Method for purifying recombined human granulocyte-macrophage stimulating factors
A technology of macrophage stimulation and purification method, which is applied in the field of purification of recombinant human granulocyte macrophage stimulation factor, can solve the problems of increasing the difficulty of wastewater pollution treatment, high price, residual solvents, etc. The effect of improving safety and simplifying the process
Active Publication Date: 2010-09-15
XIAMEN AMOYTOP BIOTECH
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[0004] Most of the above reports emphasize the expression level and biological activity of the target protein, but there is basically no mention of how to obtain a pure protein with high purity and few related impurities
U.S. Patent No. 5,391,706 reports a purification scheme, but this purification scheme includes multiple steps such as QAE, affinity chromatography, ammonium sulfate precipitation, gel filtration, and reversed-phase chromatography. This purification scheme is obviously not environmentally friendly. Economic and other requirements
In particular, the purification of proteins by reversed-phase chromatography requires the use of a large amount of organic solvents, which is not only expensive, but also has the problem of residual solvents, and proteins are easily denatured and inactivated
Purification of the organic solvent produced increases the difficulty of wastewater pollution treatment and brings potential environmental pollution risks
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[0026] The present invention will be further described below through specific examples.
[0027] A purification method of recombinant human granulocyte-macrophage-stimulating factor in this embodiment uses Escherichia coli expressing recombinant human granulocyte-macrophage-stimulating factor rhGM-CSF in the form of inclusion bodies, and first performs renaturation of inclusion bodies by ultrafiltration: ① Dissolve fermented inclusion bodies (750g±10%) in inclusion body solution (guanidine hydrochloride 6M; Tris-HCl 50mM at pH 8.5±0.05; Nacl 40mM), add reducing agent, fill with nitrogen and protect from light for 2 hours for later use. Wash the MaxCell UFP-3-C-65 ultrafiltration system with 0.5NNaOH solution for 30 minutes, and rinse it with injection water until neutral. ②Dilution and renaturation: Add the inclusion body solution into the renaturation solution (guanidine hydrochloride 4M; pH8.5±0.05Tris-HCl50mM; Nacl20mM) and mix well, pump into the storage tank of the ultraf...
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The invention discloses a method for purifying recombined human granulocyte-macrophage stimulating factors. The colibacillus adopted to express recombined human granulocyte-macrophage stimulating factors rhGM-CSF is in a form of an inclusion body. The purifying method comprises the following steps of: performing ultrafiltration and renaturation for the inclusion body; performing Capto S cation chromatography; and performing chromatography by a Superdex-75 gel filter column to obtain stock solution of purified recombined human granulocyte-macrophage stimulating factors rhGM-CSF. The invention has the advantages of simple, convenient and rapid process, fewer steps, high yield and high purity, and improves the safety, controllability and effectiveness of the medicine with consideration of environment protection and is suitable for large-scale production.
Description
technical field [0001] The invention relates to a purification method of recombinant human granulocyte macrophage stimulating factor. The invention belongs to the field of protein purification and is a simple and efficient method for preparing high-purity and high-yield medicinal rhGM-CSF stock solution. Background technique [0002] Granulocyte-macrophage colony-stimulating factor (GM-CSF) is derived from activated T cells, macrophages, fibroblasts and endothelial cells, GM-CSF promotes the proliferation and maturation of hematopoietic cells; also enhances antibody-dependent cytotoxicity activity, expression of cell surface receptor antigens, and stimulation of cytokine secretion; in particular, it can enhance the release of tumor-killing cytokines such as tumor necrosis factor (TNF)α and IL-1β, which have strong antiviral properties active. At present, recombinant human GM-CSF is clinically used in the treatment of leukopenia caused by radiotherapy and chemotherapy, and ...
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IPC IPC(8): C07K14/535C07K1/36C07K1/34C07K1/18C07K1/16C07K1/14
Inventor 孙黎蔡慧丽何艳杨美花裴广强林崧余东新
Owner XIAMEN AMOYTOP BIOTECH