阅读说明：本技术 一种环保的可逆的蛋白变性工艺 (Environment-friendly reversible protein denaturation process ) 是由 范豪 黄潇 平宪卿 刘高成 杨洋 连志福 刘筱烜 于 2021-08-27 设计创作，主要内容包括：本发明公开了一种环保的可逆的蛋白变性工艺。采用不含氮的变性液溶解需变性的蛋白,得到的变性蛋白经过复性过程,仍能得到具备生物活性的蛋白。该工艺既经济又环保,可适用于工业化大生产。(The invention discloses an environment-friendly reversible protein denaturation process. The protein to be denatured is dissolved by using the nitrogen-free denaturing solution, and the obtained denatured protein still can obtain the protein with biological activity through the renaturation process. The process is economical and environment-friendly, and is suitable for industrial mass production.)

1. A method of reversible protein denaturation, comprising: mixing the protein to be denatured with a denaturing solution to form a denaturation reaction solution, and adjusting the pH value of the denaturation reaction solution to denature the protein to obtain the denatured protein, wherein the denaturation solution does not contain a nitrogen-containing substance, and the pH value of the denaturation reaction solution is adjusted to be at least 11.

2. The method for denaturing protein according to claim 1, wherein the denaturing solution comprises 1 to 30mmol/L sodium bicarbonate buffer or 1 to 30mmol/L sodium carbonate buffer.

3. The method for denaturing protein according to claim 2, wherein the denaturing solution further comprises 0.1 to 10mmol/L EDTA.

4. A method of denaturing proteins according to any of claims 1 to 3, wherein the denaturing solution comprises a reducing agent selected from one or more of the following group: beta-mercaptoethanol, dithiothreitol, dithioerythritol, cysteine, tris (2-carboxyethyl) phosphine; the concentration of each of the reducing agents is 0.05-30 mmol/L.

5. The method for denaturing protein according to any one of claims 1 to 4, wherein a surfactant is not included in the denaturation reaction solution.

6. A protein denaturing method according to any one of claims 1 to 5, wherein an alkaline solution is added to the denaturation reaction solution to adjust the pH, the alkaline solution being one selected from the group consisting of: sodium hydroxide, potassium hydroxide; the concentration of the alkaline solution is 0.2-10 mol/L.

7. A method of denaturing proteins according to any one of claims 1 to 6, wherein the method comprises adjusting the pH to at least 11.5, at least 12, or at least 12.5.

8. A protein denaturation method as claimed in any one of claims 1 to 7, characterized in that the temperature of the denaturation reaction is controlled to be 15-40 ℃ or 15-25 ℃.

9. A method of denaturing proteins according to any one of claims 1 to 8, wherein the denaturation reaction time is controlled to be 15 minutes or more, 30 minutes or more, or 60 minutes or more.

10. The method of denaturing protein according to claim 9, wherein the denaturation reaction time is controlled to be within 24 hours, within 12 hours, within 6 hours, or within 3 hours.

11. A method for denaturing protein according to any one of claims 1 to 6, wherein the method comprises adjusting the pH to between 11 and 14 and controlling the denaturation reaction time to between 15 minutes and 8 hours.

12. A method for denaturing a protein according to claim 11, wherein the denaturation reaction temperature is controlled to 15 to 40 ℃.

13. A method according to any one of claims 1 to 12 wherein the protein to be denatured is an inclusion body protein.

14. A protein denaturation method as claimed in any one of claims 1 to 13, characterized in that the protein concentration in the denatured protein solution is 0.1-15 g/L.

15. A method of denaturing a protein according to any one of claims 1 to 14, wherein the denatured protein is renatured to obtain a renatured protein having biological activity.

16. The method for denaturing protein according to any one of claims 1 to 15, wherein the denatured protein is mixed with a renaturation solution to form a renaturation reaction solution, and the pH of the renaturation reaction solution is adjusted to 7 to 12 to renaturate the protein.

17. The method for denaturing protein according to claim 16, wherein the renaturation reaction temperature is controlled to 4 to 30 ℃ and the reaction time is 2 to 48 hours.

