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Preparation method of high-activity recombinant protein

A technology of recombinant protein and high activity, which is applied in the field of preparation of highly active recombinant protein, can solve the problems of complex process, low renaturation rate, endotoxin residue, etc., and achieve the effect of simple operation

Pending Publication Date: 2021-10-15
沈阳百发科技有限公司 +2
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Problems solved by technology

When the above method prepares IL-18, there are some problems such as complex process, low purity, low renaturation rate and low yield to a certain extent.
[0008] The preparation of recombinant proteins with Escherichia coli expression system has attracted attention because of its low cost and high yield, such as recombinant human IL-2, recombinant human interferon, etc., but one of the technical difficulties in purifying recombinant proteins from Escherichia coli is the presence of endotoxin High performance liquid phase (HPLC) reverse chromatography technology is an effective method to remove endotoxin, but the recombinant protein purified by HPLC is denatured by the influence of organic agents in the mobile phase and loses biological activity, which seriously affects the purification of recombinant protein. application, this problem has not been resolved
[0009] The applicant established a high-efficiency expression strain of IL-18 Escherichia coli in the early stage, and established the technical route of rhIL-18 renaturation, purification and endotoxin removal (patent number: ZL 201510455749.X, ZL201611146887.0; patent application number: 202010771259.1) , but after removing endotoxin by HPLC, the rhIL-18 sample is in an acidified and denatured state, losing its biological activity, and the residue of acetonitrile also affects the application of the rhIL-18 sample

Method used

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  • Preparation method of high-activity recombinant protein

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[0066] The invention discloses a method for preparing a high-activity recombinant protein. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.

[0067] The present invention takes recombinant human interleukin-18 (human recombinant Interleukin-18, rhIL-18) as an example, establishes a method for purifying recombinant protein fr...

Embodiment 1

[0077] Example 1 Purification of rhIL-18

[0078] The purification of rhIL-18 in this embodiment is specifically as follows:

[0079] 1. Protein dissolution and protein renaturation:

[0080] Add protein dissolving solution to HPLC purified sample at a ratio of 1:9, and dissolve at room temperature for 2 hours; slowly add refolding solution at a ratio of 1:8 to reduce the protein concentration to 0.2 mg / mL, and dissolve with magnetic stirring at room temperature for 4 hours; refold at 4°C More than 20h; immediately purified.

[0081] 2. Purification of rhIL-18:

[0082] Equilibrate the Capto Q chromatography column with the balance solution, load the above-mentioned refolding solution at a flow rate of 5mL / min; use the balance solution to fully wash the impurity protein to the baseline, elute with the balance solution containing 0.2M NaCl, and collect the eluted The peaks were stored at -20°C until identification.

[0083] The formula range and preparation of the above-men...

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Abstract

The invention relates to the technical field of recombinant protein purification, in particular to a preparation method of high-activity recombinant protein. The preparation method comprises the following steps: mixing an HPLC (High Performance Liquid Chromatography) purified sample of recombinant protein with a protein dissolving solution, and dissolving; mixing the dissolved sample with a renaturation solution, and carrying out renaturation treatment; and purifying the sample subjected to renaturation treatment by adopting an anion exchange chromatographic column to obtain the high-activity recombinant protein. According to the present invention, by improving the formulas of the protein dissolving liquid and the renaturation liquid, the problem that the recombinant protein purified by HPLC loses the biological activity is further solved, the high-activity and high-purity recombinant protein sample is prepared, and the method has characteristics of simple operation, no endotoxin and no organic solvent residue, and is suitable for retina neovascularization treatment.

Description

technical field [0001] The invention relates to the technical field of recombinant protein purification, in particular to a preparation method of highly active recombinant protein. Background technique [0002] Wet age-related macular degeneration (AMD) and diabetic retinopathy (DR) lack ideal treatments. Clinically, symptomatic treatment or adjuvant treatment is mainly used to control or delay the development of the disease. The commonly used treatment methods are as follows: hypoglycemic drugs, anti-inflammatory drugs, local use of TNF-α antagonists, fenofibrate, vascular endothelial growth factor (VEGF) antagonists, RAS system inhibitors, non-enzymatic glycation inhibitors, combination therapy and laser therapy. Anti-VEGF therapy has a good short-term effect, but it involves regular intraocular injection of anti-VEGF monoclonal antibody (such as Lucentis), which is not only harmful, but also expensive and only for confirmed cases. In addition, although the incidence rate...

Claims

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Application Information

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IPC IPC(8): C07K1/18C07K1/20C07K1/14C07K14/54
CPCC07K1/18C07K1/20C07K1/14C07K14/54
Inventor 张朔冯铃刘辉李雪娇赵航刘畅张鹏赵洪礼何伟
Owner 沈阳百发科技有限公司
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