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Recombination preparation method of arginine deiminase

An arginine deiminase and amino acid technology, which is applied in the field of preparation of recombinant arginine deiminase invention, can solve the problems of high industrialization cost, renaturation rate and enzyme specific activity to be improved, etc.

Inactive Publication Date: 2015-11-04
CHONGQING PEG BIO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cost of industrialization of guanidine hydrochloride is relatively high, and as a denaturant method for renaturation, the renaturation rate and the specific activity of the enzyme after renaturation need to be improved

Method used

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  • Recombination preparation method of arginine deiminase
  • Recombination preparation method of arginine deiminase
  • Recombination preparation method of arginine deiminase

Examples

Experimental program
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Effect test

example 1

[0062] Example 1. Artificial synthesis of arginine deiminase cDNA sequence

[0063] The cDNA sequence (SEQ ID NO:3) designed according to the amino acid sequence (SEQ ID NO:1) of arginine deiminase was commissioned to artificially synthesize the nucleotide sequence of the whole gene, embedded in the pMD 19-T vector, and named as

[0064] Named pMD 19-ADI.

example 2

[0065] Example 2. Construction of Arginine Deiminase Recombinant Plasmid and Engineering Strain

[0066] Using the pMD 19-ADI plasmid as a template, use NdeI endonuclease and BamHI endonuclease to double digest, recover the target fragment, and then use T4 DNA ligase and plasmid pET-3C (purchased from Invitrogen) to pass NdeI and BamHI The recovered fragments were digested and ligated, transformed into Escherichia coli cloning host strain Top10, and the recombinant plasmid pET-3c-ADI was screened by enzyme digestion and PCR verification (see attached figure 2 ). After DNA sequencing (TAKARA, Dalian) proved that the cDNA sequence of ADI in the recombinant plasmid was correct (see attached image 3 ), transformed into Escherichia coli expressing host strain BL21 Star (DE3) plysS (Novagen). Recombinant expression strains were obtained through expression screening. Construction diagram see attached figure 1 .

example 3

[0067] Example 3. Fermentation of recombinant arginine deiminase

[0068] Streak inoculate pET-3C-ADI / BL21 Star (DE3) plysS recombinant engineered bacteria with the best expression on LA agar plate and culture overnight at 37°C. Pick the bacterial lawn from the overnight cultured LA plate and inoculate it in a test tube containing LB liquid medium (tryptone 10g, yeast extract 5g, NaCl 10g, add water to 1000ml, 121°C, autoclave for 30min), 37°C Cultivate for 12 hours, then transfer to a 1000ml Erlenmeyer flask containing 200ml LB culture medium at a ratio of 1%, and cultivate overnight at 37°C to become the seed solution in the upper tank. Inoculate the seed solution in the upper tank into the YT-containing culture solution (tryptone 4g / L, yeast extract 3g / L, Na 2 HPO 4 12H 2 O 30g / L, KH 2 PO 4 3g / L, NaCl 1g / L, MgSO 4 ·7H 2 O 1g / L, glucose 8g / L, 121°C, autoclaved for 30min) in a 30L fermenter, cultured at 37°C, during the whole fermentation process, the dissolved oxygen...

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Abstract

The invention discloses a recombination preparation method of arginine deiminase (hereinafter referred to as ADI), and particularly discloses the preparation method for escherichia coli to conduct recombination expression on the ADI. The method mainly comprises a recombination expression method and an inclusion body renaturation method using urea as a denaturing agent. The N tail end of the ADI prepared through recombination expression does not contain methionine.

Description

[0001] technical field [0002] The invention belongs to the fields of biotechnology and pharmacy. More specifically, the present invention relates to a preparation method invented by recombinant arginine deiminase, including a recombinant expression method in Escherichia coli, inclusion body denaturation and renaturation method, and the arginine produced by this method recombinantly The N-terminal of the deiminase does not contain methionine, and the specific activity of the enzyme is more than 20 units. [0003] Background technique [0004] Arginine Deiminase (ADI) is a protease with a molecular weight of about 46kd, which has a variety of microbial sources, including Mycoplasma hominus, Mycoplasma arginini, Clostridium perfringens (Clostridium perfringens), Pseudomonas pudita (Pseudomonas pudita), etc., among which the activity of arginine deiminase derived from Mycoplasma is the highest. Arginine deiminase degrades L-arginine to citrulline and ammonia both in vitro a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12R1/19
Inventor 张增涛范开刘日勇
Owner CHONGQING PEG BIO BIOTECH CO LTD
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