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Gene engineering arginine deiminase reformed through site directed mutagenesis

An arginine deiminase and site-directed mutagenesis technology, applied in the field of genetic engineering, can solve the problems of weak substrate affinity, low enzymatic activity, short in vivo half-life, etc.

Active Publication Date: 2017-04-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, arginine deiminase has not been widely used as a medicinal enzyme due to the limitations of low enzyme activity under physiological conditions, short half-life in vivo, and weak substrate affinity.

Method used

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  • Gene engineering arginine deiminase reformed through site directed mutagenesis
  • Gene engineering arginine deiminase reformed through site directed mutagenesis
  • Gene engineering arginine deiminase reformed through site directed mutagenesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Construction of recombinant plasmid

[0029] Will Enterococcus faecalis SK32.001 was cultured to the mid-exponential growth stage, and 2 mL of the bacterial liquid was centrifuged at 10,000 r / min for 10 min to discard the supernatant. After lysozyme treatment for 30 min, genomic DNA was extracted according to the kit instructions.

[0030] The following primers were designed for the amplification of arcA:

[0031] FADI-2: 5'-CGCGGATCCA TGAGTCATCC AATTAATGT-3' (with Bam H I restriction site),

[0032] RADI-2: 5’-CCGCTCGAGT TAAAGATCTT CACGGT-3’ (with xho Ⅰ restriction site),

[0033] PCR amplification conditions: denaturation at 95°C for 3 minutes, 30 cycles (95°C for 30 s, 55°C for 30 s, 72°C for 210 s), and finally extension at 72°C for 2 min.

[0034] After purification of the amplified product, the Bam H I and xho Ⅰ Carry out double enzyme digestion on the PCR product and the vector pET-28a-c(+), recover the digested products separately, conne...

Embodiment 2

[0035] Example 2: Site-directed mutagenesis

[0036] according to Enterococcus faecalis Coded in SK23.001 arc The coding gene of A was designed for primers.

[0037] G264P-Forward primer: 5'-CTTGGCTTTT GATATC CCT G AACATCGTAA ATTC-3',

[0038] G264P-Reverse Primer: 5'-GATATCAAAA GCCAAGATAT TTTTGAATCC TA-3',

[0039] The underlined part represents the codon corresponding to the 264-position glycine encoded by the mutant gene. The PCR amplification system is:

[0040] 10×Reaction Buffer 5 μL dNTP mix 1 μL Forward primer (100 ng / μL) 1.25 μL Reverse primer (100 ng / μL) 1.25 μL Template pET-28a-ADI (10 ng) 2 μL pfu Turbo DNA polymerase (2.5U / μL)

[0041] After PCR amplification, add 1 μL to the reaction solution Dpn ⅠRestriction endonuclease (10 U / μL), incubate at 37°C for 1h to eliminate the template. Transform the PCR product into Escherichia coli DH5α cells, smear the plate, pick a single colony into LB medium, extract ...

Embodiment 3

[0042] Example 3: Expression and purification of wild enzyme and mutant enzyme

[0043] Pick BL21(DE3) / pET-28a-ADI and pET-28a-ADI G264PSingle colonies were respectively cultured in LB medium containing 0.5mmol / L kanamycin at 37°C and 200r / min for 12h, then transferred to LB medium containing 0.5mmol / L kanamycin and incubated at 37°C , 200r / min culture to OD 600 In the range of 0.5-0.7, add 1 mmol / L IPTG to induce 9 h at 28°C and 200 r / min.

[0044] After the fermentation broth was centrifuged at 10000r / min and 4°C for 10min, discard the supernatant, wash twice with phosphate buffer, add 15-20mL of phosphate buffer to suspend the cells, and ultrasonically break for 15min (power 22W, break for 1s , intermittent 2s). Centrifuge at 4°C and 10,000 r / min for 10 minutes, collect the supernatant, which is the crude enzyme solution, and filter it with an aqueous membrane with a pore size of 0.22 μm.

[0045] Use Binding Buffer for Ni 2 + The chelating agarose resin column was pre...

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Abstract

The invention relates to a strain of gene engineering arginine deiminase reformed through site directed mutagenesis, and belongs to the technical field of gene engineering, wherein the amino acid sequence is SEQ ID No.1, and the 264th site glycine in the amino acid sequence of the arginine deiminase reformed through site directed mutagenesis mutates into the proline relative to the amino acid sequence of the natural arginine deiminase. According to the present invention, compared to the wild enzyme, the mutant arginine deiminease has the following characteristics that the effective pH value action range is expanded to a certain degree, particularly the good enzyme activity is provided at the physiological pH value of 7.4, and the mutant enzyme has the high stability under the pH value of 5.5-7.5 along with the broadening of the effective pH value action range, such that the problems of low enzyme activity and short half-life in vivo under the physiological conditions of the clinical applications of the arginine deiminase in tumor treatment are solved, and the good conditions are created for the applications of the arginine deiminase and the coding gene thereof in the clinical treatment.

Description

technical field [0001] The invention relates to an arginine deiminase mutant whose optimal pH and pH stability shift towards physiological neutrality, and belongs to the technical field of genetic engineering. Background technique [0002] Arginine deiminase (Arginine deiminase, EC 3.5.3.6) is referred to as ADI, which catalyzes the first reaction in the arginine metabolic pathway in microorganisms, that is, the hydrolysis of arginine to generate citrulline and ammonia . Arginine deiminase has a wide range of sources and is currently found in bacteria, archaea and some eukaryotic cells. At the same time, the properties of arginine deiminase from different sources are quite different. [0003] At present, scholars at home and abroad who study arginine deiminase mainly apply the enzyme to the preparation of citrulline and the treatment of diseases. Among them, in the field of medicine, arginine deiminase has good performance in inhibiting arginine-deficient tumors, breast c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78C12N15/55C12N15/70C12N1/21C12R1/19
CPCC12N9/78C12N15/70C12N2800/101C12Y305/03006C12P13/10C12Q1/34
Inventor 张涛江波蒋航宇沐万孟缪铭
Owner JIANGNAN UNIV
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