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Method for producing human insulin growth factor-1 in recombinant Escherichia coli

A technology for recombining Escherichia coli and growth factors, applied in the field of biomedicine, can solve problems such as high cost, prevent large-scale production of hIGF-1, etc., and achieve the effects of promoting the formation of disulfide bonds, improving soluble expression, and simple separation methods

Inactive Publication Date: 2011-01-19
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recent experiments found that the production of soluble hIGF-1 reached 400 mg / L in E. coli cell-free system, but the high cost of cell-free system prevented the large-scale production of hIGF-1

Method used

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  • Method for producing human insulin growth factor-1 in recombinant Escherichia coli
  • Method for producing human insulin growth factor-1 in recombinant Escherichia coli
  • Method for producing human insulin growth factor-1 in recombinant Escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Take 5ul of the bacterial solution in the glycerol tube and add it to 5ml of LB culture solution containing ampicillin (Amp), chloramphenicol (Chl), kanamycin (Kan) and tetracycline (Tet), at 37°C, 200 rpm, Incubate overnight on a shaker. The next day, take 1ml of the bacterial liquid, add it to LB medium containing 10 times the corresponding antibiotic, and cultivate it at 37°C until OD=1.0, and the concentration of the inducer is 1.0mM. , 34°C, 37°C and 40°C for 4 hours. The optimum temperature for expression was 34°C, and the expression level of the soluble fusion protein reached 1.2g / L ( figure 2 ).

Embodiment 2

[0049] Same as in Example 1, inoculate 1ml of the bacterial solution the next day, add it to 10 times the LB medium containing the corresponding antibiotic, determine the growth curve of the engineering bacteria at 34°C, and take the early, middle, late and stable phases of the logarithmic growth phase respectively Induction was carried out, the culture temperature was 34°C during induction, the concentration of inducer was 1.0mM, and cultured for 4h after induction.

[0050] The optimum induction time was in the middle of the logarithmic growth phase, that is, after 4 hours of inoculation, the expression level of the soluble fusion protein reached 1.56g / L ( image 3 ).

Embodiment 3

[0052] Same as Example 1, inoculate 1ml of bacterial liquid the next day, add to 10 times LB medium containing corresponding antibiotics, culture at 34°C, and induce in mid-logarithmic growth phase, the concentrations of inducer IPTG are 0.1mM and 0.25mM respectively , 0.5mM, 0.75mM and 1.0mM, after adding the inducer, cultured at 34°C for 4h, the results are shown in Table 1.

[0053] The optimum concentration of inducer was 0.25mM, and the expression level of soluble fusion protein was 1.83g / L under this induction concentration.

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Abstract

The invention discloses a method for producing a human insulin growth factor-1 in recombinant Escherichia coli, which comprises: designing and synthesizing a gene associated with a hIGF-1 protein; constructing a hIGF-1 fusion protein expression vector; transforming an Escherichia coli host with the expression vector and constructing genetic engineering bacteria; expressing the hIGF-1 fusion protein by the genetic engineering bacteria; separating the hIGF-1 fusion protein; digesting the fusion lag of the hIGF-1 fusion protein by enterokinase; detecting the activity of the hIGF-1 by culturing NIH3T3 cells; and the like. In the invention, the fusion protein expressed in series is adopted, the N terminal of the fusion protein is thioredoxin, the C terminal of the fusion protein is hIGF-1, the middle of the fusion protein has 6 His lags, the expression product is more stable, the separation method is simple and cheap, and thus, the method has a promising application prospect.

Description

technical field [0001] The invention relates to the field of biomedicine in biotechnology, in particular to an application of DNA recombination technology, that is, to construct a fusion protein of thioredoxin and hIGF-1, and to express and prepare hIGF-1 by using Escherichia coli host bacterium Rosetta-gami (DE3) fusion protein, and using enterokinase to cleave the fusion protein to finally obtain the method of hIGF-1. Background technique [0002] Human insulin-like growth factor-1 (hIGF-1) is a single-chain protein consisting of 70 amino acids, containing three disulfide bonds. Its structure, biological properties and chemical properties are similar to insulin, so it is called insulin-like growth factor. Relevant studies have shown that hIGF-1 has the functions of promoting growth and material metabolism, so bone growth, cell replication and other growth-related processes are all affected by IGF-1. For this reason, IGF-1 has received much attention, and has broad applic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C12N1/21C07K19/00C07K14/475C07K1/22C07K1/18
Inventor 徐志南黄磊蔡谨张丹萍
Owner ZHEJIANG UNIV
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