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Method for dissolving inclusion body proteins

A technology for inclusion bodies and proteins, applied in the field of solubilizing inclusion body proteins and inclusion body proteins, can solve the problems of large buffer consumption, low renaturation efficiency, long renaturation time, etc., and achieve simplified operation steps and high-efficiency renaturation. , mild effect of denaturing conditions

Active Publication Date: 2014-06-04
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The object of the present invention is to provide a method for dissolving inclusion body protein, which needs to consume a large amount of buffer solution and take a long time for refolding in the process of protein refolding in the prior art. Moreover, the technical problem of low refolding efficiency

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  • Method for dissolving inclusion body proteins
  • Method for dissolving inclusion body proteins
  • Method for dissolving inclusion body proteins

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Embodiment 1

[0029] The EGFP protein gene sequence was connected to the prokaryotic expression vector pET-28a by the method of molecular cloning, transformed into Escherichia coli BL21, and the EGFP protein was expressed in the form of inclusion body after being induced and expressed at 37°C, and the method of the present invention was used to dissolve the EGFP inclusion body protein. The operation is as follows:

[0030] (1) Induce the expression of the recombinant strain pET-28a-EGFP500ml, collect the bacteria by centrifugation at 12000r for 15min at 4°C, then resuspend in 20ml of PBS, add 200ul1mg / ml lysozyme to incubate at room temperature for 2h, and then break it by ultrasonic;

[0031] (2) Centrifuge the broken bacteria in the previous step at 12000r for 15min at 4°C, and discard the supernatant;

[0032] (3) Wash the precipitate 3 times with IB buffer;

[0033] (4) After resuspending with IB buffer for the last time, take a 50ul sample as the inclusion body protein sample before fre...

Embodiment 2

[0041] The 1-180 (r-mCRT / 1-180) amino acid gene sequence of the mouse CRT protein was connected to the prokaryotic expression vector pET28a by molecular cloning, transformed into Escherichia coli BL21, and expressed in the form of inclusion bodies after induced expression at 37°C , applying the method of the present invention to dissolve r-mCRT / 1-180 inclusion body protein, its specific operation is as follows:

[0042] (1) Induce 500ml of the recombinant strain pET-28a-CRT, collect the bacteria by centrifugation at 12000r for 15min at 4°C, then resuspend in 20ml of PBS, add 200ul of 1mg / ml lysozyme and incubate at room temperature for 2h, and then break it by ultrasonic;

[0043] (2) Centrifuge the crushed bacteria in the previous step at 4°C, 12000r for 15min, and discard the supernatant;

[0044] (3) Wash the precipitate 3 times with IB buffer;

[0045] (4) After resuspending with IB buffer for the last time, take a 50ul sample as the inclusion body protein sample before f...

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Abstract

The invention discloses a method for dissolving inclusion body proteins. The method comprises the following steps: centrifugally collecting thalli for expressing the inclusion body proteins, re-suspending the centrifugal thalli by using a PBS (Phosphate Buffer Solution), adding lysozyme, keeping at the room temperature for 1-3 hours, performing ultrasonic smashing, and centrifugally removing supernatant to obtain inclusion body protein precipitate; re-suspending the inclusion body protein precipitate in IBbuffer, centrifuging, removing the supernatant, and washing the inclusion body proteins for 1-6 times repeatedly; re-suspending the washed inclusion body proteins by using a urea-containing PBS; refrigerating an inclusion body protein suspension; putting the refrigerated inclusion body protein suspension at the room temperature, slowly dissolving, and centrifuging to obtain supernatant, namely, the inclusion body proteins. The method is easy to operate, an obtained inclusion body protein solution contains a low-concentration denaturant, a renaturation step is simplified, time is saved, and the protein renaturation efficiency is increased.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to inclusion body protein, especially a method for dissolving inclusion body protein. Background technique: [0002] When exogenous genes are expressed in prokaryotic cells, especially when they are highly expressed in E. coli, they often aggregate to form protein aggregates, that is, inclusion bodies. The traditional view is that inclusion bodies are inactive and insoluble protein forms formed by protein misfolding. To dissolve inclusion body proteins, strong denaturants, such as 8M urea or 6M guanidine hydrochloride, are generally required to interact with ions. , to break various chemical bonds within and between molecules of the inclusion body protein, so that the polypeptide can be stretched and soluble. Denatured proteins restore the target protein from its denatured stretched state to its correct higher order structure by slowly removing the denaturant. Recent studies have fou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14
Inventor 熊思东齐兴梅
Owner SUZHOU UNIV
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