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Human recombinant arginase I producing strain and construction method thereof

A technology of arginase and construction method, applied in microorganism-based methods, biochemical equipment and methods, recombinant DNA technology and other directions, can solve problems such as recombinant arginase I and other problems that have never been seen, and achieve convenient purification treatment, The effect of fast growth and increased activity

Pending Publication Date: 2022-04-15
XINTAI JIAHE BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The above-mentioned expression systems for expressing human arginase I by genetic engineering mostly focus on Escherichia coli expression system and Pichia pastoris expression system, but there is no relevant report on the production of human recombinant arginase I based on the expression system of Bacillus subtilis

Method used

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  • Human recombinant arginase I producing strain and construction method thereof
  • Human recombinant arginase I producing strain and construction method thereof
  • Human recombinant arginase I producing strain and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Plasmid Transformation

[0039] Using the pHT304 plasmid (purchased from Youbao Biology) as the basic plasmid, it was transformed as follows:

[0040] The pHT304 plasmid was digested with NdeI and HincII, and then the Pg3 promoter (sequence shown in SEQ ID NO.1) was integrated into the pHT304 plasmid after double digestion to construct the plasmid pHT304-Pg3 ( figure 1 ).

[0041] The constructed plasmid pHT304-Pg3 is resistant to single ampicillin, has a lactose operon, and its nucleotide sequence is shown in SEQ ID NO.2.

[0042] The purpose of the present invention to transform the pHT304 plasmid is: one is to reduce the length of the vector and improve its expression stability in Bacillus subtilis; Therefore, after integrating the Pg3 promoter, the bacterial strain grows fast in the early stage, and the time required to enter the stable phase is short, thereby shortening the expression time of human recombinant arginase I, and improving the expression t...

Embodiment 2

[0043] Example 2: Optimization and transformation of arg1 gene

[0044] The inventor obtained the amino acid sequence of human arginase I from the existing database as shown in SEQ ID NO.3; the nucleotide sequence of its encoding gene arg1 is shown in SEQ ID NO.4.

[0045] In order to further improve the activity of human recombinant arginase I, the inventors used molecular dynamics simulation to optimize the structure of human recombinant arginase I, and the 158th asparagus of human recombinant arginase I Amino acid is mutated into glutamic acid, and the amino acid sequence of the mutated human recombinant arginase I is shown in SEQ ID NO.5.

[0046] Schematic diagram of the three-dimensional structure of human arginase I figure 2 As shown, aspartic acid (D) at the 158th position protrudes outwards. After being mutated to glutamic acid (E), it does not protrude outwards, and the angle between P and V is also reduced, so the overall structure Also more compact.

[0047] Afte...

Embodiment 3

[0055] Embodiment 3: Construction of recombinant expression vector

[0056] The transformed plasmid pHT304-Pg3 in Example 1 was treated with SphI and SacI double-enzyme digestion and double-enzyme digestion, and then the arg1 gene (arg1Δ158, whose nucleotide sequence was as SEQ ID NO. 7) was integrated into the plasmid pHT304-Pg3 after the double enzyme digestion treatment to obtain the recombinant expression vector (pHT304-Pg3-arg1); the structural diagram of the recombinant expression vector is shown in Figure 7 shown.

[0057] The constructed recombinant expression vector was verified by electrophoresis, and the results were as follows: Figure 8 shown. The results showed that the arg1 gene (shown in SEQID NO.7) had been successfully integrated into the plasmid pHT304-Pg3. After sequencing verification, the nucleotide sequence of the constructed recombinant expression vector (pHT304-Pg3-arg1) is shown in SEQ ID NO.8.

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Abstract

The invention discloses a human recombinant arginase I producing strain and a construction method thereof. Comprising the following steps: (1) carrying out double enzyme digestion treatment on a pHT304 plasmid by using NdeI and HincII, and integrating a Pg3 promoter sequence onto the pHT304 plasmid subjected to the double enzyme digestion treatment, so as to obtain a plasmid pHT304-Pg3; carrying out double enzyme digestion treatment on the plasmid pHT304-Pg3 by using SphI and Sac I, and integrating the arg1 gene onto the plasmid pHT304-Pg3 subjected to the double enzyme digestion treatment, so as to obtain a recombinant expression vector; and (2) introducing the obtained recombinant expression vector into bacillus subtilis, and constructing to obtain the human recombinant arginase I producing strain. According to the invention, the bacillus subtilis system is adopted for the first time to construct the human recombinant arginase I producing strain, and the expression quantity and activity of the human recombinant arginase I are remarkably improved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a human recombinant arginase I producing bacterium and a construction method thereof. Background technique [0002] Arginase (L-Arginase), also known as L-arginine urea hydrolase, catalyzes L-arginine to generate ornithine and urea. Arginase is widely distributed in different organisms, such as mammals, reptiles, invertebrates, plants, fungi and bacteria, etc. In mammals, two arginases with different gene coding sequences have been found so far, among which: arginase I (Arginase I) is mainly located in the cytoplasm of mammalian liver organs, and is responsible for the last step reaction of the urea cycle; Arginase II (Arginase II) is a mitochondrial enzyme that exists in large quantities in body tissues other than the urea cycle, and its main function is to regulate and balance the concentration of arginine / ornithine in cells. [0003] At present, arginase in domes...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/55C12N15/66C12N1/21C12P13/10C12R1/125
CPCY02A50/30
Inventor 曹华杰谢沛岳明瑞郭永胜
Owner XINTAI JIAHE BIOTECH CO LTD
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