CN101812115A

CN101812115A - Method for renaturing inclusion bodies of colibacillus

Info

Publication number: CN101812115A
Application number: CN201010139322A
Authority: CN
Inventors: 王志超
Original assignee: SHANGHAI REALGENE BIOTECH Inc
Current assignee: SHANGHAI REALGENE BIOTECH Inc
Priority date: 2010-04-02
Filing date: 2010-04-02
Publication date: 2010-08-25

Abstract

The invention relates to the field of bioengineering-downstream protein purification, in particular to a method for renaturing inclusion bodies formed by the protein expression of colibacillus. The method is characterized in that the specific processing steps are as follows: (1) collection of inclusion body protein: mycelia are collected by way of 6-minute centrifugation at 5000rpm/min under the temperature of 4 DEG C, and are broken up by ultrasonic, and by way of 30-minute high-speed freezing centrifugation at 8000rpm/min under the temperature of 4 DEG C, P003B protein inclusion bodies are obtained; (2) purification of inclusion bodies: the inclusion bodies are resuspended in 50mL of wash buffer, so that impurity protein and other impurities are dissolved, and thereby the P003B inclusion bodies are purified; (3) renaturation of inclusion bodies: under the temperature of 4 DEG C, the dissolved inclusion bodies are slowly diluted by renaturation buffer by means of a constant-flow pump until the pH of the buffer is 8.0, and are constantly stirred. Compared with the prior art, the mild dissolution method maintains the protein secondary structure formed in the process of inclusion body formation, increases the efficiency of inclusion body renaturation, simplifies the steps of renaturation and saves time, and thereby the cost of recombinant protein production is greatly reduced.

Description

A kind of method of renaturing inclusion bodies of colibacillus

Technical field

What the present invention relates to relate to is protein purification field, biotechnology downstream, is specifically related to e. coli protein and expresses the refolding method that forms inclusion body.

Background technology

Intestinal bacteria are widely used for expressing those does not need posttranslational modification such as glycosylation to keep active protein, yet intestinal bacteria great expression recombinant protein usually causes its gathering and forms albumen precipitation---inclusion body.Inclusion body protein does not have biological activity, needs complicated dissolving, renaturation, and purifying has obtained the active product of function.Usually, add reductive agent such as beta-mercaptoethanol simultaneously, behind the protein dissolution, come renaturation by the method for slowly removing denaturing agent existing under the condition of oxygenant with the denaturing agent (urea, Guanidinium hydrochloride) of high density dissolving inclusion body.

The shortcoming of traditional renaturing inclusion bodies method: (1) will cause the secondary protein structure forfeiture with high density denaturing agent dissolving inclusion body, peptide chain will be twined at random and hydrophobic surface is exposed, and renaturing inclusion bodies to go out to have the low major cause of bioactive protein efficiency ratio, PER be the time to have lost secondary protein structure in dissolving, the protein of sex change twines aggregate and precipitate mutually in the process of renaturation.(2) many times, the rate of recovery of renaturing inclusion bodies probably is 15-25%, and it occupies most expenses that intestinal bacteria produce recombinant protein.Therefore, a bionic very big challenge is exactly not have active and insoluble albumen is more effective is converted into the solvable and correct protein that folds with this.Therefore, if can find a kind of dissolved method to make solubilization of inclusion bodies but do not make the forfeiture of its secondary structure, this not only can make protein renaturation have higher success rate, and can significantly reduce the expense that it produces activated protein.

Summary of the invention

Need the complicated operations step in order to overcome existing renaturing inclusion bodies method, cycle is long, and the low shortcoming of the rate of recovery, the present invention adopts gentle dissolving method to keep the secondary structure of protein aggregation body when solubilization of inclusion bodies, thereby simplified protein renaturation, improved protein recovery, reduced production costs.

For achieving the above object, design a kind of method of renaturing inclusion bodies of colibacillus, it is characterized in that concrete processing step is as follows: the collection of (1) inclusion body protein, 4 ℃ of centrifugal collection thalline of 5000rpm/min 6min, with the thalline ultrasonication, 4 ℃ of 8000rpm/min 30min high speed frozen centrifugations obtain P003B albumen inclusion body; (2) purifying of inclusion body, resuspended inclusion body in 50mL lavation buffer solution (5mM EDTA, 1%Triton X-100,10% glycerine, pH 8.0 for 50mM Tris, 500mMNaCl) thus dissolving foreign protein and other impurity purifying P003B inclusion body; (3) renaturing inclusion bodies, at 4 ℃, the dissolved inclusion body is 8.0 by constant flow pump (west, Shanghai, Qingpu, BT-100 Shanghai instrument plant) with the slow dilution of renaturation buffer (50mM Tris 500mM NaCl 2M Urea0.5mM EDTA 1%Triton X-100 10% glycerine 1mM PMSF pH 8.0) (0.5mL/min) to pH of buffer, and constantly stirs.

The present invention compared with prior art, gentle dissolving method has kept the secondary protein structure that forms in the inclusion body forming process, improved the efficient of renaturing inclusion bodies, has simplified the step of renaturation, save the time, thereby greatly reduced the expense that recombinant protein is produced.

Embodiment

Embodiment 1 P003B inclusion body protein purifying

1. cultivating 37 ℃ of 240rpm/min abduction deliverings of 1L P003B recombination bacillus coli recombinant protein makes it form inclusion body.

