CN113267520A - Electron microscope carrier net using hydrophobin film as support film and preparation method thereof - Google Patents
Electron microscope carrier net using hydrophobin film as support film and preparation method thereof Download PDFInfo
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- CN113267520A CN113267520A CN202110576212.4A CN202110576212A CN113267520A CN 113267520 A CN113267520 A CN 113267520A CN 202110576212 A CN202110576212 A CN 202110576212A CN 113267520 A CN113267520 A CN 113267520A
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- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/02—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material
- G01N23/04—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material and forming images of the material
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
- G01N23/20008—Constructional details of analysers, e.g. characterised by X-ray source, detector or optical system; Accessories therefor; Preparing specimens therefor
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
- H01J37/02—Details
- H01J37/20—Means for supporting or positioning the object or the material; Means for adjusting diaphragms or lenses associated with the support
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Abstract
The invention provides an electron microscope grid which is composed of an electron microscope grid and a hydrophobin film arranged on the surface of the electron microscope grid and takes the hydrophobin film as a supporting film. The invention also provides a preparation method of the electron microscope carrier mesh by using the hydrophobin film as a support film, which comprises the following steps: dripping the hydrophobin solution on a substrate with hydrophobic property, and standing until a hydrophobin film is formed on the surface of the liquid drop; placing an electron microscope carrier net on the surface of the liquid drop, contacting the electron microscope carrier net with the hydrophobin film, and incubating until the hydrophobin film is laid on the electron microscope carrier net; removing the redundant liquid on the electron microscope carrier net paved with the hydrophobin film; dripping pure water into the electron microscope carrying net paved with the hydrophobin film for rinsing; and naturally drying the rinsed electron microscope carrying net to prepare the electron microscope carrying net. The invention provides an electron microscope grid using a hydrophobin film as a support film and a preparation method thereof, which are used for solving the problems of thicker ice layer, serious orientation advantage, protein denaturation caused by a gas-liquid interface and the like in the preparation of a frozen electron microscope sample.
Description
Technical Field
The invention relates to the technical field of preparation of electron microscope samples, in particular to an electron microscope carrier mesh taking a hydrophobin film as a supporting film and a preparation method thereof.
Background
The cryoelectron microscopy technique is a technique for performing low-dose imaging and structural analysis on a sample embedded in glassy ice at the temperature of liquid nitrogen by using a transmission electron microscope. Due to the technical breakthroughs in hardware and image processing algorithms, the cryoelectron microscope technology can analyze the structure of the biomacromolecule to the near atomic resolution, and is used for clarifying the functional mechanism exerted by the biomacromolecule. Frozen sample preparation is the rapid freezing of biological samples in amorphous ice using a freezing agent such as ethane, propane or a mixture of both, where the sample is in a near physiological state and where imaging can mitigate the damage of radiation damage to the high resolution structural information of the sample.
The following technical problems are encountered when using common uniform pore size carbon membrane meshes such as Quantifoil, GIG for frozen sample preparation: protein particles do not enter the pores, a sample is easier to remain at a thick ice position, the orientation advantage is serious, and protein denaturation is caused by a gas-liquid interface. These problems make high resolution structures of some important biological macromolecules unavailable, resulting in many structural biology studies that require a significant expenditure of time and effort in sample optimization.
Therefore, at present, the problems of thick ice layer, severe orientation advantage, protein denaturation caused by a gas-liquid interface and the like in the preparation of a cryoelectron microscope sample need to be solved, so as to improve the success rate of the preparation of the cryoelectron microscope sample.
Disclosure of Invention
The invention aims to solve the technical problem of providing an electron microscope carrier net using a hydrophobin film as a support film and a preparation method thereof, and aims to solve the problems of thicker ice layer, serious orientation advantage, protein denaturation caused by a gas-liquid interface and the like in the preparation of a frozen electron microscope sample.
In order to solve the technical problems, the invention provides an electron microscope grid using a hydrophobin film as a support film, which comprises the electron microscope grid and the hydrophobin film arranged on the surface of the electron microscope grid.
