WO2020087987A1

WO2020087987A1 - Method and kit for expressing protein in nucleic acid aptamer purification gene engineering

Info

Publication number: WO2020087987A1
Application number: PCT/CN2019/096220
Authority: WO
Inventors: 廖世奇; 田彩平; 袁红霞; 魏政丽; 栗怡
Original assignee: 廖世奇
Priority date: 2018-10-30
Filing date: 2019-07-16
Publication date: 2020-05-07
Also published as: CN109400670A

Abstract

Provided are a method and a kit for purifying protein by a nucleic acid aptamer. The method comprises: screening a target molecule-specific nucleic acid aptamer by utilizing a SELEX technique; coupling the nucleic acid aptamer to a magnetic bead to form a target molecule capturing magnetic bead; capturing molecule target protein by utilizing a molecule recognition principle of a nucleic acid ligand and the aptamer, and separating the molecule target protein from a mixture by combining magnetic separation; and then effectively separating the target molecule and the magnetic bead by means of a method of heating (or acid and alkali and the like), so as to obtain a purified target molecule. The method can separate and purify the proteins in mixed liquids such as secretory or intracellular expression proteins of bacteria, yeasts, insects and mammal cells and the like in gene engineering, a fermentation liquid, serum, body fluid or wastewater treatment.

Description

Method and kit for nucleic acid aptamer to purify protein expressed in genetic engineering

Technical field

The invention relates to a method for purifying protein expressed in genetic engineering by nucleic acid aptamers, in particular to a method for organically combining nucleic acid ligand molecular recognition principles to establish a method for purifying protein expressed in genetic engineering by nucleic acid aptamers. It also relates to a kit based on this method, which belongs to the field of molecular biology.

Background technique

The interaction of nucleic acids and proteins in cells is a common phenomenon. Nucleic acids can fold to form secondary and tertiary structures, which is very important for their interaction with proteins. Screening specific target proteins by diversifying nucleotide sequences and interacting with nucleic acid proteins can purify expressed proteins in genetic engineering. SELEX technology is used to isolate the nucleotide ligands of the selected target. These ligands are called ligands or aptamers, which means that the nucleic acid can form a certain structure and fit into the pocket of the target molecule. SELEX technology uses this Principle Separation and screening of target molecular ligands.

SELEX is an exponentially enriched ligand evolution system (Systematic Evolution of Ligands by Exponential Enrichment, SELEX), which uses a large-capacity random oligonucleotide library to interact with target proteins to screen out suitable nucleic acids that specifically bind to the target. Ligand. This method has the advantages of high sensitivity, strong specificity, fast and high efficiency. Since Tuerk et al. First used this technology to screen for specific oligonucleotide ligands that specifically adsorbed bacteriophage T4 DNA polymerase and organic dye molecules, after more than 20 years of development, SELEX technology has become an important research tool and tool , Used in biochemistry, gene regulation, disease diagnosis, and new drug research and development. The screening targets are becoming more and more extensive, gradually expanding from the initial organic dyes and enzymes to smaller ions, amino acids, toxins, and more complex cancer cells, stem cells, clinical tissue sections, serum, etc. For different target substances, use Appropriate methods can be used to find aptamers with high affinity and specificity faster and more conveniently.

After the start of SELEX technology, many target nucleotide ligands have been screened, especially many proteins known to bind nucleic acids can be used as more suitable targets for SELEX technology, such as T4 DNA polymerase, bacteriophage R17 envelope protein, E. coli rho Factors, E. coli ribosomal protein S1, phenylalanine-tRNA synthetase, autoimmune antibodies that recognize RNA, E2F transcription factor, different HIV-related proteins. The fact that SELEX technology can screen out many different protein ligands has led to the expansion of ligand applications. It can be used as a substitute for monoclonal and multi-antibody products for diagnosis and treatment. The application of ligand instead of antibody in diagnosis has shown its value. DNA polymerase ligands have been used in hot-start PCR to diagnose low-copy replicons, improving PCR sensitivity and fidelity. Ligands are also used to facilitate experimental methods. Neutral elastin is fluorescently labeled with enzyme ligands and used for flow cytometry to detect elastase concentration. Neutral elastase ligands are still used for in vivo diagnosis of rat lung inflammation diagnosis models.

In recent years, researchers have introduced different materials and instruments such as graphene, carbon nanotubes, capillary tubes, flow cytometers, atomic force microscopes, etc. on the basis of the original screening methods to improve the separation efficiency, so that the classic SELEX technology has been developed. And perfection, derived many improved SELEX technologies such as: reverse screening (counter SELEX), subtractive screening (subtractive SELEX), capillary electrophoresis screening (CE SELEX), genome SELEX (genomic SELEX), tailed screening (tailored SELEX) , Toggle SELEX and SELEX based on modified nucleotides.

