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A kind of purification method of recombinant human epidermal growth factor

A technology of epidermal growth factor and purification method, applied in the field of purification of recombinant human epidermal growth factor (rhEGF), can solve the problems of difficulty in preparation, large differences, influence on activity and safety, etc. The effect of few process steps and high practical value

Active Publication Date: 2017-08-11
山东仁瑞生物科技有限公司
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AI Technical Summary

Problems solved by technology

[0004] EGF contains 3 pairs of disulfide bonds, it is difficult to produce in large quantities by chemical synthesis, and now it can only be produced by large-scale recombination by genetic engineering.
In order to promote the correct pairing of disulfide bonds and the formation of higher-order structures, the preparation of recombinant EGF (rEGF) is mainly based on fusion expression: EGF is fused with a partner protein and / or a purification tag that can help the protein fold correctly, and the fusion protein Purification, enzyme digestion, purification of target protein (EGF) and other steps of preparation, the purification method involved is mainly for the fusion protein, EGF purification after enzyme digestion only needs to remove the chaperone protein, which is quite different from the conventional non-fused EGF purification process, and after enzyme digestion EGF protein still contains residual cleavage tags, affecting activity and safety
Chinese patent 201310651408.0 discloses the process of recombining EGF with SUMO protein and HIS tag and then using CHO cell recombination, but does not mention the purification method of EGF after digestion; After fusion, E. coli was used for recombinant expression. After enzyme digestion, based on the HIS tag remaining in the EGF structure, the PDI chaperone protein was removed using a nickel ion affinity column. Big, there is a security risk
Chinese patent 97115284.5 discloses a method for the recombinant preparation of hEGF truncated isomer—EGF (1-51) by using methylotrophic yeast. The purification steps include hydrophobic chromatography, ion exchange chromatography and gel chromatography, which are not conducive to Industrial scale-up, and the biological activity of the prepared EGF (1-51) (1.14×10 6 IU / mg) lower
At present, there is no high-activity, high-purity rhEGF preparation process with simple process, high activity yield, and easy scale-up production

Method used

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  • A kind of purification method of recombinant human epidermal growth factor
  • A kind of purification method of recombinant human epidermal growth factor
  • A kind of purification method of recombinant human epidermal growth factor

Examples

Experimental program
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Embodiment 1

[0033] Example 1. Purification of rhEGF protein

[0034] Collect the culture supernatant (1000 mL) containing rhEGF protein, adjust the pH to 8.0 with 1mol / L NaOH solution, centrifuge to remove insoluble impurities, and collect the supernatant.

[0035] Take the Ni Sepharose 6 Fast Flow affinity chromatography medium, put it into the normal pressure chromatography column, and use the equilibrium buffer (25mmol / L PB buffer, pH8.0) at a flow rate of 150cm / h (the loading and elution speed and the equilibrium The speed is the same, the same below) After equilibrating for 3 column volumes, load the centrifuged supernatant containing rhEGF, re-equilibrate with equilibration buffer (25mmol / L PB buffer, pH8.0), and detect A 280nm , the breakthrough peak was discarded, and when A 280nm After falling to the baseline, elute with equilibration buffer (25mmol / LPB buffer, pH8.0) containing 20mM imidazole, and collect the elution peak containing rhEGF protein;

[0036] Take Source 15RPC re...

Embodiment 2

[0038] Example 2. Determination of the purity of rhEGF protein SDS-PAGE

[0039] (1) 15% separation gel configuration

[0040] Double distilled water: 3.3 mL; 1.5 mol / L Tris-HCl (pH 8.8): 2.5 mL; 10% SDS: 100 μL; 30% acrylamide monomer stock solution: 4.0 mL; TEMED: 4 μL; 10% persulfuric acid Ammonium: 100 μL; total volume: 10 mL. After mixing, put it into the gap between the glass plates of the electrophoresis tank, and add about 1cm of double distilled water on the surface of the gel. After the gel naturally coagulates, remove the double distilled water and put a comb in the gap.

[0041] (2) 5% stacking gel configuration

[0042] Double distilled water: 3.4 mL; 1mol / L Tris-HCl (pH6.8): 0.63 mL; 10% SDS: 50 μL; 30% acrylamide monomer stock solution: 0.83 mL; TEMED: 5 μL; 10% persulfate Ammonium: 150 μL; total volume: 5 mL. After mixing, add to the crevice and do not pass through the comb hole. After condensation, carefully pull out the comb, and rinse the sample hole wit...

Embodiment 3

[0053] Embodiment 3. rhEGF RP-HPLC purity determination

[0054] Instrument: Aglient 1100 HPLC; Chromatographic column: Welch Ultimate XB-C18 (5μm 4.6×100mm); Mobile phase: Liquid A is 0.1% trifluoroacetic acid + aqueous solution; Liquid B is 0.1% trifluoroacetic acid + acetonitrile solution; flow rate: 1.0 mL / min; Elution mode: 0-30min, B from 5-95%; Detection wavelength: 220nm. Take 10 μL of rhEGF sample, and perform full gradient RP-HPLC purity determination according to the above conditions.

[0055] as attached figure 2 As shown, there is no obvious impurity band in the RP-HPLC spectrum of the rhEGF sample, and the RP-HPLC purity of the main peak is 99.3%, which is higher than the high-performance liquid chromatography stipulated in the Chinese Pharmacopoeia "the main peak area of ​​human epidermal growth factor should not be less than the total area 95.0%" standard.

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Abstract

The invention belongs to the field of protein purification and relates to a purification method for a recombinant human epidermal growth factor (rhEGF). According to the purification method for the recombinant human epidermal growth factor (rhEGF), high-activity high-purity rhEGF protein is obtained through Ni Sepharose affinity chromatography, Source 15RPC reversed phase chromatography and Source 30Q ion-exchange chromatography of centrifugation supernatant containing the rhEGF in sequence. According to the purification method for the recombinant human epidermal growth factor (rhEGF), the Ni Sepharose affinity chromatography is adopted for purifying a natural structure recombinant human epidermal growth factor without containing an HIS purification tag, and the purification method for the recombinant human epidermal growth factor (rhEGF) has the advantages that the technology is simple, the activity yield is high, and the purification method for the recombinant human epidermal growth factor (rhEGF) is easy for large-scale production.

Description

technical field [0001] The invention belongs to the field of protein purification and relates to a purification method of recombinant human epidermal growth factor (rhEGF). Background technique [0002] Human epidermal growth factor (hEGF) was isolated from human urine by Cohen et al. in 1975. Because it can inhibit gastric acid secretion, it is also known as Urogastrone (Urogastrone), which is a small molecule polypeptide composed of 53 amino acids. . hEGF combines with the EGF receptor (EGFR), dimerizes EGFR and activates cell pathways, and exerts various biological functions, such as promoting cell division, repairing epidermal damage, gastrointestinal ulcers, and corneal damage; preventing skin aging, and exerting whitening and tenderness Skin efficacy; target binding to tumor tissue containing high-density EGFR receptors, inhibit cancer cell growth, etc. In view of Cohen's pioneering work on EGF and the important biological role of EGF itself, he and Montabcini were a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/485C07K1/22C07K1/20C07K1/18
CPCC07K14/485
Inventor 张淳孟磊
Owner 山东仁瑞生物科技有限公司
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