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Sulfhydryl modified recombinant human arginase I, as well as preparation method and application thereof

A technology of arginase and sulfhydryl modification, applied in the field of chemical modification of recombinant proteins, can solve the problem of immunogenicity, liver function decompensation and other problems

Inactive Publication Date: 2014-02-12
BEIJING SL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this medicinal molecule is that it is a mycoplasma enzyme, and although PEG-modified, its immunogenicity remains an issue
In its phase II clinical study, it has been reported that autoantibodies can be detected in the first 5 weeks, and their titers will continue to rise later, which will lead to the ineffectiveness of subsequent treatments; secondly, ADI converts arginine into citrulline and Free amino groups, which can lead to cirrhosis and hepatic decompensation when blood ammonia levels are high, and hepatic encephalopathy in some patients

Method used

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  • Sulfhydryl modified recombinant human arginase I, as well as preparation method and application thereof
  • Sulfhydryl modified recombinant human arginase I, as well as preparation method and application thereof
  • Sulfhydryl modified recombinant human arginase I, as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Preparation of recombinant human arginase I

[0041] Human type Ⅰ arginase gene (hAI) was amplified by PCR, inserted into the expression vector pET30a of Escherichia coli, the recombinant expression plasmid pET30a-hAI was constructed, transformed into the host strain of Escherichia coli BL21 (DE3), and the genetically engineered strain of Escherichia coli BL21 (DE3 ) (pET30a-hAI), expressed after IPTG induction. SDS-PAGE analysis showed that the induced BL21(DE3)(pET30a-hAI) had obvious protein expression at about 35kD. After fermentation and purification, the final purity is over 95%. Refer to the literature for detailed steps (Masaki IKEMOTO, Masayoshi TABATA, Toshio MIYAKE, et al. Expression of human liver arginase in Escherichia coli. Biochem, J.1990, (270): 697-703)

Embodiment 2

[0042] Example 2. Preparation of PEG (40KD)-Arg whose modification site is at the 303rd cysteine ​​sulfhydryl group of Arg

[0043] Adjust the protein concentration of Arg to 4.0mg / ml, adjust the pH value of the protein solution to 7.5, add Y-type PEG-MAL (40KD) equivalent to 2 times the molar number of Arg, and stir at 4°C for 12 hours. The modification efficiency in the reaction was detected by SDS-PAGE method. After 12 hours of reaction, the SDS-PAGE results showed that more than 80% of Arg was modified, and the single modified component accounted for 50-70% of the modified components. The time was further prolonged, and the modification rate No longer improve. After the reaction was terminated, the precipitate was discarded by centrifugation, and the supernatant was directly applied to the Sephacryl-S300 column, and 50mM glycine-sodium hydroxide (pH9.8) buffer solution plus 0.15M sodium chloride was used as the eluent. After this step, the unmodified Arg molecule removal....

Embodiment 3

[0044] Embodiment 3. Preparation of PEG-Arg injection

[0045] After the PEG-Arg stock solution obtained in Example 2 was used to determine the protein concentration by the Lowry method, 5000 mg PEG-Arg was added to the liquid containing the following substances: 6.78 g of disodium hydrogen phosphate, 0.17 g of sodium dihydrogen phosphate, 0.1 ml of Tween 80, Sodium chloride 5.844g. After sterile filtration, it is divided into 2ml vials, and the filling volume of each bottle is 1ml.

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Abstract

The invention relates to a sulfhydryl modified recombinant human arginase I, as well as preparation method and application thereof. The arginase is a recombinant human arginase I obtained by recombinant technology and protein purification technology; the used polyethylene glycol is a Y-type polyethylene glycol maleimide which is a branched PEG (polyethylene glycol) derivative containing two methoxy groups, the molecular weight is 40Kd, and the active end group maleimide of the polyethylene glycol is selectively in bond connection with a free sulfhydryl (-SH) of the protein molecule in neutral and alkaline conditions; the modified polyethylene glycol recombinant human arginase I has a strong in vivo effect and a prolonged vivo half-life period, can inhibit growth of some human hepatoma cells and melanoma cells, particularly the human hepatoma cell resisting against arginase deiminase (ADI).

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to the chemical modification technology of recombinant protein. Background of the invention [0002] Arginase is an enzyme in the urea cycle, which catalyzes the hydrolysis of arginine to produce ornithine and urea, and plays an important role in the nitrogen metabolism of the body. There are two types of arginase in the human body (type I is called liver arginase, type II is called extrahepatic arginase), and the expression of type I arginase gene is induced by the substrate arginine and coenzyme Mn2+ (Brock, A.A et al, Dietary manganese deficiency decreases rat hepatic arginase activity. J. Nutr. 1994,124:340-344), the type II arginase that exists outside the liver has a wide tissue distribution, in different tissues and Play different roles in organs, including participation in polyamine metabolism, NO synthesis, etc. Compared with arginase, its activity is lower. [0003] Arginase dep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78A61K38/50A61K47/48A61P35/00
CPCC12N9/96A61K38/50A61K47/60C12N9/80C12Y305/03001
Inventor 徐明波杨仲璠徐川赵婧梁果义王俊玲连治国王喜鹤吴彦卓
Owner BEIJING SL PHARMA
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