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Arginine deiminase mutant and preparation and application thereof

A technique for arginine deiminase and mutants, which is applied in the field of arginine deiminase mutants and their preparation and application, and can solve the problems of decreased activity of modified proteins, heterogeneous mixture of products, and uncontrollable modification site and other issues, to achieve high activity retention, high product uniformity, and high in vitro activity

Active Publication Date: 2014-08-27
JIANGSU T MAB BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Conventional PEG modification not only leads to a decrease in the activity of the modified protein, but also results in an inhomogeneous mixture of products due to the inability to control the modified site

Method used

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  • Arginine deiminase mutant and preparation and application thereof
  • Arginine deiminase mutant and preparation and application thereof
  • Arginine deiminase mutant and preparation and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1: Expression, purification and identification of recombinant arginine deiminase mutants

[0043] 1.1 Expression and renaturation of recombinant arginine deiminase mutants

[0044] In order to obtain a recombinant arginine deiminase with a partial mutation of lysine with high biological activity, the researchers of the present invention screened out a mutant of arginine deiminase with a random point mutation of lysine. A mutant of arginine deiminase with partial mutation of lysine, compared with the original sequence of SEQ ID NO: 1, the mutant of arginine deiminase with partial point mutation only retains a part that does not significantly affect the activity Lys residues.

[0045] Sequencing shows that the amino acid sequence of the above mutant is shown in SEQ ID NO: 2, in which 15 lysines are replaced by other amino acids, named raADI-Lys -15 , the specific mutation sites are: K9N, K59Q, K66R, K93E, K111R, K119Q, K121Q, K122E, K126E, K178I, K196I, K209G, K...

Embodiment 2

[0055] According to the mutation site in the mutant obtained in Example 1, the protein after the single point mutation of 15 mutation points was prepared with reference to the method of Example 1, and the activity residue after the single mutation of each mutation point was checked. The results are shown in the following table:

[0056] Table 1 Activity residues of arginine deiminase mutants after single point mutation

[0057] amino acid

[0058] The results showed that the arginine deiminase mutant with the above single point mutation can also retain more than 85% of the residual activity. In view of the results of Examples 1 and 2, those skilled in the art know that no matter whether these mutations are single mutations or multiple mutations, they have basically no effect on the arginine degradation activity of arginine deiminase, so even if only Partial mutations in these 15 lysines, the obtained mutants can still maintain the enzyme activity of degrading arginin...

Embodiment 3

[0060] According to the results of Example 2, another arginine deiminase mutant was artificially synthesized. The amino acid and DNA sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 6 respectively. Compared with SEQ ID NO: 2, this mutation Only K9N, K59Q, K66R, K93E, K111R, K119Q, K121Q, K122E, K126E, K178I, K196I, K209G mutations occurred in the body sequence, a total of 12, named raADI-Lys -12 . Its expression, renaturation, purification and activity determination are the same as in Example 1, and the specific activity is still about 30 U / mg. It is proved that the 15 lysine mutations in Example 1 can be combined at random without significantly affecting their activity.

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Abstract

The invention relates to an arginine deiminase mutant with partial mutated lysine and preparation and application thereof. The arginine deiminase mutant of the invention has enzymatic activity of degrading arginine into citruline; compared with the arginine deiminase with the amino acid sequence of SEQ IDNO:1, the amino acid sequence comprises mutants of K9N, K59Q, K66R, K93E, K111R, K119Q, K121Q, K122E, K126E, K178I, K196I and one or more of K209G, K243E, K249D and K279Y. Compared with natural arginine deiminase, after the arginine deiminase mutant of the invention is modified by PEG, the retention rate of active residues is improved; and as the quantity of lysine is reduced, products are more uniform and can be applied to clinical treatment of hepatoma, melanoma and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an arginine deiminase mutant and its preparation and application. Background technique [0002] Arginine is not required for normal cell growth because they can be synthesized from guanidine through a two-step reaction catalyzed by argininosuccinate synthetase (ASS) and argininosuccinate lyase (AL). Arginine: However, hepatocellular carcinomas (HCC), melanomas, and some other sarcomas do not express argininosuccinate synthetase, so they are arginine auxotrophs and can only develop in an environment containing arginine. Growth; in the presence of enzymes that degrade arginine, the removal of arginine makes these tumor cells enter a "starved" state, and their growth is inhibited. Therefore, arginine-degrading enzymes can be used as potential clinical drugs for diseases such as liver cancer and melanoma. Arginine deiminase (arginine deiminase, ADI) can catalyze the conversion of arg...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/78C12N15/55C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K38/50A61P31/12A61P35/00A61P25/28
Inventor 黄岩山裘霁宛凌建群温晓芳尚玉栓傅晓瑜范敏王玉姣王叶飞
Owner JIANGSU T MAB BIOPHARMA
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