The invention also provides a preparation method of the electron microscope carrier mesh by taking the hydrophobin film as the support film, which comprises the following steps:
dripping the hydrophobin solution on a substrate with hydrophobic property, and standing until a hydrophobin film is formed on the surface of the liquid drop;
placing an electron microscope carrier net on the surface of the liquid drop, contacting the electron microscope carrier net with the hydrophobin film, and incubating until the hydrophobin film is laid on the electron microscope carrier net;
removing the redundant liquid on the electron microscope carrier net paved with the hydrophobin film;
dripping pure water into the electron microscope carrying net paved with the hydrophobin film for rinsing;
and naturally drying the rinsed electron microscope carrying net to prepare the electron microscope carrying net.
Furthermore, the electron microscope carrying net is a Quantifoil carrying net, a GIG carrying net, a NiTi alloy carrying net or a graphene carrying net and the like.
Further, the concentration of the hydrophobin solution is 1-1000 mug/mL.
Further, the hydrophobins in the hydrophobin solution are all kinds of hydrophobins or hydrophobins after modification.
Further, the preparation method of the hydrophobin solution comprises the following steps:
transforming the linearized plasmid pPIC9-hfbI into Pichia pastoris GS 115;
selecting positive transformants for shake flask fermentation and induced expression;
purifying hydrophobin by hollow fiber column ultrafiltration and acetonitrile extraction two-step method;
lyophilizing hydrophobin to hydrophobin powder;
dissolving hydrophobin powder in a solvent and carrying out ultrasonic oscillation to prepare a hydrophobin solution;
and diluting the hydrophobin solution and carrying out ultrasonic oscillation to obtain the hydrophobin solution with the concentration of 1-1000 mug/mL.
Further, the solvent is ultrapure water, PBS buffer, Tris buffer, HEPES buffer, or the like.
Further, the volume of the hydrophobin solution dropped onto the substrate having hydrophobic properties is 10-500. mu.L.
Further, the substrate is a sealing film or a glass slide or the like.
Further, the incubation time after the electron microscope mesh is contacted with the hydrophobin membrane is more than 5 min.
The electron microscope carrier mesh using the hydrophobin film as the supporting film is suitable for a common electron microscope carrier mesh in the preparation of a conventional frozen electron microscope sample, only the hydrophobin film is covered on the electron microscope carrier mesh to serve as the supporting film, and the electron microscope carrier mesh is simple in structure and easy to manufacture. Because the hydrophobin film has natural hydrophilicity, when the hydrophobin film is used for preparing a frozen electron microscope sample, glow discharge treatment is not needed to be carried out on an electron microscope carrier net, the sample can be directly frozen, the use is simple and convenient, thinner ice can be prepared, the problems of protein denaturation and the like caused by serious orientation advantage and gas-liquid interface are well solved, the success rate of preparing the frozen electron microscope sample is improved, and the reconstructed resolution can reach the highest
The following. Meanwhile, the preparation method of the electron microscope carrier mesh by using the hydrophobin film as the support film has the advantages of simple preparation process and easy and convenient operation, and the hydrophobin film used as the support film of the electron microscope carrier mesh can use all kinds of hydrophobins, so that the requirements on raw materials are lower, and the preparation cost is lower.
Drawings
FIG. 1 is a flow chart of a method for preparing an electron microscope carrier using a hydrophobin film as a support film according to an embodiment of the present invention;
FIG. 2 is a plasmid map for heterologous expression of Pichia pastoris used in the method for preparing an electron microscope mesh using a hydrophobin membrane as a support membrane according to an embodiment of the present invention;
FIG. 3 is a SDS-PAGE gel image after hydrophobin purification in the preparation method of the electron microscope carrier using the hydrophobin membrane as the support membrane according to the embodiment of the present invention;
FIG. 4 is a cryo-electron micrograph of apoferritin on a hydrophobin mesh in a method for preparing an electron-microscope mesh using a hydrophobin membrane as a support membrane according to an embodiment of the present invention;
fig. 5 is a three-dimensional reconstructed electron density map of the human apoferritin on the hydrophobin mesh in the method for preparing the electron microscope mesh using the hydrophobin film as the support film according to the embodiment of the present invention;
FIG. 6 is a diagram showing the electron density of the side chain of a representative amino acid of human apoferritin on a hydrophobin carrier in a method for preparing an electron microscope carrier using a hydrophobin membrane as a support membrane according to an embodiment of the present invention;
FIG. 7 is a cryo-electron micrograph of humanized catalase on a hydrophobin loaded mesh in a method for preparing the electron-microscopy loaded mesh using the hydrophobin membrane as a support membrane according to an embodiment of the present invention;
FIG. 8 is a three-dimensional reconstructed electron density map of humanized catalase on a hydrophobin carrier network in a preparation method of an electron microscope carrier network using a hydrophobin film as a support film according to an embodiment of the present invention;
FIG. 9 is a diagram showing the electron density of the side chain of the amino acid of humanized catalase on the hydrophobin carrier in the preparation method of the electron microscope carrier using the hydrophobin membrane as the support membrane according to the embodiment of the present invention.
