A method for increasing the expression level of soluble recombinant prokinein 2β in Escherichia coli

A protokinin, Escherichia coli technology, applied in the field of genetic engineering biology, can solve the problems of complex process, cumbersome operation, and high purification cost, and achieve the effects of improving solubility, simple and easy process operation, and improving soluble expression.

Active Publication Date: 2019-04-02
ANHUI IPROCOM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the type (1) strategy requires more attempts to explore the appropriate medium formula and engineering bacteria culture conditions; the type (2) strategy needs to select the appropriate molecular chaperone protein, and then use genetic engineering technology to construct the corresponding expression vector , to introduce the expression vector into engineering bacteria, the process is complicated and the operation is cumbersome; after the (3) type of strategy obtains the recombinant fusion protein, it must be processed with expensive protease to obtain the desired target protein, and the later purification cost is higher

Method used

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  • A method for increasing the expression level of soluble recombinant prokinein 2β in Escherichia coli
  • A method for increasing the expression level of soluble recombinant prokinein 2β in Escherichia coli
  • A method for increasing the expression level of soluble recombinant prokinein 2β in Escherichia coli

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Experimental program
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Embodiment 1

[0022] Demo strain used E.coli BL21(DE3)pET-PK2β (Wen Chongwei et al. Study on optimization of induction conditions for recombinant protokinesin 2β by response surface analysis. Biotechnology Bulletin. 2012. Pages 173-178) The engineering bacteria are self-constructed. The LB liquid medium used contained 10 g of peptone, 5 g of yeast extract, and 10 g of sodium chloride per liter. The prepared LB medium was sterilized in an autoclave at 121°C for 20 minutes. Then, kanamycin was added to the sterilized LB medium to a final concentration of 50 μg / mL to obtain the fresh LB medium for expression used in the examples. At the same time, lactose is used as the inducer, and lactose is added to the final concentration of 1g / L in the examples. EDTA broken bacteria liquid contains 0.05 mol•L -1 Tris•HCl pH 8.0, 0.10 mol•L -1 NaCl, 0.025mol•L -1 EDTA; hypertonic liquid is 0.02 mol•L -1 Tris•HCl, 0.025 mol•L -1 EDTA, pH 8.0, 35% (w / v) sucrose, hypotonic solution is 0.02 mol•L -1 T...

Embodiment 2

[0029] Demo strain used E.coli BL21 (DE3) pET-PK2β (Wen Chongwei et al. Study on optimization of induction conditions for recombinant protokinesin 2β by response surface analysis. Biotechnology Bulletin. 2012. Pages 173-178) The engineering bacteria are self-constructed. The LB liquid medium used contained 10 g of peptone, 5 g of yeast extract, and 10 g of sodium chloride per liter. The prepared LB medium was sterilized in an autoclave at 121°C for 20 minutes. Then, kanamycin was added to the sterilized LB medium to a final concentration of 50 μg / mL to obtain the fresh LB medium for expression used in the examples. At the same time, lactose is used as an inducer, and lactose is added to the final concentration of 1 g / L in the examples. EDTA broken bacteria liquid contains 0.05 mol•L -1 Tris•HCl pH 8.0, 0.10 mol•L -1 NaCl, 0.025mol•L -1 EDTA; hypertonic liquid is 0.02 mol•L for hypotonic liquid -1 Tris•HCl, 0.025 mol•L -1 EDTA, pH 8.0, 35% (w / v) sucrose, hypotonic solut...

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Abstract

The invention discloses a process method for effectively increasing the expression level of soluble recombinant proteins in escherichia coli. The method comprises the following steps: inoculating 0.1% of engineering bacterium frozen stock liquid expressing the recombinant proteins into a fresh LB liquid culture medium for expressing, carrying out shake cultivation at 37 DEG C for one night to obtain seed liquid, then inoculating 1% of seed liquid into the fresh LB liquid culture medium for expressing, continuing to carry out shake cultivation at 37 DEG C until OD600 of culture liquid reaches 75%-90% of a preset induction starting point, heating until the culture temperature is 40 DEG C, continuing to culture for 0.5-1.5 hours, adding inducers into the culture liquid when the OD600 of culture liquid reaches the preset induction starting point, and continuing to carry out shake cultivation at 37 DEG C or 40 DEG C for 6 hours, so as to produce remarkable soluble recombinant proteins. The method is simple, convenient and effective, low in cost, easy to amplify and suitable for expressing various soluble non-homologous recombinant proteins by virtue of escherichia coli in bioengineering or genetic engineering.

Description

Technical field [0001] The invention relates to the field of genetic engineering biotechnology, and more specifically to a process method for effectively increasing the expression level of soluble recombinant proteins in Escherichia coli. Background technique [0002] The E. coli expression system has the advantages of clear genetic background, short growth cycle, low culture cost, high expression level and easy operation. It is currently the most preferred recombinant protein expression system in the field of genetic engineering biotechnology. Due to the structural instability and distribution instability of the recombinant target gene, it is difficult to continuously cultivate recombinant E. coli. In order to solve this problem, people divide the cultivation of recombinant E. coli into seed growth stage and induced expression stage. The seed growth stage is used for the proliferation of bacterial species and the amplification of recombinant genes; the induced expression stage ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C12R1/19
Inventor 闻崇炜赵烨清周慧莲欧阳臻王晓燕
Owner ANHUI IPROCOM BIOTECH CO LTD
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