CN111378030A - Method for separating and purifying nano antibody - Google Patents
Method for separating and purifying nano antibody Download PDFInfo
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- CN111378030A CN111378030A CN201811636872.1A CN201811636872A CN111378030A CN 111378030 A CN111378030 A CN 111378030A CN 201811636872 A CN201811636872 A CN 201811636872A CN 111378030 A CN111378030 A CN 111378030A
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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Abstract
The invention discloses a method for separating and purifying a nano antibody, which relates to the technical field of biology and comprises the following steps: 1) low pH precipitation of heteroproteins: adjusting the pH of a sample to be purified to acidity by using an acidic solution, standing, centrifuging to remove precipitates, and reserving a supernatant solution; 2) and (3) high-temperature precipitation of foreign protein: heating the supernatant solution obtained in the step 1), keeping the temperature, standing, centrifuging to remove precipitates, and reserving the supernatant solution; 3) removing foreign proteins by an ultrafiltration method: carrying out ultrafiltration on the supernatant solution obtained in the step 2), and obtaining a protein solution which permeates through an ultrafiltration membrane, namely the high-purity nano antibody. The method provided by the invention does not influence the activity of the nano antibody, and has the advantages of simple operation, low cost and wide application range.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for separating and purifying a nano antibody.
Background
With the rapid development of biotechnology, antibodies play an indispensable role in the diagnosis and treatment of human diseases, and in recent years, "new and expensive" nanobodies in the antibody world have been rapidly developed and have received much attention. A unique antibody, the heavy chain antibody, is present in the blood of camels and alpacas, and scientists have cloned the variable region of the heavy chain antibody, called VHH antibody, which is 2.5nm in diameter and 4nm in length and is therefore also called nanobody, the smallest fragment that can bind to antigens that exists in nature.
The nano antibody has stable property and still maintains biological activity under the condition of high temperature, which can thoroughly solve the difficult problems of cold chain transportation and storage of biological agents. 1) The nano antibody is stable under strong acid and strong alkali conditions, so that the nano antibody can be used for preparing oral preparations, and the traditional antibody is easy to decompose under high temperature, strong acid and strong alkali conditions and needs to be stored at low temperature; 2) the nano antibody can penetrate into the interior of an antigen due to the structural characteristics of the nano antibody, so that the nano antibody can recognize the antigen which cannot be recognized or approached by the traditional antibody; 3) the nano antibody is very small, can enter the tumor tissue to achieve the aim of thoroughly eliminating the tumor, and can also enter the brain tissue through the blood brain barrier, the molecular weight of the traditional antibody is ten times that of the nano antibody, and the tissue permeability is poor; 4) the nano antibody has a simple structure, can be produced in a large scale in simpler microbial systems such as escherichia coli, saccharomycetes and the like, remarkably reduces research and development and production costs, and solves the problem of large-scale preparation of antibody products. Therefore, the nanobody technology is the first of ten new-generation biotechnology platforms that are considered most interesting in 2012, and is receiving more and more attention in the fields of immunity, diagnosis, medical imaging, detection, protein crystallization, antibody-targeted diagnosis and treatment of tumors, and the like.
The purification of the nano-antibody still depends on fusing an affinity tag (such as His) at the N terminal or the C terminal6GST, MBP) using affinity chromatography, but protease cleavage of the affinity tag increases downstream purification pressure. The traditional chromatographic techniques such as ion exchange chromatography, hydrophobic chromatography and the like are directly utilized to purify the nano antibody, so that the cost is high, the purity is low, and the nano antibody generally needs to be purifiedTwo or more chromatography steps are required to achieve higher purity and rely on expensive protein chromatography systems and chromatography media. Therefore, there is a need to find a simpler and more effective purification method for separating and purifying nanobodies.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for separating and purifying a nanobody, comprising the following steps:
1) low pH precipitation of heteroproteins: adjusting the pH of a sample to be purified to acidity by using an acidic solution, standing, centrifuging to remove precipitates, and reserving a supernatant solution;
2) and (3) high-temperature precipitation of foreign protein: heating the supernatant solution obtained in the step 1), keeping the temperature, standing, centrifuging to remove precipitates, and reserving the supernatant solution;
3) removing foreign proteins by an ultrafiltration method: carrying out ultrafiltration on the supernatant solution obtained in the step 2), and obtaining a protein solution which permeates through an ultrafiltration membrane, namely the high-purity nano antibody.
The solution in the step 1) is one of acetic acid and HCl, and the concentration is 1-2 mol/L.
Step 1) adjusting the pH to acidity, and adjusting the pH to 2.0-3.0.
The standing time of the step 1) is 1-2 h.
Step 2) heating, namely heating to 70-80 ℃.
And 2) preserving the heat, wherein the temperature is 70-80 ℃, and the standing time is 1-2 h.
Step 3), performing ultrafiltration, wherein the molecular weight cut-off is 30 kDa.
