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Recombinant arginine deiminase, industrial preparation method and applications

An arginine deiminase and pre-purification technology, which is applied in the field of industrial preparation, recombinant arginine deiminase and its encoding gene, can solve the problem of reducing the specific activity rate of recombinant protein, unqualified product quality and precipitation. Precipitation and other problems, to achieve the effect of easy amplification and repetition, reduction of content, and clear composition

Active Publication Date: 2018-07-10
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the renaturation conditions are not suitable, there will be covalent bonding or hydrophobic bonding between molecules to form aggregates, which will reduce the specific activity rate of the recombinant protein, resulting in unqualified product quality, and at the same time, it is easy to produce precipitation and affect the yield.

Method used

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  • Recombinant arginine deiminase, industrial preparation method and applications
  • Recombinant arginine deiminase, industrial preparation method and applications
  • Recombinant arginine deiminase, industrial preparation method and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 rADI gene optimization design

[0063] According to the cDNA sequence of arginine deiminase published by GenBank (GenBank accession number: BAA02571), the inventor determined the rADI gene sequence before the gene optimization of the present invention, and obtained the rADI of the present invention after codon optimization of the gene. Gene, as shown in SEQ ID NO:1.

[0064] The following is the codon optimization of rADI. The parameters before and after optimization are compared as follows:

[0065] 1. Codon Adaptation Index (CodonAdaptation Index, CAI)

[0066] Depend on Figure 2-a It can be seen that before codon optimization, the codon adaptation index (CAI) of rADI natural gene in Escherichia coli was 0.60. Depend on Figure 2-b It can be seen that after codon optimization, the codon adaptation index (CAI) of the rADI-optimized gene in E. coli was 0.90. Usually, when CAI=1, it is considered that the target gene is in the most ideal high-efficiency...

Embodiment 2

[0071] Example 2 rADI strain construction and expression identification

[0072]The optimized recombinant arginine deiminase whole gene (as shown in SEQ ID NO: 1) was introduced upstream and downstream into restriction endonuclease sites Xba I and Xho I, respectively, and constructed into pUC57 plasmid, A long-term preserved cloning plasmid was obtained, which was designated as pUC57-rADI plasmid (gene synthesis and cloning, and construction of the plasmid were entrusted to Nanjing GenScript Co., Ltd.). In order to clone the rADI gene from the pUC57 vector into the pET28a vector, and then transform it into an expression host, see the following steps:

[0073] 1. Take the glycerol tube of DH5α-pET28a (preserved in our laboratory) frozen in the refrigerator at -80°C, inoculate it in LB liquid medium after thawing, and culture it overnight on a shaker at 220 rpm at 37°C. Extraction (purchased from Tiangen Biochemical Technology Co., Ltd.).

[0074] 2. Use primers to amplify t...

Embodiment 3

[0081] Example 3 Establishment of rADI Escherichia coli Tertiary Seed Bank

[0082] Step 1: Establishment of the original seed bank

[0083] 1. Pick a single colony of recombinant rADI Escherichia coli that has been verified, streak it on an LB plate containing 50 μg / mL kanamycin (0408, purchased from Amresco), and culture overnight at 37°C.

[0084] 2. Pick a single colony and inoculate it in 5 mL of LB liquid medium containing 50 μg / ml kanamycin, culture at 37°C and 220 rpm when the OD600 reaches 1.0-1.2, then stop the culture.

[0085] 3. Take 700 μl of the bacterial solution into a sterile pyrogen-free cell cryopreservation tube, then add 500 μL of 50% glycerol solution, and store it in aliquots as the original seed bank.

[0086] Step 2 Establishment of the main seed bank

[0087] 1. Streak the original seed bank bacterial liquid on the LB plate containing 50ug / mL kanamycin, culture overnight at 37°C.

[0088] 2. Pick a monoclonal colony and inoculate it in 5 mL of L...

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Abstract

The invention provides a recombinant arginine deiminase, a coding gene, a preparation method and applications thereof. According to the present invention, based on an Escherichia coli expression system, the coding gene, the vector, the strain, the preparation process and the buffer system are completely optimized and screened; the expression amount and the activity of the arginine deiminase obtained through the recombinant expression of the optimized arginine deiminase encoding gene are substantially improved compared to the unoptimized codon; by optimizing the recombinant arginine deiminase recombinant strain fermentation process, the high-yield and high-activity target protein can be stably obtained; the invention further provides an excellent recombinant arginine deiminase activity determination system, which can truly reflect the enzyme activity of the recombinant protein; and the in vitro activity results indicate that the recombinant arginine deiminase can effectively inhibit thegrowth of a variety f tumor cells, and has broad medicinal prospects and industrial production possibilities.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to recombinant arginine deiminase (hereinafter referred to as rADI) and its coding gene, industrial preparation method and application. Background technique [0002] Arginine is a non-essential amino acid in the human body, which can be obtained through the urea cycle. Arginine-deficient tumors cannot synthesize arginine due to the lack of ASS (Argininosuccinate Synthetase), so their growth requires the intake of sperm from the external environment. acid. Studies have shown that arginine deiminase (Arginine deiminase, EC3.5.3.6, referred to as ADI) can degrade arginine in the environment, thereby inhibiting the growth of these arginine-deficient tumors and killing cancer cells The purpose of treating cancer. This novel "arginine deprivation therapy" can be used in the treatment of certain arginine auxotrophic cancer cells, such as liver cancer cells and melanoma cells...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/78C12N15/70C12N1/21A61K38/50A61P35/00
CPCA61K38/50C12N9/78C12N15/70C12Y305/03006
Inventor 马永赵百学王俊
Owner ZONHON BIOPHARMA INST
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