Detailed Description
The embodiment of the invention provides an electron microscope grid using a hydrophobin film as a supporting film, which comprises an electron microscope grid and the hydrophobin film arranged on the surface of the electron microscope grid.
The electron microscope carrying net can be a currently common Quantifoil carrying net, a GIG carrying net, a NiTi alloy carrying net or a graphene carrying net, and the like, and the hydrophobin film can be all kinds of hydrophobins or modified hydrophobins.
Referring to fig. 1, the method for preparing an electron microscope carrier mesh using a hydrophobin film as a support film provided by the invention comprises the following steps:
step 1) dripping a hydrophobin solution on a substrate with hydrophobic property, and standing until a hydrophobin film is formed on the surface of the liquid drop;
step 2) taking an electron microscope carrying net and placing the electron microscope carrying net on the surface of the liquid drop, enabling the electron microscope carrying net to be in contact with the hydrophobin film, and incubating until the hydrophobin film is laid on the electron microscope carrying net;
step 3) removing redundant liquid on the electron microscope carrier net paved with the hydrophobin film;
step 4), dripping pure water into the electron microscope carrying net paved with the hydrophobin film for rinsing;
and 5) naturally drying the moistened electron microscope carrier net to prepare the electron microscope carrier net which can be used for preparing subsequent electron microscope samples.
The electron microscope carrying net is a Quantifoil carrying net, a GIG carrying net, a NiTi alloy carrying net or a graphene carrying net and the like.
Wherein the concentration of the hydrophobin solution is 1-1000 mug/mL.
Wherein, the hydrophobin in the hydrophobin solution is all kinds of hydrophobins or hydrophobins after modification.
The preparation method of the hydrophobin solution comprises the following steps:
1) transforming the linearized plasmid pPIC9-hfbI into Pichia pastoris GS 115;
2) selecting positive transformants for shake flask fermentation and induced expression;
3) purifying hydrophobin by hollow fiber column ultrafiltration and acetonitrile extraction two-step method;
4) lyophilizing hydrophobin to hydrophobin powder;
5) dissolving hydrophobin powder in a solvent and carrying out ultrasonic oscillation to prepare a hydrophobin solution;
6) and diluting the hydrophobin solution and carrying out ultrasonic oscillation to obtain the hydrophobin solution with the concentration of 1-1000 mug/mL.
Wherein the solvent is ultrapure water, PBS buffer, Tris buffer or HEPES buffer, etc.
Wherein the volume of the hydrophobin solution dropped onto the substrate having hydrophobic properties is 10-500. mu.L.
Wherein the substrate is a sealing film or a glass slide and the like.
Wherein the incubation time after the electron microscope carrier mesh is contacted with the hydrophobin film is more than 5 min.
Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings.
Unless otherwise specifically indicated, the sample pictures and values provided in these examples are not intended to limit the scope of the invention, and it should be understood that the dimensions of the various parts shown in the figures are not drawn to scale in nature for ease of description.
The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses.
Techniques, methods, and data known to those of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate.
Example 1
The plasmid pPIC9-hfbI (plasmid map shown in FIG. 2) was linearized with restriction enzyme Stu I and incubated at 37 ℃ for 5 hours. Then purified with a DNA gel recovery kit. The linearized pPIC9-hfbI was then electroporated into Pichia pastoris GS 115. And finally, carrying out colony PCR verification by using AOX 1F/R as a universal primer to obtain a positive transformant.