And 3) performing ultrafiltration through an ultrafiltration tube or a tangential flow filtration system.
Step 1) before the sample to be purified is treated, filtering and sterilizing the sample by a 0.22 mu m filter membrane.
The sample to be purified in the step 1) is cell fermentation liquor or a protein sample extracted from cells.
The cell fermentation liquid is culture liquid obtained by fermenting yeast, and the protein sample extracted from the cells is intracellular protein or periplasmic protein solution extracted from escherichia coli cells.
Advantageous effects
Compared with the prior art, the invention has the following advantages:
(1) the method is simple to operate, the high-purity nano antibody can be obtained only through simple precipitation and centrifugation, the time required by purification is greatly reduced, and the purification period is short.
(2) Does not depend on a professional protein chromatographic system and a chromatographic medium, and has wider application range.
(3) The economic cost is low, and the requirements of conventional industrial mass production are met.
At present, the purification of nanobodies still depends on the fusion of an affinity tag (such as His) at the N-terminal or C-terminal6GST and MBP), based on which the field generally believes that the nano antibody needs to be purified by using an affinity chromatography technology, the invention does not follow the conventional method, adopts a simpler method which overturns the technical bias in the field, does not influence the activity of the nano antibody, and has the advantages of simple operation, low cost and wide application range.
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FIG. 1 shows the SDS-PAGE results of the precipitation of intracellular proteins of E.coli under different pH conditions.
FIG. 2 shows SDS-PAGE results of E.coli intracellular proteins precipitated under different temperature conditions.
FIG. 3 shows SDS-PAGE results of E.coli intracellular proteins after low pH precipitation, high temperature precipitation and ultrafiltration purification.
FIG. 4 shows SDS-PAGE results of E.coli periplasmic proteins after low pH precipitation, high temperature precipitation and ultrafiltration purification.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention. The experimental procedures in the examples, unless otherwise specified, were carried out using techniques conventional in the art. All chemical reagents are commercially available, conventional analytical reagents without specific indication.
Example 1 purification of Nanobodies in E.coli periplasmic proteins.
Obtaining the sample to be purified (Large intestine)Bacillus intracellular protein): coli expression in 5L
B, producing the nano antibody A in a stirring type glass bioreactor, inducing the nano antibody to perform periplasm expression by using 1mM IPTG, centrifuging the cell culture solution at 4 ℃ and 8000rpm for 20min, collecting thalli, carrying out heavy suspension extraction on the thalli, crushing cells by using ultrasonic waves, centrifuging at 12000rpm for 20min, collecting supernatant, filtering and sterilizing through a 0.22 mu m filter membrane, and then purifying the nano antibody serving as a sample to be purified.
1) Taking 30mL × 3 parts of sample to be purified, respectively adjusting the pH to 2.0, 3.0 and 4.0 by using 2mol/L acetic acid solution, as three experiments, respectively carrying out the following steps of standing for 2h at 4 ℃, centrifuging at 12000rpm for 20min, separating supernatant and precipitate, adjusting the pH of the supernatant to be neutral by using 2mol/L LTris solution, carrying out heavy suspension on the precipitate by using 30mL LPBS solution, respectively taking 20 mu L of supernatant and precipitate solution, and carrying out SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), wherein the results are shown in figure 1, and the effect of removing impure proteins is best at pH 2.0 and pH 3.0.
2) And (3) high-temperature precipitation of foreign protein: taking 10ml of protein supernatant solution treated by pH3.0 in the step 1), respectively placing the protein supernatant solution in water baths at 60 ℃, 70 ℃ and 80 ℃ for standing for 2h, and performing the following steps as three experiments: centrifuging at 12000rpm for 20min, separating supernatant and precipitate, resuspending the precipitate with 10mL PBS solution, and performing SDS-PAGE on 20 μ L of the supernatant and the precipitate respectively, the results are shown in FIG. 2, and the effect of removing the impurity proteins at 70 ℃ and 80 ℃ is the best.
3) Removing foreign proteins by an ultrafiltration method: and (3) carrying out ultrafiltration on the protein supernatant solution treated at the temperature of 70 ℃ in the step 2) by using an ultrafiltration tube with the molecular weight cutoff of 30kDa, collecting the lower protein solution passing through the ultrafiltration membrane, and taking 20 mu L of the protein solution to carry out SDS-PAGE, wherein the result is shown in figure 3, and the nano antibody A is pure, has no visible impurity band and does not influence the affinity of the nano antibody A.
Example 2 purification of Nanobodies in E.coli periplasmic proteins.