18. A method of denaturing proteins as claimed in claim 16, wherein said renaturation solution is sodium carbonate, sodium bicarbonate or glycine.

19. A method of denaturing proteins according to any one of claims 15 to 18, wherein a protease inhibitor is included in the renaturation solution.

20. A method of denaturing proteins as claimed in claim 19, wherein the protease inhibitor is selected from one or more of the group consisting of: EDTA, PMSF, pepsin inhibitors, leupeptin, trypsin inhibitors, serine protease inhibitors, metalloproteinase inhibitors, acid protease inhibitors, and broad-spectrum protease inhibitors.

21. A protein denaturation method as claimed in claim 20, characterized in that the renaturation solution comprises 1-30 mmol/L sodium bicarbonate buffer or sodium carbonate buffer, 1-50 mmol/L glycine buffer, and 0.1-10 mmol/L EDTA.

22. A reversible inclusion body protein denaturation and renaturation method, characterized in that it comprises: mixing the inclusion body protein with a denaturation solution to form a denaturation reaction solution, and adjusting the pH value of the denaturation reaction solution to denature the protein to obtain denatured protein, wherein the denaturation solution does not contain nitrogen-containing substances, and the pH value of the denaturation reaction solution is adjusted to be at least 11; renaturing the denatured protein to obtain the renaturated protein.

23. Renaturation protein obtainable by the process according to claim 22.

24. The renaturation protein of claim 23 in a concentration of at least 0.1 g/L.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an environment-friendly reversible protein denaturation process.

Background

Coli expression systems are widely used in research or production because of their advantages of easy operation, clear genetic background, low production cost, high expression level, etc. However, high levels of expression of recombinant proteins in E.coli often result in protein aggregation to form insoluble, inactive inclusion bodies. Therefore, there is a need to convert inclusion body proteins into recombinant proteins having biological activity through an efficient denaturation and renaturation process.

Inclusion body proteins typically require the addition of denaturing solutions to disrupt the intramolecular and intermolecular forces that maintain the structure of the inclusion body protein, thereby allowing the polypeptide chains to stretch to achieve the effect of solubilizing the inclusion bodies. Commonly used denaturing solutions contain chaotropic agents or detergents. The conventional chaotropic agent such as 6-8 mol/L guanidine hydrochloride or urea can destroy hydrogen bonds between the inclusion body proteins to solubilize the proteins. Guanidine hydrochloride is generally used for dissolving some drug protein molecules with higher added values, and is not suitable for industrial production. Urea is widely used due to low price, but in the industrial denaturation process of the inclusion body protein, the use of a large amount of urea can cause two problems, firstly, the dissolution of high-concentration urea is a continuous heat absorption process, the temperature of equipment needs to be continuously controlled, and certain requirements are designed on a denaturation tank body; secondly, compounds with high nitrogen content (46% of urea and 44% of guanidine hydrochloride) such as urea or guanidine hydrochloride are used, and nitrogen elements cannot be utilized in the denaturation and subsequent purification processes of original core inclusion protein, so that the generated nitrogen-containing waste liquid causes environmental pollution and needs to be recycled, which is a great limiting factor for environment-friendly examination and environmental evaluation work of enterprises. The commonly used detergent is SDS, which has the major disadvantage of being difficult to remove, thus interfering with subsequent purification and renaturation steps and is not generally used for large scale production.

Summary of the invention

The invention aims to provide an environment-friendly and reversible protein denaturation process.

The purpose of the invention can be realized by the following technical scheme:

an industrialized environment-friendly reversible protein denaturation method specifically comprises the following steps:

mixing the protein to be denatured with a denaturing solution to form a denaturation reaction solution, and adjusting the pH value of the denaturation reaction solution to denature the protein to obtain the denatured protein, wherein the denaturation solution does not contain a nitrogen-containing substance, and the pH value of the denaturation reaction solution is adjusted to be at least 11.

The deformation solution comprises 1-30 mmol/L sodium bicarbonate buffer solution or 1-30 mmol/L sodium carbonate buffer solution.

The denaturing solution comprises 0.1-10 mmol/L EDTA.