5 positive transformants were selected and simultaneously subjected to shake flask fermentation, and after enrichment culture in BMG medium (containing 1% glycerol) at 30 ℃ for 2 days, expression was induced in BMM medium at 30 ℃ for 4 days with 0.7% methanol. Hydrophobins are present in the fermentation supernatant by secretory expression. Through polyacrylamide gel electrophoresis (SDS-PSGE) and Western Blot verification, 1 strain with the highest expression level in 96h is selected and then high-density fermentation is carried out. Then purifying the protein by a two-step method of hollow fiber column ultrafiltration and acetonitrile extraction. Wherein, the protein purification process firstly adopts a hollow fiber column to carry out ultrafiltration impurity removal, concentration and purification on the fermented supernatant containing hydrophobin, and then freeze-drying the supernatant into crude protein powder. The crude protein powder was then redissolved in 40% acetonitrile in water containing 0.01% trifluoroacetic acid (TFA) to a final concentration of 40mg/ml, and subjected to 10min vortexing and 10min sonication, respectively. Hydrophobins are stable in acetonitrile due to their amphiphilic nature, whereas most heteroproteins are insoluble in acetonitrile or denature to form precipitates during processing. Hydrophobin and heteroprotein were separated by high speed low temperature centrifugation (8000rpm, 30 min) and the supernatant was lyophilized to a pure protein powder. The purity of hydrophobin in the protein which was crudely extracted by acetonitrile extraction was checked by SDS-PAGE and Coomassie staining. Coomassie staining indicated successful isolation of the hydrophobin from the crude protein. Referring to FIG. 3, it can be seen that it shows two states of monomer (7.5kDa) and trimer (22.5kDa) with most of the unwanted proteins removed.
Dissolving the hydrophobin freeze-dried powder in ultrapure water, a PBS (phosphate buffer solution), a Tris (Tris buffer solution) or a HEPES (high efficiency particulate ES) buffer again, fully dissolving the hydrophobin by ultrasonic oscillation to avoid the hydrophobin from forming aggregates to obtain a hydrophobin solution, then diluting the hydrophobin solution to 1-1000 mu g/mL, and simultaneously carrying out ultrasonic oscillation again.
Cutting a sealing film or a glass slide with a proper size, sucking 10-500 mu L of hydrophobin solution by using a pipette, dripping the hydrophobin solution on the sealing film or the glass slide, and standing for about 1-3 h until a macroscopic hydrophobin film is formed on the liquid drop.
And clamping the NiTi alloy carrying net by using tweezers, lightly placing the NiTi alloy carrying net on the surface of the liquid drop of the hydrophobin solution, contacting the front surface of the NiTi alloy carrying net with the hydrophobin solution film, and incubating for about 10min until the hydrophobin solution film covers the front surface of the NiTi alloy carrying net.
And lightly clamping the NiTi alloy carrying net paved with the hydrophobin film by using forceps, and absorbing redundant liquid on the NiTi alloy carrying net by using filter paper.
And (3) dripping ultrapure water on the NiTi alloy grid for rinsing, washing off impurities remained on the hydrophobin film of the NiTi alloy grid, and then absorbing the remained liquid by using filter paper.
In order to fully wash off the impurities remained on the hydrophobic protein film of the NiTi alloy mesh, the operation can be repeated to carry out 1-3 times of rinsing on the NiTi alloy mesh.
And finally, after the NiTi alloy grid covered with the hydrophobin film is naturally dried, the preparation of the electron microscope grid using the hydrophobin film as a supporting film is finished, and the electron microscope grid is placed in a grid box for storage and used for preparing a subsequent frozen electron microscope sample.
The human apoferritin cryoelectron microscope sample is prepared by the electron microscope mesh with the hydrophobin film as the support film prepared by the invention, and the superiority of the electron microscope mesh with the hydrophobin film as the support film prepared by the embodiment of the invention is illustrated.
Based on a general preparation process of a sample for a cryoelectron microscope, a suitable freezing device is selected, freezing instruments such as Vitrobot, EMGP, CP3 and the like can be selected, and the Vitrobot Mark IV freezing instrument is taken as an example for preparing the sample for the cryoelectron microscope.
1) Firstly, the filter paper is replaced, and parameters such as the temperature and the humidity in the freezing sample chamber, the waiting time before the filter paper absorbs water, the water absorption time of the filter paper and the like are set.
2) Liquid nitrogen is added into the sample freezing device for precooling, and then the gaseous ethane as the refrigerant is liquefied until the liquid ethane is changed into a state of solid-liquid coexistence, wherein the sample freezing temperature is about-183.3 ℃.