Secreted or intracellularly expressed genetically engineered proteins of bacteria, yeast, insects, and mammalian cells are a mixture of various molecules. Protein purification is performed on these complexes, and known proteins can be purified based on known target molecule nucleic acid aptamers. Unknown proteins can be purified by screening unknown target nucleic acid aptamers to provide technical support for nucleic acid aptamers to purify proteins expressed in genetic engineering and drug research. Therefore, the establishment of a rapid extraction, trace and high-purity expression protein platform is of great significance for the separation and purification of expressed proteins in genetic engineering and drug research.

Summary of the invention

The purpose of the present invention is to provide a method for purifying proteins expressed in genetic engineering by nucleic acid aptamers with advantages of high purification efficiency, trace quantity, rapidity, etc., which can be used for bacteria, yeast, insects and mammalian cells Purification of expressed proteins in genetic engineering such as secretion or intracellular expression.

The method for purifying protein expressed in genetic engineering by the nucleic acid aptamer of the present invention includes the following steps:

(1) Preparation of crude protein samples: centrifuge the fermentation broth of the specifically expressed protein in genetic engineering at 4 ° C and 12000r / min for 30min, discard the supernatant (express the extracellularly expressed protein and discard the precipitate), dilute with Binding buffer, and resuspend the cells ， Using ice bath to lyse the cells, which is the crude protein sample.

The target molecule-expressed protein fermentation broth is one of proteins expressed in genetic engineering secreted or intracellularly expressed by bacteria, yeast, insects and mammalian cells.

(2) Extract target molecules: mix the crude protein sample with the capture agar magnetic beads at a volume ratio of 4: 1, and incubate at 37 ± 0.3 ° C for 30 min to form a magnetic bead-target molecule nucleic acid aptamer-target molecule complex , Washed once with 3 × SSC, washed 3 times with 0.4 mM Binding buffer, magnetic separation and discarded unbound fermentation broth to obtain magnetic beads-target molecule nucleic acid aptamer-target molecule complex;

The capture agar magnetic beads are: composite magnetic beads coated with nucleic acid aptamers of target molecules on agar magnetic beads, which is a ligand evolution system (Systematic Evolution of Ligands by Exponential Enrichment) enriched exponentially for specific expressed proteins. SELEX) Screen the specific target nucleic acid aptamers obtained.

Among them, the agar magnetic beads are independently developed and provided by this research group. The agar magnetic beads are combined with biomolecules such as proteins through chemical bonds, and through the combined biomolecules and other molecules, enzyme catalysis, immunity, ligand ligand and other reactions occur, non-specific adsorption Very small, when the buffer ionic strength> 0.05mol / L, there is almost no specific adsorption of proteins, and it has good hydrophilicity, which can make biomolecules easy to approach and interact with ligands without causing Biomolecules lose their activity.

(3) Preparation of target molecules: add elution buffer to the magnetic bead-target molecule aptamer-target molecule complex, shake for 1 min, collect the elution buffer, and immediately add Tris-HCL with pH 8.0 and 1M to neutralize ; Repeat adding elution buffer three times for elution, mixing the elution neutralization solution three times, dialysis and desalting to obtain purified target molecule-expressed protein.

The formulation of the elution buffer: 0.1M glycine, 0.5M NaCL, 0.05% Tween 20 at pH 3.0. The elution can dissociate the protein molecules bound to the magnetic beads from the magnetic beads to the supernatant. The elution efficiency of eluting and separating the aptamers and target molecules is more than 98%.

2. Kit for nucleic acid aptamer purification of protein expressed in genetic engineering

Including the following reagents:

Reagent 1: 5-10 mL of 0.4 mM 1 × Binding Buffer buffer with a pH value of 7.4, internally dissolved 50% agar magnetic beads with a particle size of 5 to 150 microns and specific nucleic acid aptamer composite capture magnetic beads Among them, the pH value is 7.4 1 × Binding buffer (preservative containing 0.01% sodium azide), and the preparation of 1L 1 × Binding buffer: 138mmol / L NaCl, 2.7mmol / L KCl, 8.1mmol / L Na ₂ HPO ₄ , 1.1mmol / L KH ₂ PO ₄ , 5nmol / L MgCl ₂ , adjust the pH value of the solution to 7.4 with HCl, add water to bring the volume to 1L, autoclave for 20min, and store at room temperature.

Reagent 2: Elution buffer 3 × SSC, including citric acid, sodium chloride; preparation of 1L 3 × SSC: 25.04g NaCl, 12.6g trisodium citrate, add water to volume to 1L, store at room temperature.

