 1 Forward primer (100 ng/L) 1.25 Reverse primer (100 ng/L) 1.25 Template pET-28a-ADI (10 ng) 2 PfuTurbo DNA polymerase (2.5 U/L) 1 ddH.sub.2O 38.5 Total volume 50

(15) After PCR amplification, 1 L of Dpn I restriction enzyme (10 U/L) was added into a reaction solution, and a template was eliminated by incubation at 37 C. for 1 h. A PCR product was transformed into E. coli DH5 cells and coated on a plate. Single colonies were picked to an LB medium, a plasmid was extracted, and a correct mutant plasmid was obtained by sequencing. The successfully constructed mutant plasmid was transformed into E. coli BL21(DE3) to obtain a mutant strain BL21(DE3)/pET-28a-ADIG264P.

EXAMPLE 3: EXPRESSION AND PURIFICATION OF WILD ENZYME AND MUTANT ENZYME

(16) BL21(DE3)/pET-28a-ADI and pET-28a-ADIG264P single colonies were picked up and cultured in an LB medium containing 0.5 mmol/L kanamycin at 37 C. and 200 r/min for 12 h, then transferred into an LB medium containing 0.5 mmol/L kanamycin and cultured at 37 C. and 200 r/min until OD.sub.600 fell within in the range of 0.5-0.7. 1 mmol/L IPTG was added and the mixture was induced under the conditions of 28 C. and 200 r/min for 9 h.

(17) Fermentation broth was centrifuged at 10000 r/min and 4 C. for 10 min, and then supernatant was discarded. Washing with a phosphate buffer was performed twice, thallus was suspended by adding 15-20 mL of phosphate buffer, and ultrasonicated for 15 min (power 22 W, ultrasonication for 1 s, and intermittence for 2 s). Centrifugation was performed under the conditions of 4 C. and 10000 r/min for 10 min, and supernatant (namely crude enzyme solution) was collected, and filtered through a hydrophilic membrane with a pore size of 0.22 m.

(18) An Ni.sup.2+chelate agarose resin column was pre-equilibrated with a Binding Buffer; the crude enzyme solution was added, and the column was equilibrated with the Binding Buffer and a Washing Buffer respectively; enzyme was eluted with an Elution Buffer and recovered; the recovered enzyme solution was dialyzed in a dialysis buffer and then stored in a refrigerator at 4 C.

Formulation of the Involved Buffers

(19) 1. phosphate buffer (PB): 50 mmol/L, pH 5.5;

(20) 2. Binding Buffer: 50 mmol/L PB, 500 mmol/L NaCl, pH 7.0;

(21) 3. Washing Buffer: 50 mmol/L PB, 500 mmol/L NaCl, pH 7.0, 50 mmol/L imidazole;

(22) 4. Elution Buffer: 50 mmol/L PB, 500 mmol/L NaCl, pH 7.0, 500 mmol/L imidazole;

(23) 5. Dialysis buffer: 50 mmol/L PB, pH 5.5, 10 mmol/L EDTA.

EXAMPLE 4: DETERMINATION OF OPTIMUM pH AND pH STABILITY OF WILD ENZYME AND MUTANT ENZYME

(24) Enzyme activity assay method: 0.49 mL of 50 mmol/L PB buffer was added into 0.5 mL of a substrate L-arginine (concentration: 10 g/L) and heat-insulated at 45 C. for 5 min, and then 0.01 mL of an enzyme solution was added. A reaction was carried out at 45 C. for 10 min and terminated by boiling for 10 min. A reaction solution was centrifuged, supernatant was removed, and the content of citrulline was determined.

(25) Definition of enzyme activity: The amount of enzyme required to produce 1 mol L.sup.1 citrulline within 1 min is defined as one enzyme activity unit (U).

(26) Determination of citrulline content: high performance liquid chromatography: Angilent 1200; chromatographic column: Hypersil ODS (5 m, 4.0 mm250 mm); mobile phase A: 2 L of water, 13 g of sodium acetate trihydrate, 0.4 mL of triethylamine, 5 mL of tetrahydrofuran, pH 7.20.5; mobile phase B: 2 L, 15 g of sodium acetate trihydrate, water/methanol/acetonitrile (volume ratio 1:2:2), pH 7.20.5; gradient elution with mobile phases A and B, total flow rate: 1.0 mL/min; column temperature: 40 C.; injection volume: 10 L; detector: ultraviolet detector; detection wavelength: 338 nm, emission wavelength 360 nm.

(27) Optimum pH: Wild enzyme or mutant enzyme pre-preserved in 50 mM of PB buffer at pH 4.0-7.5 was placed in a water bath at 45 C. A reaction was carried out for 10 min and then terminated immediately upon boiling. PH stability: The same concentration of wild enzyme and mutant enzyme were pre-preserved in a buffer at pH 4.0-7.5 and 4 C. for 12 h, and then the enzyme activity was measured under the condition of 45 C.

(28) Results obtained are shown in FIG. 2: the effective pH range of the mutant enzyme is broadened and shifted to neutral compared to the wild enzyme. As shown in FIG. 3: the mutant enzyme is more stable when preserved at near-neutral pH compared to the wild enzyme.