The denaturing solution comprises a reducing agent selected from one or more of the following group: beta-mercaptoethanol, dithiothreitol, dithioerythritol, cysteine, tris (2-carboxyethyl) phosphine; the concentration of each of the reducing agents is 0.05-30 mmol/L.

The denaturing reaction solution does not include a surfactant.

Adding an alkaline solution to the denaturation reaction solution to adjust the pH value, wherein the alkaline solution is selected from one of the following groups: sodium hydroxide, potassium hydroxide; the concentration of the alkaline solution is 0.2-10 mol/L.

The temperature of the denaturation reaction system is 15-40 ℃, and the preferable temperature is 20-25 ℃.

The time of the denaturation reaction is more than 15 minutes and/or within 24 hours. The concentration of the protein in the denatured protein solution is 0.1-15 g/L.

Renaturing the denatured protein to obtain the renatured protein with biological activity.

And mixing the denatured protein with a renaturation solution to form a renaturation reaction solution, and adjusting the pH value of the renaturation reaction solution to 7-12 to ensure that the protein is renatured.

The renaturation reaction temperature is 4-30 ℃, and the reaction time is 2-48 hours.

The renaturation solution comprises 1-30 mmol/L sodium bicarbonate buffer solution or sodium carbonate buffer solution and 1-50 mmol/L glycine buffer solution.

The renaturation solution comprises a protease inhibitor, and the protease inhibitor is selected from one or more of the following groups: EDTA, PMSF, pepsin inhibitors, leupeptin, trypsin inhibitors, serine protease inhibitors, metalloproteinase inhibitors, acid protease inhibitors, and broad-spectrum protease inhibitors.

A reversible inclusion body protein denaturation and renaturation method, characterized in that it comprises: mixing the inclusion body protein with a denaturation solution to form a denaturation reaction solution, and adjusting the pH value of the denaturation reaction solution to denature the protein to obtain denatured protein, wherein the denaturation solution does not contain nitrogen-containing substances, and the pH value of the denaturation reaction solution is adjusted to be at least 11; renaturing the denatured protein to obtain the renaturated protein.

The concentration of the renaturation protein is at least 0.1 g/L.

The invention relates to a reversible protein denaturation method, wherein a denaturation solution is not added with a nitrogen-containing compound, so that protein is completely dissolved in a short time in a high pH environment, the process has no special design requirement on denaturation equipment, simplifies operation steps, greatly reduces the industrial production cost and time, does not generate nitrogen-containing waste liquid in the production process, is economic and environment-friendly, and is suitable for industrial mass production.

Drawings

FIG. 1A is the HPLC chromatogram of the sample after enzyme digestion after denaturation and renaturation of the inclusion body protein of recombinant human insulin precursor protein in example 1.

FIG. 1B is an HPLC chromatogram of standard recombinant human insulin (3.47 mg/mL).

FIG. 2 is the HPLC chromatogram of the sample after denaturation and renaturation of the inclusion body protein of recombinant human insulin precursor protein and enzyme digestion in example 2.

FIG. 3 is the HPLC chromatogram of the sample after denaturation and renaturation of the inclusion body protein of recombinant human insulin precursor protein and enzyme digestion in example 3.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 renaturation of recombinant human insulin precursor

1) Suspending 2g of inclusion body protein expressed by recombinant human insulin precursor protein in escherichia coli in 998mL of 10mmol/L sodium bicarbonate and 0.2mmol/L EDTA basic buffer solution, adding 0.1mmol/L beta-mercaptoethanol, and adjusting the pH to 11.9 by using 2mol/L sodium hydroxide solution; the temperature of the reaction system is controlled at 20 ℃, and the mixture is stirred for 20 minutes to completely dissolve the inclusion body protein, so as to obtain 2g/L denatured protein solution.