Reagent 3: acid eluent: 0.5mL elution buffer (0.1M glycine, 0.5M NaCL, 0.05% Tween20 at pH 3.0);

Reagent 4: alkaline eluent: pH 8.0, 1M Tris-HCL. The preparation of Tris-HCL with pH 8.0 and 1M: 121g Tris base, add 800mL of pure water to dissolve, adjust the pH value to 8.0 with concentrated hydrochloric acid, and then make the volume to 1L.

The preparation of agar magnetic beads is as follows:

Preparation of Fe3O4 magnetic fluid with oleic acid as dispersant

Under nitrogen protection, 19.48g of FeCl2 · 4H2O and 52.98g of FeCl3 · 6H2O were placed in a flask and dissolved in 600mL of distilled water, magnetically stirred at 80 ° C and slowly added 30mL of ammonia containing 25% Tween-20, and magnetically stirred at 80 ° C 30min. After the reaction was completed, 30mL of oleic acid was slowly added to the suspension, magnetically stirred at 80 ° C for 30min, pH was adjusted to neutral with hydrochloric acid, Fe3O4 in the solution was separated with a magnet, excess oleic acid was repeatedly washed away with three distilled water and then dried naturally . Take 3g of oleic acid-modified Fe3O4 particles in a wide-mouth bottle, add 10mL of distilled water and 120μL Tween-20 ultrasound for 10 min, prepare a magnetic fluid, and store it in a refrigerator at 4 ℃ for use.

Preparation of Magnetic Agarose Microspheres (AMM) by Reverse Phase Polymerization

Organic phase: Add 2g Span-80, 50mL liquid paraffin to a 250mL three-necked flask, preheat to 80 ℃. Aqueous phase: Add 0.125g of agarose in 4mL of distilled water, add 1mL of self-made magnetic fluid after heating to dissolve, and mix thoroughly. Under mechanical stirring at 850r / min, the aqueous phase was quickly dropped into the organic phase, and the reaction was cooled to room temperature after reacting at 80 ° C for 10min, and stirred at 250r / min for 5min to form the microspheres. The microspheres were washed with 10 mL petroleum ether and 1 mL isopropanol, filtered with suction, then washed twice with petroleum ether and three distilled water to remove the organic phase, and then placed in 20% ethanol to prepare agarose magnetic microspheres, stored at 4 ℃ Refrigerator spare.

Compared with the prior art, the present invention has the following advantages:

1. The present invention organically combines the principle of nucleic acid ligand molecule recognition, without the need to perform multiple long-term high-speed centrifugation and filter filtration on the expressed crude protein, the target protein can be directly purified from the crude sample, and the purification time is greatly shortened. By adjusting the volume of the elution buffer, the target protein concentration and volume can be controlled without protein loss, and there is no risk of protein denaturation and precipitation. One-step purification can obtain high-purity target protein, no need to control the flow rate, and no need for expensive chromatography equipment; the specific binding of the magnetic bead-aptamer-target molecule complex and the acid elution operation are simple, fast, and also improve The efficiency and purity of nucleic acid aptamers for the isolation and purification of expressed proteins in genetic engineering;

2. Agar magnetic beads are small spherical particles with a superparamagnetic and protective shell. Its structure is that the core is superparamagnetic particles, the outer layer of the core is wrapped with polymer, and the outermost layer is nucleic acid ligand. Agar magnetic beads are magnetic, which can be conveniently and simply separated and magnetically guided. They have good biocompatibility, chemical stability, and dispersibility. The non-specific adsorption is small, and they can maintain the spatial structure and other characteristics of the bound molecules. Groups, such as reactive groups such as carboxyl group, epoxy group and amino group, can make biomolecules easy to approach and interact with ligands without deactivating biomolecules. The steps of incubation, elution and separation can be completed mechanically, reducing the error of human operation.

3. Bacterial, yeast, insect and mammalian cells secreted or intracellularly expressed genetically engineered protein is a mixture of multiple molecules, protein purification of these complexes, known proteins can be purified according to known target nucleic acid aptamers It can also purify unknown proteins by screening unknown target nucleic acid aptamers to provide technical support for nucleic acid aptamers to purify proteins expressed in genetic engineering and drug research.

detailed description

The method and kit for purifying the protein expressed in genetic engineering by the nucleic acid aptamer of the present invention will be further described below through specific examples.