3) The sample is initially frozen and, as an embodiment of the invention, human apoferritin is used as the sample. Clamping the hydrophobin carrying net by using a frozen sample forceps, fixing the hydrophobin carrying net at a specific position of a frozen sample rod of a frozen sample device, sucking 3 mu L of a human apoferritin sample by using a liquid transfer gun, adding the human apoferritin sample to one surface of the carrying net on which the hydrophobin supporting membrane is paved, and waiting for a certain time to allow the human apoferritin sample to be fully adsorbed on the hydrophobin supporting membrane.
4) And (3) sucking excessive liquid by using filter paper to form a nano-scale ultrathin liquid film on the carrying net, and then quickly putting the nano-scale ultrathin liquid film into liquid ethane to form glassy ice.
5) The frozen electron microscope carrier net is put into liquid nitrogen with the temperature of 196 ℃ below zero, and can be stored for a long time.
Referring to fig. 4, a cryoelectron micrograph of the human apoferritin prepared from the alloy mesh using the hydrophobin film as the support film according to the present invention is shown in fig. 5, and the final three-dimensional reconstructed electron density map of the human apoferritin has the overall resolution up to the point where the entire resolution is obtained
Referring to fig. 6, representative amino acid types were selected, and it can be seen from their electron densities that the reconstructed results reached near atomic resolution levels. In addition, the human apoferritin sample prepared by the alloy mesh with the hydrophobin film as the support film in the embodiment of the invention reaches the highest resolution of the current analysis.
Example 2
This example expresses hydrophobins fused to other proteins such as antibodies. The method comprises the following specific steps:
1) the linearized plasmid containing the hydrophobin gene and other protein genes (e.g.antibodies) was transformed onto Pichia pastoris GS 115.
2) Positive transformants were selected for shake flask fermentation and induced expression.
3) The hydrophobin is purified by hollow fiber column ultrafiltration and acetonitrile extraction.
4) Hydrophobin was lyophilized to powder and then dissolved in ultrapure water, PBS buffer, Tris buffer or HEPES buffer to give a hydrophobin solution.
5) The hydrophobin solution is diluted to a concentration of 5 mug/ml, and ultrasonic oscillation is carried out in the dilution process to fully dissolve the hydrophobin, so that aggregates are prevented from being formed.
6) And (3) sucking 25 mu L of hydrophobin solution by using a pipette gun, dripping the hydrophobin solution on the sealing film at a certain distance, and standing for 2.5 hours until a macroscopic hydrophobin film is formed on the liquid drop.
7) The electron microscope carrier mesh is clamped by tweezers and placed above the surface of the liquid drop, the carrier mesh is contacted with the hydrophobin membrane, and incubation is carried out for 8 min.
8) The carrier web on which the hydrophobin membrane had been laid was gently gripped with tweezers and excess liquid was aspirated off with filter paper.
9) Dropping ultrapure water on the hydrophobin carrier net, rinsing, washing off residual impurities, and absorbing excess liquid by using filter paper.
10) And (4) after the hydrophobin net is naturally dried, preparing a subsequent electron microscope sample.
11) The preparation process of the sample for the cryo-electron microscope is based on the preparation process of a common sample for the cryo-electron microscope, but after sample adding, the sample needs to be incubated on a carrying net for a certain time to be fully adsorbed on a hydrophobin film, and at the moment, the sample can be washed for a plurality of times by using a buffer solution to remove the uncombined impurity protein, and then the subsequent steps are carried out.
In the embodiment, the hydrophobin is subjected to fusion expression with other proteins such as antibodies, and the hydrophobin is subjected to specific binding with protein molecules of interest due to the existence of the antibodies on the surface of the hydrophobin carrier net, so that the hydrophobin carrier net is changed into an affinity carrier net, and the protein purification can be directly carried out on the carrier net.
Example 3
In this example, hydrophobins were engineered to mutate hydrophilic amino acids to other amino acids, as follows.
1) The hfbI gene was subjected to amino acid mutation, and the linearized plasmid pPIC9-hfbI (plasmid map see fig. 2) was transformed into pichia pastoris GS 115.
2) Positive transformants were selected for shake flask fermentation and induced expression.
3) The hydrophobin is purified by hollow fiber column ultrafiltration and acetonitrile extraction.
4) Hydrophobin was lyophilized to powder and then dissolved in ultrapure water, PBS buffer, Tris buffer or HEPES buffer to give a hydrophobin solution.
