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Protein and polypeptide protection agent and application thereof

A protective agent and protein technology, applied in the field of biopharmaceuticals, can solve problems such as low success rate, huge R&D investment, and difficulty in realization, and achieve the effect of simplifying the purification process, increasing production costs, and promoting successful expression

Active Publication Date: 2020-02-21
BEIJING SL PHARMA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is very difficult to obtain defective engineering strains suitable for industrial production, which requires huge investment in research and development, and in many cases the success rate is not high, so it is extremely difficult to achieve, and the research and development results are not universal
The second method and the third method have obvious defects and problems, such as the introduction of a large number of non-single impurities, difficulty in controlling the quality uniformity, increasing the complexity and cost of purification operations, or limited control over the degradation of the target protein Wait

Method used

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  • Protein and polypeptide protection agent and application thereof
  • Protein and polypeptide protection agent and application thereof
  • Protein and polypeptide protection agent and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 Amino acid sequence is the solid-phase synthesis of the protecting agent of Arg-Gly-Arg-Gly-Arg-Gly

[0032] The polymer resin Wang Resin was selected, and the solid-phase synthesis was carried out from the carboxyl terminal Gly according to the characteristics of the amino acid sequence Arg-Gly-Arg-Gly-Arg-Gly. Weigh 500g of Wang Resin resin, add it to the synthesis reactor, fully swell with an appropriate amount of DMF for 1 hour, and then wash it with DMF for 5 times. Then use 20% piperidine / DMF (V / V) to remove the Fmoc group, the temperature is 20-30°C, and the reaction time is 30 minutes. Afterwards, wash with DMF 5 times, 5 min each time. Complete removal of the Fmoc protecting group from the solid phase resin.

[0033] Add 297.31g (1mol) of Fmoc-Gly-OH, 36.5g of HOBt Hydratel, 445.2g of BOP Reagent, and 150mL of NMM, shake or stir the reaction in the reactor for 1h, and the reaction temperature is 20-30°C. Afterwards, wash with DMF 5 times, each t...

example 2

[0037] Example 2 Liraglutide Fermentation Experiment Adding a Protective Agent with the Amino Acid Sequence Arg-Gly-Arg-Gly-Arg-Gly

[0038] The engineering strains of Pichia pastoris carrying the precursor of liraglutide that were frozen in the ultra-low temperature freezer were taken out, and after thawing, they were streaked and inoculated on YPG solid medium, and activated at 30°C.

[0039] Put the activated strains into the seed medium in the shaker flask, place them in a shaker for cultivation, the rotation speed is 250-300rpm, the temperature is 30°C, the cultivation time is 16-24h, until the OD 600 = 2 ~ 6, that is, the first-grade seed liquid is obtained. Then the primary seed liquid is inoculated into the medium of the secondary shake flask according to the inoculum amount of 1%, and the expansion culture is carried out.

[0040] Inoculate the amplified strains into the BSM+PTM1 medium in the fermenter according to the inoculum amount of 5% to 10%, use glycerol as t...

example 3

[0044] Example 3 Liraglutide Fermentation Experiment Adding Amino Acid Sequence Arg-Gly-Arg-Gly-Gly-Arg-Gly-Arg-Gly Protective Agent

[0045] The engineering strains of Pichia pastoris carrying the precursor of liraglutide that were frozen in the ultra-low temperature freezer were taken out, and after thawing, they were streaked and inoculated on YPG solid medium, and activated at 30°C.

[0046] Put the activated strains into the seed medium in the shaker flask, place them in a shaker for cultivation, the rotation speed is 250-300rpm, the temperature is 30°C, the cultivation time is 16-24h, until the OD 600 = 2 ~ 6, namely the first grade seed solution. Then the primary seed liquid is inoculated into the medium of the secondary shake flask according to the inoculum amount of 1%, and the expansion culture is carried out.

[0047] Inoculate the amplified strains into the BSM+PTM1 medium in the fermenter according to the inoculum amount of 5% to 10%, use glycerol as the carbon sou...

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Abstract

The invention discloses a protein and polypeptide protection agent and application thereof, and aims at providing the target-protein (or polypeptide) protection agent during fermentation in the fieldsof genetic engineering and biopharmaceutics. The protein and polypeptide protection agent belongs to a polypeptide, and an amino acid sequence of the protection agent is (-Arg-Gly-)X and is abbreviated as (RG)X, wherein X=2, 3, 4, 5 or 6. The protein and polypeptide protection agent can be prepared by adopting a solid-phase synthesis method. The protein and polypeptide protection agent has effects of being capable of inhibiting hydrolysis of recombinant proteins or polypeptides which contain amino acid sequences of -Arg-Gly- or are easily hydrolyzed by proteases at Arg sites, promoting successful expression of target products, increasing the expression quantity of the target products, and reducing the content of related impurities. The protein and polypeptide protection agent can also reduce purification difficulty and increase the preparation yield of the target products. Therefore, high-efficiency industrial production of products of the recombinant proteins or the polypeptides canbe promoted.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals and relates to a protein and polypeptide protective agent. It is used for the protection of the target product during the fermentation process when protein or polypeptide products are prepared by gene recombination technology. After adding the protective agent, it can inhibit the degradation of the target product and maintain its stability in the production process. It helps to realize the preparation of proteins and polypeptides that are difficult to express in microorganisms (such as yeast, Escherichia coli) or animal cells and insect cells, and helps to increase their yield and reduce the difficulty of purifying target products. Background technique [0002] Since the first genetically engineered product recombinant human insulin came out in the 1880s, more than 200 genetically engineered drugs have been put on the market. Mainly include: interferon, growth hormone, coagulation factor, inter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/11C07K7/06C07K7/08C07K1/04C07K1/06C07K14/605
CPCC07K5/1019C07K7/06C07K7/08C07K14/605
Inventor 牛罡苑德禄袁宛君秦静超陈晶晶李阳任小飞陶真民郭志慧李昭英舒艳萍徐明波
Owner BEIJING SL PHARMA
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