1. Kits for protein expression

The kit for nucleic acid aptamer purification of protein expressed in genetic engineering includes the following reagents:

Reagent 1: 5 ～ 10mL, 0.4mM 1 × Binding Buffer buffer (preservative containing 0.01% sodium azide), pH 7.4, 50% agar magnetic beads-specific nucleic acid with 50% particle size The aptamer compound captures magnetic beads. Preparation of 1L1 × Binding buffer: 138mmol / L NaCl, 2.7mmol / L KCl, 8.1mmol / L Na ₂ HPO ₄ , 1.1mmol / L KH ₂ PO ₄ , 5nmol / L MgCl ₂ , adjust the pH value of the solution with HCl to 7.4, add water to volume to 1L, autoclave for 20min, store at room temperature;

Reagent 2: Elution buffer 3 × SSC includes citric acid and sodium chloride. Preparation of 1L 3 × SSC: 25.04g NaCl, 12.6g trisodium citrate, add water to volume to 1L, store at room temperature;

Reagent 3: acid eluent: 0.1M glycine, 0.5M NaCL, 0.05% Tween20 at pH 3.0

Reagent 4: alkaline eluent: pH 8.0, 1M Tris-HCL. Preparation of 1L Tris-HCL with pH 8.0 and 1M: 121g Tris base, add 800mL of pure water to dissolve, adjust the pH value to 8.0 with concentrated hydrochloric acid, and then make up to 1L.

3 × SSC elution buffer is used to separate the oligonucleotide molecules bound to agar magnetic beads,

2. Method for nucleic acid aptamer to purify protein expressed in genetic engineering

Example 1. Method for purifying Pichia pastoris to express human placental growth factor 2 protein using nucleic acid aptamer purification protein expression kit in genetic engineering

(1) Pichia pastoris expresses human placental growth factor 2 protein fermentation broth at 4 ° C, 12000r / min, centrifuge for 30min, discard the supernatant, dilute with 500μL of Binding Buffer, resuspend the cells, and lyse the cells by ice bath sonication. Crude protein sample;

(2) Mix 400 μL crude protein sample with i00 μL capture agar magnetic beads (composite magnetic beads coated with growth factor 2 protein-specific nucleic acid aptamers), rotate and incubate at 37 ± 0.3 ℃ for 30 min to form magnetic Bead-human placental growth factor 2 nucleic acid aptamer-placental growth factor 2 complex was washed once with 3 × SSC and washed 3 times with 20 times the volume of 0.4 mM Binding Buffer, and the supernatant was discarded by magnetic separation;

(3) Add 0.5 mL of elution buffer (0.1 M glycine, 0.5 M NaCl, 0.05% Tween 20 at pH 3.0) to the complex obtained by magnetic separation, shake for 1 min, collect the elution buffer, and immediately add 250 μL, pH 8.0, 1M Tris-HCL neutralization; repeat elution three times, mix the elution neutralization solution three times, dialyze to remove salt ions, that is, purified human placental growth factor 2 protein.

Example 2. Method for purifying expression protein of His fusion protein in E. coli cells by using His nucleic acid aptamer purification protein expression kit in genetic engineering

(1) The E. coli cells express the fusion protein bacterial solution at 4 ° C, 12000r / min, centrifuge for 30min, discard the supernatant, dilute with 500μL Binding Buffer, resuspend the cells, and lyse the cells by ice bath sonication, which is the crude protein sample;

(2) Mix 400 μL of crude protein sample with 100 μL of capture agar magnetic beads (agar magnetic beads coated with composite magnetic beads of His nucleic acid aptamer), rotate and incubate at 37 ± 0.3 ℃ for 30 min to form magnetic beads-His nucleic acid The ligand-His fusion protein complex was washed once with 3 × SSC and washed three times with 20 times the volume of 0.4 mM Binding Buffer, and the supernatant was discarded by magnetic separation;

(3) Add 0.5 mL of elution buffer (0.1 M glycine, 0.5 M NaCl, 0.05% Tween 20 at pH 3.0) to the complex obtained by magnetic separation, shake for 1 min, collect the elution buffer, and immediately add 250 μL, pH 8.0, 1M Tris-HCL neutralization, repeat elution three times, mix the elution neutralization solution three times, and dialyze to remove salt ions to obtain purified His fusion protein.

Example 3. Method for purifying extracellular expression of human-derived EC-SOD protein by using multiple nucleic acid aptamers to purify protein expression kits in genetic engineering

This method is mainly used for: the method that the nucleic acid aptamer has a large non-specificity, which seriously affects the purity of the extracted protein, or a method for separating and purifying multiple aptamers to obtain a high-purity protein.