5) The hydrophobin solution is diluted to the concentration of 11 mug/mL, and ultrasonic oscillation is carried out in the dilution process to fully dissolve the hydrophobin, so that aggregates are prevented from being formed.
6) And (3) sucking 30 mu L of hydrophobin solution by using a pipette gun, dripping the hydrophobin solution on the sealing film at a certain distance, and standing for 1h until a macroscopic hydrophobin film is formed on the liquid drop.
7) The electron microscope carrier mesh is clamped by tweezers and placed above the surface of the liquid drop, the carrier mesh is contacted with the hydrophobin membrane, and incubation is carried out for 15 min.
8) The carrier web on which the hydrophobin membrane had been laid was gently gripped with tweezers and excess liquid was aspirated off with filter paper.
9) Dropping ultrapure water on the hydrophobin carrier net, rinsing, washing off residual impurities, and absorbing excess liquid by using filter paper.
10) And after the hydrophobin grid is naturally dried, placing the hydrophobin grid into a grid box for storage, and preparing a subsequent electron microscope sample.
11) The frozen sample preparation process is based on a general frozen electron microscope sample preparation process, after sample adding, the sample needs to be incubated on a carrying net for a certain time to be fully attached to the hydrophobin film, and then subsequent steps are carried out.
In some cases, the problem of orientation advantage still exists to a certain extent when hydrophobin is used for carrying out sample preparation, and in order to solve the problem, the embodiment of the invention modifies hydrophobin, so that hydrophilic amino acids are fully or partially mutated into other amino acids, and the orientation distribution of protein particles on the surface of the hydrophobin film is changed, thereby solving the problem of orientation advantage.
Example 4
In this embodiment, a hydrophobin film is laid on a graphene net-carrying surface, and the specific steps are as follows.
1) The linearized plasmid pPIC9-hfbI (plasmid map see FIG. 2) was transformed onto Pichia pastoris GS 115.
2) Positive transformants were selected for shake flask fermentation and induced expression.
3) The hydrophobin is purified by hollow fiber column ultrafiltration and acetonitrile extraction.
4) Hydrophobin was lyophilized to powder and then dissolved in ultrapure water, PBS buffer, Tris buffer or HEPES buffer to give a hydrophobin solution.
5) The hydrophobin solution is diluted to the concentration of 8 mug/mL, and ultrasonic oscillation is carried out in the dilution process to fully dissolve the hydrophobin, so that aggregates are prevented from being formed.
6) And (3) sucking 40 mu l of hydrophobin solution by using a pipette gun, dripping the hydrophobin solution on the sealing film at a certain distance, and standing for 1.5 hours until a macroscopic hydrophobin film is formed on the liquid drop.
7) And clamping the graphene carrying net by using tweezers, placing the graphene carrying net above the surface of the liquid drop, contacting the carrying net with the hydrophobin membrane, and incubating for 12 min.
8) The carrier web on which the hydrophobin membrane had been laid was gently gripped with tweezers and excess liquid was aspirated off with filter paper.
9) Dropping ultrapure water on the hydrophobic opal graphene carrying net, rinsing, washing off residual impurities, and absorbing excess liquid by using filter paper.
10) And after the hydrophobic protein graphene net is naturally dried, placing the hydrophobic protein graphene net in a net carrying box for storage, and preparing a subsequent electron microscope sample.
11) The frozen sample preparation process is based on a general frozen electron microscope sample preparation process, after sample adding, the sample needs to be incubated on a carrying net for a certain time to be fully attached to the hydrophobin film, and then subsequent steps are carried out.
In some cases, the hydrophobin film is easy to break, so that the hydrophobin film is laid on the graphene grid surface to change the hydrophobic graphene surface into hydrophilic, so that glow discharge treatment is not needed, and the hydrophobin film can be directly used for preparing a frozen electron microscope sample.
Example 5
This example is the preparation of a human catalase cryoelectron microscope sample using an electron microscope mesh with a hydrophobin membrane as a support membrane according to the present invention. The method comprises the following specific steps:
1) the linearized plasmid pPIC9-hfbI (plasmid map as shown in FIG. 2) was transformed onto Pichia pastoris GS 115.
2) Positive transformants were selected for shake flask fermentation and induced expression.
3) The hydrophobin is purified by hollow fiber column ultrafiltration and acetonitrile extraction.
