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Preparation method and application of recombinant allosteric collagenase

A technology of collagenase and collagenase, which is applied in the field of purification of recombinant allosteric collagenase, can solve the problems of affecting the expression of protein solubility, inability to enter the market, and low protein purity.

Active Publication Date: 2018-12-07
科笛生物医药(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Xiaflex, which was launched in 2015, is a mixture of two collagenases, ColH and ColG, and the purity is low
[0008] CN101678088 discloses the application of a recombinant allosteric collagenase in lipolysis. The allosteric enzyme sequence contains a GST tag, and a peptide motif is attached before CoIH (Glu451Asp), and the purity is obtained by nickel column affinity chromatography. It is 90% protein product, but in the process of practical application, it is found that this product is difficult for pharmaceutical industrialization and cannot enter the market
Mainly there are following two problems: (1) protein purity is low
Affinity chromatography is currently the most commonly used method for protein separation and purification, and GST is one of the most commonly used affinity chromatography purification tags. Recombinant proteins with this tag can be purified with cross-linked glutathione chromatography media, but the protein on the The GST must be properly folded to form a spatial structure combined with glutathione to be purified by this method; moreover, the GST tag is up to 220 amino acids, and such a large tag may affect the solubility of the expressed protein, causing it to form inclusions body, which will destroy the natural structure of the protein, making it difficult to carry out structural analysis, sometimes even after purification and enzyme digestion to remove the GST tag may not be able to solve the problem. , Peking University Medical Press, 2015.08, page 18)

Method used

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  • Preparation method and application of recombinant allosteric collagenase
  • Preparation method and application of recombinant allosteric collagenase
  • Preparation method and application of recombinant allosteric collagenase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Construction of Recombinant Allosteric Collagenase Strains

[0053] Instruments and materials

[0054] The ColH allosteric collagenase gene sequence was artificially synthesized. pET-30a(+), host bacteria BL21(DE3), BL21(DE3)playS, and Transetta were purchased from Merck, and endonuclease was purchased from Thermo.

[0055] experimental method

[0056] figure 1 The E451D single point mutation ColH allosteric collagenase sequence is described. figure 2 The E451D single point mutation ColH allosteric collagenase protein sequence is described. The synthesized plasmid and pET-30a(+) empty vector were digested with NdeI / XhoI, detected by electrophoresis, and the target fragment and vector fragment were recovered by slicing gel. Ligate the recovered two fragments with T4 DNA ligase, transform 10ul of the ligation product into 100ul of competent cells, plate and pick a single clone, and the clone with the correct sequencing result is the target strain.

[0057]...

Embodiment 2

[0058] Embodiment 2 Recombinant Allosteric Collagenase Strain Fermentation

[0059] Instruments and materials

[0060] BIOFLO 610 65.0L fermentation tank was purchased from Eppendorf Company, high-speed refrigerated centrifuge was purchased from Thermo Company, working seed lot, peptone and yeast extract were purchased from OXID Company, and various reagents were purchased from Sinopharm Chemical Reagent Company.

[0061] experimental method

[0062] After the shake flask seeds are cultivated overnight, they are inoculated into the seed tank in a suitable state, and after a certain period of cultivation, the seed liquid is injected into the production tank connected to it.

[0063] Cultivate at 37°C for a certain period of time, the production medium formula is peptone 13.5051g / L, yeast powder 7g / L, magnesium sulfate 0.4g / L; 4 hours after inoculation in the production tank, add IPTG with a final concentration of 0.5mM for induction, and the induction time is 7-8 Hours, feedi...

Embodiment 3

[0065] Embodiment 3 Purification method of recombinant allosteric collagenase

[0066] Instruments and materials

[0067] Fillers such as Capto Phenyl HS, Capto Q, CaptoOctyl, and Phenyl HP were purchased from GE Company, AktaPurifier chromatography system (GE Company), and hollow fiber column ultrafiltration system (Pall).

[0068] experimental method

[0069] 1) Bacterial harvest and clarification

[0070] After the fermentation, the bacterial cells are collected by centrifugation; further, the bacterial cells can be collected by membrane treatment and other methods after amplification. The fresh bacteria can be preserved by freezing, or directly crushed to enter the next step. The bacteria to be crushed are resuspended uniformly in Tris buffer at a resuspension concentration of 10-20%, crushed by a high-pressure homogenizer at 600-700 bar pressure, passed through a high-pressure valve for 3 times, and the temperature is controlled at 2-8°C during the crushing process.

...

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Abstract

The invention relates to a purification method and an application of recombinant allosteric collagenase and discloses a method for preparing high-purity single-mutant collagenase ColH and a product prepared by virtue of the method. The method for preparing high-purity single-mutant collagenase ColH comprises the steps: expressing an E451D single-point-mutation ColH allosteric collagenase protein by virtue of specific host bacteria BL32(DE3), and carrying out low-temperature culture induction so as to increase the yield of the target protein. By virtue of five-step purification as follows: Capto Phenyl HS hydrophobic chromatography, Capto Q anion exchange chromatography, CaptoOctyl hydrophobic chromatography, Phenyl HP hydrophobic chromatography and Source 15Q anion exchange chromatography,the target protein with a purity over 98% is acquired. A biological experimental result shows that allosteric collagenase produced by virtue of the method is high in purity and good in stability; andcompared with collagenase commodities in the prior art, allosteric collagenase has obvious superiorities, and the specific enzyme activity is remarkably increased.

Description

technical field [0001] The invention belongs to the technical field of biological products and pharmaceuticals, and relates to a purification method and application of recombinant allosteric collagenase. Background technique [0002] Collagenase has a wide range of applications in medical health, production practice and scientific research. It is expected to develop new drugs for fat melting, scar removal, and skin micro-shaping, which can be used in food softening in production, cell separation, and processing of archaeological samples in scientific research and experiments. [0003] With the improvement of people's living standards, obesity is becoming more and more common, and obesity is a big problem for people who love beauty. At present, there are many products in the weight loss market, and liposuction is a method of fat reduction that is widely used. Liposuction is a physical method with the help of instruments, which has certain damage to the body. Other tissues i...

Claims

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Application Information

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IPC IPC(8): C12N9/52A61K38/48A61P3/04A61P17/02A61P35/00A61K8/66A61Q19/00A23L33/195
CPCA61P3/04A61P17/02A61P35/00A61Q19/00A23L33/195A61K8/66C12N9/52C12Y304/24003A23V2002/00A61K38/00A23V2200/308A23V2200/332A23V2200/318A23V2250/546C12N9/6491A61Q19/06Y02A50/30A61K38/4886
Inventor 仓勇张震杜刚陈福鼎
Owner 科笛生物医药(上海)有限公司
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