Recombinant human insulin-like growth factor-2 protein and production method thereof

A production method and recombinant protein technology, applied in insulin-like growth factors, animal/human proteins, recombinant DNA technology, etc., can solve problems such as low protein yield, reduced active protein yield, and inactivation of target products

Inactive Publication Date: 2013-02-20
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Claims
  • Application Information

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Problems solved by technology

However, these methods using E. coli to produce recombinant human IGF2 protein have obvious defects
The human IGF2 expression product of Escherichia coli usually exists in the form of inclusion bodies, and the active form of hIGF2 must be obtained through complex operations such as denaturation and renaturation, thereby reducing the yield of active protein; if one wants to obtain soluble

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  • Recombinant human insulin-like growth factor-2 protein and production method thereof
  • Recombinant human insulin-like growth factor-2 protein and production method thereof
  • Recombinant human insulin-like growth factor-2 protein and production method thereof

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Embodiment Construction

[0039] In the present invention, the Escherichia coli expression strain BL21 CodonPlus (DE3), the vector amplification strain TOP10 and the expression vector pET32-a(+) were all purchased from Invritrogen Company of the United States.

[0040] The medium formulation used is as follows:

[0041] 1) LB liquid medium: NaCl 10g, peptone 10g, yeast extract 5g, distilled water 1L, autoclave, store at room temperature;

[0042] 2) LB / Amp plate: NaCl 10g, peptone 10g, yeast extract 5g, distilled water 1L, agar powder 15g, after autoclaving, cool to below 70°C, add 1mL Ampicillin (100mg / ml), fully Invert the plate after mixing, and store in the dark at 4°C;

[0043] 3) LB / Amp medium: NaCl 10g, peptone 10g, yeast extract 5g, distilled water 1L, after autoclaving, cool to below 70°C, add 1mL ampicillin (100mg / ml), mix well, store at 4°C ;

[0044] 4) 50×TAE agarose gel electrophoresis buffer: Tris base 121g, glacial acetic acid 28.6mL, 0.5mol / LEDTA (pH 8.0) 50mL, add distilled water t...

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Abstract

The invention discloses a recombinant human insulin-like growth factor-2 (hIGF2) protein and production method thereof. An expression host is BL21 (DE3), and a vector is pET32-a(+). The method comprises the steps that: hIGF2 gene is cloned; a prokaryotic expression vector is constructed; expression of soluble Trx-hIGF2 fusion protein with an N terminal with a His*6 mark is carried out; Trx-hIGF2 fusion protein purification is carried out; and enterokinase with the His*6 mark is used for carrying out enzyme digestion upon the Trx-hIGF2 protein, such that high-purity hIGF2 is obtained. According to the invention, BL21 (DE3) and pET32-a(+) are adopted for a first time for realizing hIGF2 high-efficiency soluble fusion expression; His*6 mark and enterokinase cutting technologies are adopted, such that the preparation of a recombinant hIGF2 completely same with hIGF2 on a protein primary structure is realized; and an expression product has bioactivity.

Description

technical field [0001] The present invention relates to the application of recombinant DNA technology to produce genetic engineering protein drug technology, in particular to a recombinant human insulin-like growth factor-2 protein and its production method. Background technique [0002] Insulin-like growth factor (insulin-like growth factor, IGF) is a kind of multifunctional cell proliferation regulator, named for its chemical structure similar to insulin. The human insulin-like growth factor-2 (Human Insulin Like Growth Factor-2, hIGF2) gene consists of 9 exons and 8 introns, its transcription and expression are regulated by 4 promoters, and it is a single-copy gene. The full length is 3kb. The mature human IGF2 protein consists of 67 amino acid residues, and it plays an important regulatory role in fetal development, tumor cell proliferation, and muscle growth. IGF2 has structural homology to the mitogenically active insulin and has been shown to be a major mitogenic gr...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K14/65
Inventor 吴东海李洪波胡兴徐爱民李鹏金守光李娜
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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