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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 complete dissolution, difficulty in renaturation, poor effect, etc., and achieve the effect of improving yield and simple operation.

Active Publication Date: 2015-01-21
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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  • Method and reagent for preparing soluble interleukin recombinant protein from inclusion body
  • Method and reagent for preparing soluble interleukin recombinant protein from inclusion body
  • Method and reagent for preparing soluble interleukin recombinant protein from inclusion body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Comparing the dissolution of IL10 recombinant protein inclusion bodies in different solutions:

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

[0041] B. Inclusion body dissolving 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), 6MGuanidine;

[0044] The IL10 inclusion bodies expressed by E. coli were washed and centrifuged and divided into 4 equal aliquots, then added to the above solution, mixed well, resuspended the protein inclusion bodies, suspended with ultrasonic for 10 seconds, centrifuged to collect the supernatant, and measured each supernatant The dissolved IL10 protein content. The results are as follows:

[0045]

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

Embodiment 2

[0047] Example 2: Comparison of the dissolution and renaturation of interleukin recombinant protein inclusion bodies in different denaturing agents:

[0048] The IL10 inclusion bodies expressed in E. coli were washed and centrifuged and divided into 4 groups in equal amounts, and then respectively added the following dissolving solutions of groups A, B, C, and D, mixed well, resuspended the protein inclusion bodies, and suspended 10 by ultrasonic Second, centrifuge to collect the supernatant, and then dialyze the obtained supernatant to remove the denaturant, recover the solution in each dialysis bag, and collect the supernatant by centrifugation (to remove insoluble protein) to detect the dissolved IL10 protein content in each supernatant. The results are as follows:

[0049] Group A:

[0050] Dissolving solution without denaturant: 50mM Tris-HCl (pH8);

[0051] Dialysate: 50mM Tris-HCl (pH8), 50mM NaCL, 5% Glycerol, 5mM EDTA, 3mM 2-mercaptoethanol.

[0052] Group B:

[0053] Inclusio...

Embodiment 3

[0067] The soluble interleukin recombinant protein preparation reagent of the present invention is used to prepare the interleukin IL9 recombinant protein.

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

[0069] B. Harvesting: 3000g, centrifugation at 4°C for 10 minutes, and discard the supernatant.

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

[0071] D Add the cell lysis solution, fully resuspend the cells, lyse the cells on ice 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 washing solution, mix well with the precipitate, resuspend the protein incl...

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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 reagents for preparing soluble interleukin recombinant protein from inclusion bodies. Background technique [0002] Interleukin, abbreviation: Interleukin, (English name: Interleukin, abbreviation: IL), also known as lymphocyte activating factor is the most critical protein molecule in the regulation of the human immune system. At present, 35 interleukins have been found. Interleukins transmit information, activate and regulate immune cells, mediate T and B cell activation, proliferation and differentiation, inflammatory response, and play a very important regulatory role in the process of tumorigenesis. Interleukin has attracted much attention in medical research, new drug development, etc., and its application is getting more and more attention. For example, interleukin-2 (IL-2) has been used as an antitumor drug, according to Global Information Inc. last year. Among...

Claims

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Application Information

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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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