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Method for preparing heterodimer protein

A heterodimer and protein technology, applied in the field of biological macromolecular connection, can solve the problems such as the unfixed ratio of two proteins and the difficulty in controlling the molecular weight of the protein connection product, and achieve the effect of low molecular weight and good repeatability.

Pending Publication Date: 2020-07-28
润方(长春)生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems in the prior art that the molecular weight of the protein-linked product is not easy to control and the ratio of the two proteins in the product is not fixed, the present invention provides a method for preparing heterodimeric proteins

Method used

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  • Method for preparing heterodimer protein
  • Method for preparing heterodimer protein
  • Method for preparing heterodimer protein

Examples

Experimental program
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Effect test

Embodiment 1

[0022] like image 3 Shown, a kind of method for preparing heterodimer protein, concrete steps are as follows:

[0023] Step 1: Activate protein:

[0024] Take SPDP (3-(2-pyridyldimercapto)propionic acid N-hydroxysuccinimide ester) and protein 1 according to the amount of substances 1.2:1 and mix in buffer P2, pH = 7.4, at room temperature (25 ℃ ) reacted overnight, reacted for 10 hours, and kept shaking during the reaction to obtain activated protein 1;

[0025] Take HS-PEG-NHS (mercapto PEG-N-hydroxysuccinimide) and protein 2 according to the amount of substances 1.2:1, mix in buffer PB, pH=7.4, react overnight at room temperature (25°C), and react 10 hours, during the reaction, keep shaking to obtain activated protein 2;

[0026] Step 2: Mix the activated protein 1 and activated protein 2 obtained in step 1, and react at room temperature (25° C.) for 2 days, and keep shaking well during the reaction, wherein the amount of the activated protein 2 is at least the amount of...

Embodiment 2

[0029] A method for preparing heterodimeric proteins, the specific steps are as follows:

[0030] Step 1: Activate protein:

[0031] Prepare HRP with a mass fraction of 1%: take 5 mg of horseradish peroxidase (HRP) and add 500 μL of 10 mmol / L PB (pH 7.4) to dissolve;

[0032] Preparation of 10mmol / L SPDP: Take 10mg of SPDP and add 3.2mL of 10mmol / L PB (pH 7.4) to dissolve;

[0033] Preparation of 10mmol / L HS-PEG-NHS: Take 10mg of HS-PEG-NHS and add 0.5mL of 10mmol / LPB (pH 7.4) to dissolve;

[0034] Mix the samples according to the table below, react overnight at room temperature (25°C), and keep shaking during the reaction

[0035]

[0036] Step 2: Connexin:

[0037] Mix samples 1 and 2, react at room temperature (25°C) for 2 days, and keep shaking during the reaction

[0038] Step three:

[0039] The product obtained in step 2 was purified and stored at low temperature.

[0040] Labeled product SDS-PAGE reduction electrophoresis such as Figure 4 shown. The molecul...

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Abstract

The present invention discloses a method for preparing heterodimer protein. The specific steps are as follows: firstly activating proteins: taking SPDP (3-(2-pyridinedimercapto) propionate N-hydroxysuccinimide ester) to be mixed with a protein 1 in a buffer and taking HS-PEG-NHS (sulfhydryl PEG-N-hydroxysuccinimide) to be mixed with a protein 2 in a buffer to separately obtain an activated protein1 and an activated protein 2; and mixing the activated protein 1 and the activated protein 2, purifying the obtained product and conducting preservation at low temperature. The protein product prepared by the method is an oligomer with low molecular weight, the product is mostly heterodimers, all used reagents can stably exist in a water phase, and the method has good repeatability, stability andreliability.

Description

technical field [0001] The invention relates to a connection method of biological macromolecules, which can be used for preparing enzyme-labeled proteins, protein-protein linkers, protein-small molecule markers and the like. Background technique [0002] The commonly used protein linking methods in the prior art include NHS / EDC linking protein and sodium periodate oxidation; EDC is a common crosslinking agent that utilizes carboxyl and amino groups to realize mutual crosslinking. EDC first reacts with carboxyl groups to form O - An acylisourea intermediate that reacts rapidly with an amino group to form an amide bond and releases an isourea by-product. The intermediate is unstable in aqueous solution, and N-hydroxysuccinimide can stabilize the intermediate, and the intermediate that has not reacted with the amino group will be hydrolyzed to form a carboxyl group and N-substituted urea; another commonly used method is high iodine Sodium acid oxidation method, common applicat...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N9/08C07K1/08C07K1/02
CPCC07K14/4737C12N9/0065C12Y111/01007C07K2319/00
Inventor 王旭东焦虎平李文亮杨文魁夏志平游可为陈浩源
Owner 润方(长春)生物科技有限公司
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