(1) Pichia pastoris extracellularly expressing human-derived EC-SOD protein bacterial solution was centrifuged at 4 ℃, 12000r / min for 30min, and the precipitate was discarded. The extracellular expression supernatant was taken as the crude protein sample;

(2) A composite magnetic bead of 400 μL crude protein sample and 100 μL capture agar magnetic beads 1 (agar magnetic beads coated with nucleic acid aptamer 1 of EC-SOD protein, nucleic acid aptamer 1 is multiple nucleic acids of EC-SOD protein One of the aptamers) was mixed together, and incubated at 37 ± 0.3 ° C for 30 min, to form magnetic beads-human EC-SOD nucleic acid aptamer 1-human EC-SOD protein, washed once with 3 × SSC, 20 times the volume The 0.4mM Binding Buffer was washed 3 times, and the supernatant was discarded by magnetic separation.

(3) Add 0.5 mL of elution buffer (0.1 M glycine, 0.5 M NaCl, 0.05% Tween 20 at pH 3.0) to the complex obtained by magnetic separation, shake for 1 min, collect the elution buffer, and immediately add 250 μL, pH 8.0, 1M Tris-HCL neutralization, repeat elution three times, mix the elution neutralization solution three times, dialyze to remove salt ions, that is, the first purified human EC-SOD protein (human EC-SOD protein 1 );

(4) Use the capture magnetic beads coated with EC-SOD protein nucleic acid aptamer 2 to bind to the purified human-derived EC-SOD protein 1, and repeat steps (2) and (3) to obtain the second purified human Source EC-SOD protein (Human EC-SOD protein 2).

(5) After N times, the human EC-SOD protein is purified by using the capture agar magnetic beads constructed with N human-source EC-SOD nucleic acid aptamers to obtain high-purity human-source EC-SOD protein.

Claims

A method for nucleic acid aptamer purification of protein expressed in genetic engineering, characterized in that it includes the following steps:

(1) Preparation of crude protein samples: centrifuge the fermentation broth of the specifically expressed protein in genetic engineering at 4 ° C and 12000r / min for 30min, discard the supernatant (express the extracellularly expressed protein and discard the precipitate), dilute with Binding Buffer, and resuspend the cells , The ice bath ultrasonically lyses the cells, which is the crude protein sample;

(2) Extract target molecules: mix the crude protein sample with the capture agar magnetic beads at a volume ratio of 4: 1, and incubate at 37 ± 0.3 ° C for 30 min to form a magnetic bead-target molecule nucleic acid aptamer-target molecule complex , Washed once with 3 × SSC, washed 3 times with 0.4 mM Binding buffer, magnetic separation and discarded unbound fermentation broth to obtain magnetic beads-target molecule nucleic acid aptamer-target molecule complex;

(3) Preparation of target molecules: add elution buffer to the magnetic bead-target molecule aptamer-target molecule complex, shake for 1 min, collect the elution buffer, and immediately add Tris-HCL with pH 8.0 and 1M to neutralize ; Repeat adding elution buffer three times for elution, mixing the elution neutralization solution three times, dialysis and desalting to obtain purified target molecule-expressed protein.
The method for purifying proteins expressed in genetic engineering by nucleic acid aptamers according to claim 1, characterized in that: in step (1), the fermentation liquid of the protein expressed by the target molecule is bacteria, yeast, insects, mammalian cells, etc. One of the proteins expressed in secretion or intracellular expression in genetic engineering.
The method for purifying a protein expressed in genetic engineering by a nucleic acid aptamer according to claim 1, wherein in step (2), the capture agar magnetic beads are: agar magnetic beads coated with target molecule nucleic acid adaptation The composite magnetic bead refers to a magnetic bead that can bind to a specific expressed protein; the target molecule nucleic acid aptamer is a specific nucleic acid aptamer obtained by screening the target molecule using the ligand evolution system enriched exponentially.
The method for purifying protein expressed in genetic engineering by nucleic acid aptamer according to claim 1, characterized in that: in step (3), the formulation of the elution buffer is: 0.1M glycine, 0.5M NaCL, pH 0.05 and 0.05 % Tween20.
A kit for purifying protein expressed in genetic engineering based on the nucleic acid aptamer according to claim 1, comprising the following reagents:

Reagent 1: 5-10 mL, 0.4 mM 1 × Binding buffer buffer with pH 7.4, internally dissolved 50% composite agar magnetic beads-specific nucleic acid aptamer capture magnetic beads with a particle size of 5 to 150 microns;

Reagent 2: Elution buffer 3 × SSC, containing citric acid and sodium chloride;

Reagent 3: acid eluent: formula: 0.1M glycine, 0.5M NaCL, 0.05% Tween20 at pH 3.0;

Reagent 4: alkaline eluent: pH 8.0, 1M Tris-HCL.