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Preparation method of FGF1 (fibroblast growth factor 1) full-length protein by utilizing escherichia coli

A technology of Escherichia coli and full length, applied in the field of preparing FGF1 full length protein from Escherichia coli, can solve the problems of low solubility and low product purity, and achieve the effect of high solubility and high product purity

Inactive Publication Date: 2013-03-20
赵谦
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  • Claims
  • Application Information

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Problems solved by technology

There are many ways to produce FGF1 by recombinant DNA technology, for example, some improved recombinant expression systems have been constructed, and some FGF1 analogs or mutants with improved biological activity and stability, but the integrated form of the whole The activity of long-length FGF1 is higher than that of other forms of FGF1 that have removed the N-terminal part of the amino group, and some of these prior art FGF1 proteins contain fusion tags, the solubility is not high, and the product purity is also low

Method used

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  • Preparation method of FGF1 (fibroblast growth factor 1) full-length protein by utilizing escherichia coli
  • Preparation method of FGF1 (fibroblast growth factor 1) full-length protein by utilizing escherichia coli

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Embodiment

[0046] Transform the recombinant plasmid with correct sequencing into Escherichia coli BL21 according to the above steps, cultivate 2L of the bacteria at 37°C, induce the expression of the bacteria after 4 hours under the condition of OD500 of 0.6 and IPTG of 1mM, centrifuge and resuspend the bacterial pellet in Centrifuge at 5500rpm at 4°C for 15 min in 50 mL of 4°C pre-cooled cell washing buffer, then resuspend the pellet in 25 mL of 4°C pre-cooled cell lysate, add PMSF to a final concentration of 1 mmol / L, stir at room temperature for 15 min, and After centrifuging at 12000rpm for 10min at 4°C, collect the bacterial pellet; resuspend the collected bacterial pellet again, add 0.1mmol / L MgCl pre-cooled at 4°C 2 The solution was 200mL, stirred at 4°C for 10min, centrifuged at 12000rpm for 10min, and then added 10mmol / L Tris-Cl (pH7.1) to the supernatant obtained by centrifugation until the pH reached 7.1. -Cl (pH7.1), dialysis at 4°C to obtain the full-length protein of FGF1 c...

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Abstract

The invention relates to recombinant human acidic fibroblast growth factor (FGF1), in particular relates to a preparation method of FGF1 full-length protein by utilizing escherichia coli, which comprises the following steps of amplifying by utilizing a PCR (polymerase chain reaction) method to obtain a human FGF1 full-length gene PCR product; and digesting the PCR product by using incision enzyme. According to the preparation method, the full-length gene of the human acidic fibroblast growth factor is cloned to a pMAL-p5X expression carrier; the recombinant plasmid expresses the FGF1 full-length protein containing MBP (myelin basic protein) fusion label at high efficiency in the escherichia coli, wherein the protein in soluble expression; and the protein with MBP infusion label is purified by utilizing an MBP affinity column, and a complete FGF1 protein without the fusion label is prepared by utilizing TEV (tobacco etch virus) enzyme binding site and using TEV digested and purified infusion protein. The FGF1 full-length protein has high solubility and higher purity.

Description

technical field [0001] The invention relates to recombinant human acidic fibroblast growth factor (FGF1), specifically a method for preparing the full-length protein of FGF1 by using Escherichia coli. Background technique [0002] Human acidic fibroblast growth factor (FGF1) is a member of the known FGF family. It is a mitogen derived from mesoderm and neuroectoderm. It has the functions of promoting angiogenesis, neuron regeneration and survival, bone or cartilage tissue repair and myoblast Differentiation and other biological functions. Generally speaking, the chemical feature of FGF1 is that it can be tightly combined with heparin, and its isoelectric point is acidic. It is known that heparin can regulate the function of FGF molecules in several ways, for example, heparin or heparin-like molecules can directly act on FGF to activate or enhance the activity of FGF1. In addition, it was found that the synergistic effect of soluble heparin appears to be selective for FGF1,...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/70C07K14/50
Inventor 潘艺熊丹刘金蕾
Owner 赵谦
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