Genetically Engineered Arginine Deiminase Modified by Site-Directed Mutagenesis

Patent metadata

Field	Value
Publication number	US-2019136219-A1
Application number	US-201916245881-A
Country	US
Kind code	A1
Filing date	Jan 11, 2019
Priority date	Jan 23, 2017
Publication date	May 9, 2019
Grant date	—

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.

First claim

What is claimed is:

1 . An arginine deiminase mutant, with an amino acid sequence as set forth in SEQ ID No. 1.

2 . A gene encoding the arginine deiminase mutant of claim 1 , with a nucleotide sequence as set forth SEQ ID No. 2.

3 . A cell, which is characterized by capable of expressing the arginine deiminase mutant of claim 1 .

4 . The cell of claim 3 , with Escherichia coli as a host strain.

5 . The cell of claim 4 , with E. coli BL21(DE3) as a host strain.

6 . A method of using the arginine deiminase mutant of claim 1 , comprising adding an amount of the arginine deiminase mutant in an assay for medicinal antitumor activity and pharmacological activity studies.

7 . A method of using the arginine deiminase mutant of claim 1 , comprising adding an amount of the arginine deiminase mutant in preparation of a medicament for inhibiting arginine-deficient tumor, breast cancer and liver cancer cells.

8 . A method of using the arginine deiminase mutant of claim 1 , comprising adding an amount of the arginine deiminase mutant in preparation of a medicament for treating leukemia.

9 . A method of using the arginine deiminase mutant of claim 1 , comprising adding an amount of the arginine deiminase mutant in production of citrulline.

Assignees

• Univ Jiangnan

Inventors

• Zhang Tao
• Jiang Bo
• Jiang Hangyu
• Mu Wanmeng
• Miao Ming

Classifications

• C12P13/10

  Citrulline; Arginine; Ornithine · CPC title

• C12N9/78 Primary

  acting on carbon to nitrogen bonds other than peptide bonds (3.5) · CPC title

• C12Y305/03006

  Arginine deiminase (3.5.3.6) · CPC title

• C12Q1/34

  involving hydrolase · CPC title

• C12N2800/101

  for bacteria · CPC title