Example 1 was repeated, differing from example 1 in that the sample to be purified (E.coli periplasmic protein) was obtained in this example by the following preparation method: escherichia coliThe expression strain is 5L
B, producing a nano antibody B in a stirring type glass bioreactor, inducing the nano antibody to perform periplasmic expression by using 1mM IPTG, centrifuging a cell culture solution at 4 ℃ for 20min under the condition of 4000 × g, collecting thalli, then re-suspending the thalli to extract periplasmic protein, filtering and sterilizing the periplasmic protein through a 0.22-micron filter membrane, and then purifying the nano antibody serving as a sample to be purified, wherein the pH precipitation condition is 3.0, the high-temperature precipitation condition is 70 ℃, the rest operations are the same as those in the example 1, the result is shown in figure 4, the nano antibody B has a good purification effect, and purer target protein can be separated.
Claims (10)
1. A method for separating and purifying a nano antibody is characterized by comprising the following steps: the method comprises the following steps:
1) low pH precipitation of heteroproteins: adjusting the pH of a sample to be purified to acidity by using an acidic solution, standing, centrifuging to remove precipitates, and reserving a supernatant solution;
2) and (3) high-temperature precipitation of foreign protein: heating the supernatant solution obtained in the step 1), keeping the temperature, standing, centrifuging to remove precipitates, and reserving the supernatant solution;
3) removing foreign proteins by an ultrafiltration method: carrying out ultrafiltration on the supernatant solution obtained in the step 2), and obtaining a protein solution which permeates through an ultrafiltration membrane, namely the high-purity nano antibody.
2. The method for separating and purifying nanobody according to claim 1, wherein: the solution in the step 1) is one of acetic acid and HCl, and the concentration is 1-2 mol/L.
3. The method for separating and purifying nanobody according to claim 1, wherein: step 1) adjusting the pH to acidity, and adjusting the pH to 2.0-3.0.
4. The method for separating and purifying nanobody according to claim 1, wherein: the standing time of the step 1) is 1-2 h.
5. The method for separating and purifying nanobody according to claim 1, wherein: step 2) heating, namely heating to 70-80 ℃.
6. The method for separating and purifying nanobody according to claim 1, wherein: and 2) preserving the heat, wherein the temperature is 70-80 ℃, and the standing time is 1-2 h.
7. The method for separating and purifying nanobody according to claim 1, wherein: step 3), performing ultrafiltration, wherein the molecular weight cut-off is 30 kDa.
8. The method for separating and purifying nanobody according to claim 1, wherein: and 3) performing ultrafiltration through an ultrafiltration tube or a tangential flow filtration system.
9. The method for separating and purifying nanobody according to claim 1, wherein: the sample to be purified in the step 1) is cell fermentation liquor or a protein sample extracted from cells.
10. The method for separating and purifying nanobody according to claim 9, wherein: the cell fermentation liquid is culture liquid obtained by fermenting yeast, and the protein sample extracted from the cells is intracellular protein or periplasmic protein solution extracted from escherichia coli cells.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110386978A (en) * | 2018-04-19 | 2019-10-29 | 上海多米瑞生物技术有限公司 | The extracting method of the albumen of colibacillus periplasm space expression |
| CN114106154A (en) * | 2022-01-25 | 2022-03-01 | 上海健士拜生物科技有限公司 | Chromatography method of nano antibody |
| CN114409787A (en) * | 2022-02-18 | 2022-04-29 | 南京英瀚斯生物科技有限公司 | Separation and purification method for HER3 nano antibody |
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| CN1450169A (en) * | 2003-05-08 | 2003-10-22 | 福建师范大学 | Separation purification method for preparing sex-gene recombination spider dragline silk protein |
| US20130005948A1 (en) * | 2010-03-10 | 2013-01-03 | Vinzenz Binder | Method for purifying polypeptide solutions |
| CN105734097A (en) * | 2016-04-28 | 2016-07-06 | 东南大学 | MIM-I-BAR protein extracting and purifying method |
| CN107794254A (en) * | 2017-09-30 | 2018-03-13 | 昆明理工大学 | The method for quickly purifying of recombinant high temperature lyases |
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- 2018-12-29 CN CN201811636872.1A patent/CN111378030A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1450169A (en) * | 2003-05-08 | 2003-10-22 | 福建师范大学 | Separation purification method for preparing sex-gene recombination spider dragline silk protein |
| US20130005948A1 (en) * | 2010-03-10 | 2013-01-03 | Vinzenz Binder | Method for purifying polypeptide solutions |
| CN105734097A (en) * | 2016-04-28 | 2016-07-06 | 东南大学 | MIM-I-BAR protein extracting and purifying method |
| CN107794254A (en) * | 2017-09-30 | 2018-03-13 | 昆明理工大学 | The method for quickly purifying of recombinant high temperature lyases |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110386978A (en) * | 2018-04-19 | 2019-10-29 | 上海多米瑞生物技术有限公司 | The extracting method of the albumen of colibacillus periplasm space expression |
| CN114106154A (en) * | 2022-01-25 | 2022-03-01 | 上海健士拜生物科技有限公司 | Chromatography method of nano antibody |
| CN114409787A (en) * | 2022-02-18 | 2022-04-29 | 南京英瀚斯生物科技有限公司 | Separation and purification method for HER3 nano antibody |
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