2) Adding the denatured protein solution into a dilution buffer solution containing 10mmol/L sodium bicarbonate and 0.2mmol/L EDTA to form a renaturation reaction solution, wherein the concentration of the recombinant human insulin precursor protein in the renaturation reaction solution is 1g/L, then adjusting the pH value of the solution to 11 by adding a hydrochloric acid solution, controlling the temperature of the renaturation system to be 4 ℃, and reacting for 24 hours to renaturate the protein. And adding carboxypeptidase B and trypsin into the solution after the renaturation reaction, carrying out enzyme digestion activation on the human insulin precursor protein, taking 5 mu L of enzyme digestion solution, carrying out HPLC analysis and detection, wherein the sample map is shown in figure 1A, and the obtained map corresponds to the HPLC map (shown in figure 1B) of the standard recombinant human insulin, thereby confirming that the obtained product is the recombinant human insulin. The retention time of HPLC (high Performance liquid chromatography) spectrum of the recombinant human insulin is 23.6 minutes, and the concentration of the actually obtained recombinant human insulin is 0.15g/L calculated according to the peak area normalization method of the recombinant human insulin

Example 2 renaturation of recombinant human insulin precursor protein

1) Suspending the inclusion body protein expressed by 6g of recombinant human insulin precursor protein in escherichia coli in 994mL of 2mmol/L sodium carbonate and 0.2mmol/L EDTA basic buffer solution, adding 0.2mmol/L beta-mercaptoethanol, and adjusting the pH to 12.1 by using 2mol/L sodium hydroxide solution; the temperature of the reaction system is controlled at 22 ℃, and the mixture is stirred for 20 minutes to completely dissolve the inclusion body protein, so as to obtain 6g/L denatured protein solution.

2) Adding the denatured protein solution into a dilution buffer solution containing 2mmol/L sodium bicarbonate and 0.2mmol/L EDTA to form a renaturation reaction solution, wherein the concentration of the recombinant human insulin precursor protein in the renaturation reaction solution is 1g/L, then adjusting the pH of the solution to 11.5 by adding a hydrochloric acid solution, controlling the temperature of a renaturation system to be 4 ℃, and reacting for 24 hours to renaturate the protein. And adding carboxypeptidase B and trypsin into the solution after the renaturation reaction, carrying out enzyme digestion activation on the human insulin precursor protein, taking 5 mu L of enzyme digestion solution, carrying out HPLC analysis and detection, wherein the sample map is shown in figure 2, and the obtained map corresponds to the HPLC map (shown in figure 1B) of the standard recombinant human insulin, thereby confirming that the obtained product is the recombinant human insulin. The retention time of the HPLC chromatogram of the recombinant human insulin is 23.6 minutes, and the concentration of the actually obtained recombinant human insulin is 0.19g/L according to the peak area normalization method of the recombinant human insulin.

Example 3 renaturation of recombinant human insulin precursor protein

1) Suspending 12g of inclusion body protein expressed by recombinant human insulin precursor protein in escherichia coli in 988mL of 2mmol/L sodium carbonate and 0.2mmol/L EDTA basic buffer solution, adding 0.2mmol/L beta-mercaptoethanol, and adjusting the pH to 12.5 by using 2mol/L sodium hydroxide solution; the temperature of the reaction system is controlled at 22 ℃, and the mixture is stirred for 20 minutes to completely dissolve the inclusion body protein, so as to obtain 12g/L denatured protein solution.

2) Adding the denatured protein solution into a dilution buffer solution containing 2mmol/L sodium bicarbonate and 0.2mmol/L EDTA to form a renaturation reaction solution, enabling the concentration of the recombinant human insulin precursor protein in the renaturation reaction solution to be 1g/L, then adjusting the pH of the solution to 11.5 by adding a hydrochloric acid solution, controlling the temperature of a renaturation system to be 4 ℃, and reacting for 24 hours to renaturate the protein. And adding carboxypeptidase B and trypsin into the solution after the renaturation reaction, carrying out enzyme digestion activation on the human insulin precursor protein, taking 5 mu L of enzyme digestion solution, carrying out HPLC analysis and detection, wherein the sample map is shown in figure 3, and the obtained map corresponds to the HPLC map (shown in figure 1B) of the standard recombinant human insulin, thereby confirming that the obtained product is the recombinant human insulin. The retention time of the HPLC map of the recombinant human insulin is 23.6 minutes, and the concentration of the actually obtained recombinant human insulin is 0.18g/L according to the peak area normalization method of the recombinant human insulin.

