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Method for separation and purification of nanobody

A technology for separation and purification of nanobodies, applied in the biological field, can solve the problems of increasing downstream purification pressure, low purity and high cost of nanobodies

Pending Publication Date: 2020-07-07
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the purification of nanobodies still relies on the fusion of affinity tags (such as His 6 , GST, MBP), using affinity chromatography to purify, but the downstream purification pressure is increased after the protease cuts off the affinity tag
However, direct use of traditional chromatography techniques such as ion exchange chromatography and hydrophobic chromatography to purify nanobodies has high cost and low purity. It usually requires two or more steps of chromatography to achieve high purity, and relies on expensive protein chromatography systems. and chromatography media

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  • Method for separation and purification of nanobody
  • Method for separation and purification of nanobody

Examples

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Embodiment 1

[0030] Example 1 Purification of Nanobodies from E. coli periplasmic proteins.

[0031] Obtain the sample to be purified (Escherichia coli intracellular protein): Escherichia coli expression strain in 5L B. Nanobody A was produced in a stirred glass bioreactor, and the nanobody was induced by 1mM IPTG for periplasmic expression. The cell culture medium was centrifuged at 4°C and 8000rpm for 20min to collect the bacteria, resuspend the bacteria, and use ultrasonic The cells were broken, centrifuged at 12000 rpm for 20 min, and the supernatant was collected, filtered and sterilized through a 0.22 μm filter membrane, and then used for subsequent nanobody purification as a sample to be purified.

[0032] 1) Precipitation of impurity proteins at low pH: Take 30mL×3 parts of the sample to be purified, and adjust the pH to 2.0, 3.0, and 4.0 with 2mol / L acetic acid solution respectively. As three experiments, the following steps were carried out separately: Set aside for 2h, centrif...

Embodiment 2

[0035] Example 2 Purification of Nanobodies from Escherichia coli periplasmic proteins.

[0036] Repeat Example 1, the difference from Example 1 is that the preparation method for obtaining the sample to be purified (Escherichia coli periplasmic protein) in this example is: the Escherichia coli expression strain is B Produce nanobody B in a stirred glass bioreactor, use 1mM IPTG to induce periplasmic expression of nanobody, centrifuge the cell culture medium at 4°C and 4000×g for 20min, collect the bacteria, and then resuspend and extract the bacteria The periplasmic protein was filtered and sterilized by a 0.22 μm filter membrane and used for subsequent nanobody purification as a sample to be purified. The pH precipitation condition is 3.0, the high temperature precipitation condition is 70 ℃, all the other operations are the same as in Example 1, the results are shown in Figure 4 , the purification effect of Nanobody B is good, and a relatively pure target protein can be ...

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Abstract

The invention discloses a method for separation and purification of a nanobody, and relates to the technical field of biology. The method comprises the following steps: (1) precipitating impure protein at low pH: regulating pH of a to-be-purified sample to acidity with an acid solution, after standing, performing centrifugation to remove precipitates, and keeping a supernatant solution; (2) precipitating the impure protein at high temperature: heating the supernatant solution obtained in step (1), after heat-preservation standing, performing centrifugation to remove precipitates, and keeping asupernatant solution; and (3) removing the impure protein with an ultrafiltration method: performing ultrafiltration on the supernatant solution obtained in step (2), wherein a protein solution permeating through an ultrafiltration membrane is the high-purity nanobody. The method provided by the invention does not affect the activity of the nanobody, and has simple operation, low cost and a wideapplication range.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating and purifying nanobodies. Background technique [0002] With the vigorous development of biotechnology, antibodies play an indispensable role in the diagnosis and treatment of human diseases. In recent years, the "upstart" nanobodies in the antibody field have developed rapidly and have received widespread attention. In the blood of camels and alpacas, there is a unique antibody-heavy chain antibody. Scientists have cloned the variable region of the heavy chain antibody, which is called VHH antibody. The VHH crystal has a diameter of 2.5nm and a length of 4nm, so it is also called Nanobodies are the smallest fragments that exist in nature that can bind to antigens. [0003] Nanobodies are very stable in nature and still maintain biological activity under high temperature conditions, which will completely solve the problem of cold chain transportation and stor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K1/34C07K1/30
CPCC07K16/00C07K2317/569
Inventor 刘文帅樊喜英年锐孙粤包子娴咸漠
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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