2.4 the centrifugal collection thalline of ℃ 5000rpm/min 6min (CR21G II FDAC).

3. the resuspended bacterial sediment of supernatant discarded is in the resuspended damping fluid of 50mL (5mMEDTA, pH 8.0 for 50mM Tris, 500mM NaCl).

4. ultrasonication (400 watts were worked a breath 1 second, total duration 5 minutes 1 second) (JY96-II supersonic cell crusher NingBo XinZhi Biology Science Co., Ltd).

5.4 ℃ 8000rpm/min 30min high speed frozen centrifugation (CR21G II FDAC).

6. the resuspended 50mL lavation buffer solution (5mM EDTA, 1%Triton X-100,10% glycerine, pH 8.0 for 50mM Tris, 500mM NaCl) that is deposited in of supernatant discarded.

7. ultrasonic dissolution (400 watts were worked a breath 1 second, total duration 5 minutes 1 second)

8.4 ℃ 8000rpm/min 30min high speed frozen centrifugation (CR21G II FDAC).

9. resuspended 37 ℃ of overnight incubation of 50mL lavation buffer solution (5mM EDTA, 1%Triton X-100,10% glycerine, pH 8.0 for 50mM Tris, 500mM NaCl) that are deposited in of supernatant discarded.

10. ultrasonic dissolution (400 watts were worked a breath 1 second, total duration 5 minutes 1 second)

11.4 ℃ 8000rpm/min 30min high speed frozen centrifugation (CR21G II FDAC).

Be deposited in 50mL lavation buffer solution (5mM EDTA, 1%Triton X-100,10% glycerine, pH 8.0 for 50mM Tris, 500mM NaCl) incubated at room 30min 12. supernatant discarded is resuspended.

13. ultrasonic dissolution (400 watts were worked a breath 1 second, total duration 5 minutes 1 second)

14.4 ℃ 8000rpm/min 30min high speed frozen centrifugation (CR21G II FDAC).(obtaining purified inclusion body)

15. the resuspended 100mL solubilization of inclusion bodies damping fluid (50mM Tris, 500mM NaCl, 2M Urea, 1mM PMSF pH 8.0) that is deposited in of supernatant discarded.

16. resuspended inclusion body is divided into 11 parts, regulates solubilization of inclusion bodies pH of buffer value to 3.0 respectively, 4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0,12.0,13.0.

17. difference ultrasonic dissolution (400 watts were worked a breath 1 second, total duration 1 minute 1 second).

18. difference 4 ℃ of 8000rpm/min 30min high speed frozen centrifugations (CR21G II FDAC).

Carry out the SDS-PAGE analysis 19. get variant pH value ultrasonic dissolution liquid supernatant liquor 20ul respectively, the amount of observing solubilization of inclusion bodies under which potential of hydrogen is big, filters out the gentle dissolving of inclusion body Optimum pH pH 12.

20. at 4 ℃, the dissolved inclusion body is 8.0 by constant flow pump (west, Shanghai, Qingpu, BT-100 Shanghai instrument plant) with the slow dilution of renaturation buffer (50mM Tris 500mM NaCl 2M Urea 0.5mM EDTA 1%Triton X-100 10% glycerine 1mM PMSF pH 8.0) (0.5mL/min) to pH of buffer, and constantly stirs.

21. the inclusion body that renaturation is good makes its clarification with the 0.22um membrane filtration.

Claims

1. the method for a renaturing inclusion bodies of colibacillus is characterized in that concrete processing step is as follows: the resuspended 100mL of the being deposited in solubilization of inclusion bodies of (1) supernatant discarded damping fluid, 50mM Tris, 500mMNaCl, 2M Urea, 1mM PMSF pH 8.0; (2) resuspended inclusion body is divided into 10 parts, regulates solubilization of inclusion bodies pH of buffer to 3.0 respectively, 4.0,5., 0,6.0,7.0,8.0,9.0,10.0,11.0,12.0; (3) difference ultrasonic dissolution, was worked a breath 1 second, total duration 1 minute 1 second by 400 watts; (4) 4 ℃ of 8000rpm/min 30min high speed frozen centrifugations of difference; (5) get variant pH value ultrasonic dissolution liquid supernatant 20ul respectively and carry out the SDS-PAGE analysis, the amount of observing solubilization of inclusion bodies under which potential of hydrogen is big, promptly filters out the gentle dissolving of inclusion body Optimum pH; (4) at 4 ℃, the dissolved inclusion body is by the constant flow pump renaturation buffer, 50mM Tris500mM NaCl 2M Urea 0.5mM EDTA 1%Triton X-10010% glycerine 1mM PMSF pH8.0, slowly being diluted to pH of buffer with 0.5mL/min is 8.0, and constantly stirs.

2. the method for a kind of renaturing inclusion bodies of colibacillus according to claim 1, the good inclusion body that it is characterized in that described purifying are resuspended in the damping fluid of the denaturing agent 2M Urea that contains lower concentration.

3. the method for a kind of renaturing inclusion bodies of colibacillus according to claim 1 is characterized in that screening suitable solubilization of inclusion bodies liquid potential of hydrogen, and 3.0,4.0,5., 0,6.0,7.0,8.0,9.0,10.0,11.0,12.0,13.0.

4. the method for a kind of renaturing inclusion bodies of colibacillus according to claim 1 is characterized in that in protein renaturation the pH value of dissolved inclusion body solution is converted into physiology acidity.