A method and reagent for preparing soluble interleukin recombinant protein from inclusion body

A recombinant protein and inclusion body technology, applied in the field of recombinant proteins, can solve the problems of difficult to completely dissolve, difficult to renature, and insoluble of interleukin recombinant proteins, and achieves the effect of simple operation and improved yield.

Active Publication Date: 2018-08-03
承功(厦门)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies have shown that there is a big problem in the production of interleukin in the E. coli expression system: most interleukin recombinant proteins exist in the form of insoluble inclusion bodies in the E. coli expression system
However, the inclusion body of the recombinant interleukin protein is difficult to completely dissolve even with 8M urea, so it is necessary to use more harsh 6M guanidine hydrochloride to dissolve, but the protein dissolved by 6M guanidine hydrochloride is difficult to refold and the effect is very poor

Method used

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  • A method and reagent for preparing soluble interleukin recombinant protein from inclusion body
  • A method and reagent for preparing soluble interleukin recombinant protein from inclusion body
  • A method and reagent for preparing soluble interleukin recombinant protein from inclusion body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment one: compare interleukin IL10 recombinant protein inclusion body dissolution situation in different solutions:

[0040] A. No denaturant: 50mM Tris-HCl (pH8);

[0041] B. Inclusion body solution of the present invention: 50mM Tris-NaOH (pH12);

[0042] C. Urea denaturant: 50mM Tris-HCl (pH8), 8M-Urea;

[0043] D. Guanidine hydrochloride denaturant: 50mM Tris-HCl (pH8), 6M Guanidine;

[0044]Wash and centrifuge the IL10 inclusion body expressed by Escherichia coli and divide it into 4 equal parts, then add the above solution respectively, mix well, resuspend the protein inclusion body, use ultrasonic wave to suspend for 10 seconds, collect the supernatant by centrifugation, and measure each supernatant The content of IL10 protein dissolved in the The results are as follows:

[0045]

[0046] Obviously, the inclusion body lysate of the present invention can significantly dissolve IL10 recombinant protein in the inclusion body.

Embodiment 2

[0047] Example 2: Comparing the dissolution and renaturation of recombinant interleukin protein inclusion bodies in different denaturants:

[0048] After washing and centrifuging, the IL10 inclusion bodies expressed by Escherichia coli were divided into 4 groups in equal amounts, and then the lysates described in the following groups A, B, C, and D were added, mixed well, and the protein inclusion bodies were resuspended. second, centrifuge to collect the supernatant, then dialyze each supernatant to remove the denaturant, reclaim the solution in each dialysis bag, and centrifuge to collect the supernatant (remove insoluble protein) to detect the dissolved IL10 protein content in each supernatant. The results are as follows:

[0049] Group A:

[0050] Denaturing agent-free solution: 50mM Tris-HCl (pH8);

[0051] Dialysate: 50 mM Tris-HCl (pH 8), 50 mM NaCL, 5% Glycerol, 5 mM EDTA, 3 mM 2-mercaptoethanol.

[0052] Group B:

[0053] Inclusion body solution of the present inv...

Embodiment 3

[0067] The interleukin IL9 recombinant protein is prepared by using the reagent for preparing the soluble interleukin recombinant protein of the present invention.

[0068] A. The overnight cultured IL9-expressing strains were inoculated with new culture medium at a 1:10 dilution, cultured on a shaker at 37°C at 200rpm for 3 hours, added IPTG (final concentration 1mM) to induce expression, and continued to grow at 37°C at 200rpm on a shaker for 4 hours.

[0069] B. Bacteria collection: 3000g, centrifuge at 4°C for 10 minutes, discard the supernatant.

[0070] C, add washing solution, fully suspend the bacteria, centrifuge at 3000g, 4°C for 10 minutes, and remove the supernatant;

[0071] D Add the cell lysate, fully resuspend the cells, lyse the cells on ice with ultrasound for 5 seconds, with an interval of 10 seconds, repeat the ultrasonic lysis for 5 minutes, centrifuge at 8000g, 4°C for 20 minutes, and remove the supernatant;

[0072] E Add the washing solution, mix with ...

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Abstract

The invention discloses a method and a reagent for preparing soluble interleukin recombinant protein from inclusion body. The reagent comprises a washing liquid, a thalli lysate, an inclusion body dissolving liquid, a protein renaturation liquid I and a protein renaturation liquid II. The employed inclusion body dissolving liquid is capable of substantially improving the yield of soluble interleukin recombinant protein from inclusion body, thereby solving the problem existed in a process of using an escherichia coli expression system to produce interleukin. Compared with the prior art, the reagent does not contain any protein denaturant, thereby facilitating subsequent protein renaturation. The method is simple in operation, is capable of improving the yield of soluble interleukin recombinant protein by 90% or more, has obvious advantage compared with a routine method which employs a protein denaturant and only has the yield of 5-20%, and has value of popularization and application.

Description

technical field [0001] The invention relates to a recombinant protein technology, in particular to a method and reagent for preparing soluble interleukin recombinant protein from inclusion bodies. Background technique [0002] Interleukin, referred to as: Interleukin, (English name: Interleukin, abbreviated: IL), also known as lymphocyte activating factor, is the most critical protein molecule in the regulation of the human immune system, and 35 interleukins have been discovered so far. Interleukins play a very important regulatory role in transmitting information, activating and regulating immune cells, mediating T and B cell activation, proliferation and differentiation, inflammatory response, and tumorigenesis. Interleukin has attracted much attention in medical research and new drug development, and its application has been paid more and more attention. For example, interleukin-2 (IL-2) has been used as an anti-tumor drug, according to GlobalInformationInc. It was report...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/54C07K1/14
CPCC07K14/54
Inventor 邹潮
Owner 承功(厦门)生物科技有限公司
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