Example 4 renaturation of recombinant porcine carboxypeptidase B precursor protein

1) Suspending 6g of inclusion body protein expressed by recombinant porcine carboxypeptidase B precursor protein in escherichia coli in 994mL of 5mmol/L sodium carbonate buffer solution, adding 10mmol/L beta-mercaptoethanol #, and adjusting the pH value to 11.5 by using 2mol/L sodium hydroxide solution; the temperature of the reaction system is controlled at 25 ℃, and the mixture is stirred for 30 minutes until the inclusion body protein is completely dissolved, so that 6g/L denatured protein solution is obtained.

2) Adding the denatured protein solution into a dilution buffer solution containing 5mmol/L glycine to form a renaturation solution, enabling the concentration of the recombinant carboxypeptidase B precursor protein of the renaturation solution to be 0.2g/L, then adjusting the pH of the solution to 9.5 by adding a hydrochloric acid solution, controlling the temperature of the renaturation system to be 20 ℃, and reacting for 24 hours. Adding trypsin into the solution after the renaturation reaction, carrying out enzyme digestion activation on carboxypeptidase B precursor protein, taking 100 mu L of enzyme digestion solution, and measuring the activity of carboxypeptidase B by using an ultraviolet spectrophotometer. The measurement method is as follows:

diluting 100 μ L of enzyme digestion solution by 3 times, taking a clean quartz cuvette, adding 100 μ L of the dilution solution and 2.9mL of substrate solution (0.1mol/L of the hippuroyl-L-arginine solution), immediately mixing uniformly, and immediately adjusting the light absorption value to zero at the wavelength of 254 nm. The ambient temperature was maintained at about 25 ℃ and absorbance was recorded every 1 minute. The measurement was continued for 5 to 10 minutes, and the measurement results are shown in the following table. Taking the measured value of 1-6min to calculate the enzyme activity. The activity of the recombinant carboxypeptidase B obtained by enzyme digestion of 1L of renaturation solution is 4250IU by calculation.

Example 5 renaturation of recombinant porcine Trypsin precursor protein

1) Suspending 992mL of inclusion body protein expressed by recombinant porcine trypsin precursor protein in escherichia coli into 2mmol/L sodium carbonate and 0.2mmol/L EDTA basic buffer solution, adding 4mmol/L beta-mercaptoethanol, and adjusting the pH value to 12.0 by using 2mol/L sodium hydroxide solution; the temperature of the reaction system is controlled at 25 ℃, and the mixture is stirred for 35 minutes until the inclusion body protein is completely dissolved, so as to obtain 8g/L denatured protein solution.

2) Adding the denatured protein solution into a dilution buffer solution containing 5mmol/L glycine to form a renaturation solution, enabling the concentration of the recombinant porcine trypsin precursor protein of the renaturation solution to be 0.4g/L, then adjusting the pH of the solution to 10.5 by adding a hydrochloric acid solution, controlling the temperature of the renaturation system to be 15 ℃, and reacting for 24 hours. Adding trypsin into the solution after the renaturation reaction, carrying out enzyme digestion activation on trypsin precursor protein, taking 100 mu L of enzyme digestion solution, and measuring the activity of the trypsin by using an ultraviolet spectrophotometer. The measurement method is as follows:

diluting 100 μ L of enzyme digestion solution by 50 times, taking a clean quartz cuvette, adding 200 μ L of the dilution solution and 3mL of substrate solution (0.025mmol/L N-benzoyl-L-arginine ethyl ester hydrochloride), immediately mixing, and immediately setting the light absorption value to zero at the wavelength of 253 nm. The ambient temperature was maintained at about 25 ℃ and absorbance was recorded every 30 seconds. The measurement was continued for 3 minutes, and the measurement results are shown in the following table. The activity of the recombinant trypsin obtained by enzyme digestion of each 1L of renaturation solution is calculated to be 3583000 IU.

Time	0s	30s	60s	90s	120s	150s	180s
								A253nm	0.002	0.024	0.045	0.067	0.088	0.114	0.131