In-vitro synthesis and purification integrated preparation method for protein, kit and application thereof

An in vitro synthesis and protein synthesis technology, applied in the field of protein synthesis and in vitro protein synthesis, can solve the problems of reduced binding performance of purification media, large labor and time, and reduced purification effect, so as to shorten the preparation cycle, increase efficiency, and reduce retention Effect of Time and Retention Ratio

Pending Publication Date: 2021-10-29
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the covalent coupling method can ensure that the purification medium is firmly fixed on the carrier, after multiple uses of the purification column, the binding performance of the purification medium will be reduced, and the purification effect will be reduced
Therefore, in order to ensure high purification efficiency and quality, operators need to update or replace all the packing materials in the affinity chromatography column in time. This process not only consumes a lot of consumables, but also consumes a lot of labor and time, resulting in high purification costs.

Method used

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  • In-vitro synthesis and purification integrated preparation method for protein, kit and application thereof
  • In-vitro synthesis and purification integrated preparation method for protein, kit and application thereof
  • In-vitro synthesis and purification integrated preparation method for protein, kit and application thereof

Examples

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Effect test

preparation example Construction

[0317] The integrated protein synthesis and purification method in vitro at least includes the following steps:

[0318] Step i: At least provide an in vitro protein synthesis system, a nucleic acid template encoding the target protein, and a protein immobilization system, and place them in the same reaction vessel to form a reaction purification mixed system (intePure system or coPure system);

[0319] Wherein, the in vitro protein synthesis system can provide translation-related elements required for synthesizing the target protein together with the nucleic acid template encoding the target protein; the target protein can specifically bind to the protein immobilizing system (protein immobilizing system, PIS )superior;

[0320] Step ii: perform an incubation reaction under appropriate conditions to obtain a target protein product; during the incubation reaction, that is, during the in vitro protein synthesis reaction, the target protein product can specifically bind to the pr...

Embodiment 1

[0884] Embodiment 1. prepares the magnetic microsphere C of acrylic polymer modification

[0885] First, measure the aqueous solution of ferroferric oxide magnetic microspheres (the particle diameter of magnetic microsphere is about 1 μm) that 50mL silicon dioxide wraps, its solid content 20% (v / v), settle magnetic microsphere with magnet, remove For the liquid phase, the magnetic microspheres were washed with 60 mL of absolute ethanol each time, for a total of 5 times. Add 100 mL of an excess of 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) in ethanol (25%, v / v) to the above-mentioned washed magnetic microspheres, at 50 ° C Stir mechanically in a water bath for 48 hours, then mechanically stir in a water bath at 70°C for 2 hours, settle the magnetic microspheres with a magnet, remove the liquid phase, wash the magnetic microspheres with 60 mL of absolute ethanol each time, wash twice in total, and then wash each time with The magnetic microspheres were washed with 60 m...

Embodiment 2

[0888] Example 2. Preparation of biotin-modified biomagnetic microspheres D

[0889] Transfer the synthesized magnetic microspheres C to 10mL solution X, add 0.004mol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.004mol N-hydroxysuccinyl amine, stirred and mixed at room temperature, stirred and reacted for 15min, settled the magnetic microspheres with a magnet, removed the liquid phase, and washed 3 times with 10mL distilled water each time; pipette 4.0×10 -4 mol 1,3-propanediamine, dissolved in 10mL PBS buffer solution, added to the washed magnetic microspheres, mechanically stirred in a water bath at 30°C for 20 hours, settled the magnetic microspheres with a magnet, and removed the liquid phase, each time Wash 6 times with 10mL distilled water, add 10mL PBS buffer solution; weigh 2.5×10 -4 mol biotin, add 10mL solution X, then add 1.0×10 -3 mol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.001mol N-hydroxysuccinimide, stirred and mi...

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Abstract

The invention discloses an in-vitro synthesis and purification integrated preparation method for a protein, a kit and application thereof and belongs to the technical field of protein synthesis. According to the preparation method, an in-vitro protein synthesis system and a nucleic acid template for encoding a target protein form a reaction system, a protein immobilization system capable of being combined with the target protein is added into the reaction system, and in an in-vitro protein synthesis reaction process, a target protein product can be specifically combined to the protein immobilization system. According to the preparation method, the kit and the application thereof, the traditional strategy of two steps of synthesis and purification is broken through, a brand new synthesis and purification coupled method is adopted, the traditional protein synthesis and purification steps are combined into one, the time cost for purifying protein products is greatly reduced, the purification efficiency is greatly increased, the preparation period is remarkably shortened, the process is simple, the operation is convenient and fast, and thus, the method can be widely applied to in-vitro protein synthesis and detection; and and the preparation period is remarkably shortened, and the practicability is greatly improved.

Description

technical field [0001] The invention relates to the technical field of protein synthesis, in particular to the technical field of in vitro protein synthesis, in particular to an integrated preparation method for protein in vitro synthesis and purification, a kit and applications thereof. Background technique [0002] Proteins are important molecules in cells and are involved in almost all functions of cells. Protein synthesis mainly includes traditional intracellular synthesis technology and new generation in vitro synthesis technology. The traditional protein expression system refers to a molecular biology technique that expresses foreign genes through model organisms such as bacteria, fungi, plant cells or animal cells. In vitro protein synthesis system, also known as cell-free expression system, came into being in the 1960s. It uses exogenous mRNA or DNA as a template for protein synthesis, and artificially controls the addition of substrates, energy, and transcription a...

Claims

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Application Information

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IPC IPC(8): C12P21/00C07K1/14
CPCC12P21/00C07K1/14
Inventor 郭敏吴亮徐丽琼于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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