4) Hydrophobin was lyophilized to powder and then dissolved in ultrapure water, PBS buffer, Tris buffer or HEPES buffer to give a hydrophobin solution.
5) The hydrophobin solution is diluted to the concentration of 10 mug/mL, and ultrasonic oscillation is carried out in the dilution process to fully dissolve the hydrophobin, so that aggregates are prevented from being formed.
6) And sucking 25 mu L of hydrophobin solution by using a pipette gun, dripping the hydrophobin solution onto a sealing film or a glass slide at a certain distance, and standing for about 2 hours until a hydrophobin film visible to naked eyes is formed on the liquid drop.
7) The electron microscope carrying net is clamped by tweezers and placed above the surface of the liquid drop, the carrying net is contacted with the hydrophobin membrane, and the incubation is carried out for about 10 min.
8) The hydrophobin film can be only attached to a specific position of the carrying net by designing the carrying net.
9) The carrier web on which the hydrophobin membrane had been laid was gently gripped with tweezers and excess liquid was aspirated off with filter paper.
10) Dropping ultrapure water on the hydrophobin carrier net, rinsing, washing off residual impurities, and absorbing excess liquid by using filter paper.
11) And after the hydrophobin grid is naturally dried, the preparation of the electron microscope grid using the hydrophobin film as a support film is finished, and the electron microscope grid is placed in a grid box for storage and used for preparing a subsequent frozen electron microscope sample.
12) Based on the general preparation process of the cryo-electron microscope sample, a Vitrobot Mark IV freeze-sampling instrument is selected to prepare the humanized catalase cryo-electron microscope sample. The preparation process of the sample for the cryoelectron microscope is the same as the preparation process of the sample for the cryoelectron microscope, and is not repeated herein.
Referring to FIG. 7, the cryoelectron micrograph of the humanized catalase prepared from the alloy mesh using the hydrophobin membrane as the support membrane of the invention is shown in FIG. 8, the final three-dimensional reconstructed electron density map of the humanized catalase has the overall resolution reaching that of the humanized catalase
Referring to FIG. 9, representative amino groups are selectedThe acid type, by its electron density, can be seen that the reconstruction results reach a near atomic resolution level.
The problem of strong orientation advantage caused by a gas-liquid interface can be solved in the conventional preparation of a cryoelectron microscope sample, so that the three-dimensional structure cannot be analyzed by a single-particle method, but the preparation of the cryoelectron microscope sample of the human catalase by using the alloy supported mesh with the hydrophobin film as the support film, which is prepared by the method, can successfully obtain the near-atomic resolution three-dimensional structure of the human catalase without the orientation advantage.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to examples, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. An electron microscope carrier net using a hydrophobin film as a support film is characterized in that: comprises an electron microscope carrier net and a hydrophobin film arranged on the surface of the electron microscope carrier net.
2. The method for preparing the electron microscope carrier net by using the hydrophobin film as the support film according to claim 1, which is characterized by comprising the following steps:
dripping the hydrophobin solution on a substrate with hydrophobic property, and standing until a hydrophobin film is formed on the surface of the liquid drop;
placing an electron microscope carrier net on the surface of the liquid drop, contacting the electron microscope carrier net with the hydrophobin film, and incubating until the hydrophobin film is laid on the electron microscope carrier net;
removing the redundant liquid on the electron microscope carrier net paved with the hydrophobin film;
dripping pure water into the electron microscope carrying net paved with the hydrophobin film for rinsing;
and naturally drying the rinsed electron microscope carrying net to prepare the electron microscope carrying net.
3. The method for preparing an electron microscope mesh using a hydrophobin film as a support film according to claim 2, wherein: the electron microscope carrying net is a Quantifoil carrying net, a GIG carrying net, a NiTi alloy carrying net or a graphene carrying net.
4. The method for preparing an electron microscope mesh using a hydrophobin film as a support film according to claim 2, wherein: the concentration of the hydrophobin solution is 1-1000 mug/mL.
5. The method for preparing an electron microscope mesh using a hydrophobin film as a support film according to claim 4, wherein: the hydrophobin in the hydrophobin solution is all kinds of hydrophobin or hydrophobin after modification.
6. The method for preparing an electron microscope carrier net by using the hydrophobin film as a supporting film according to claim 5, wherein the preparation method of the hydrophobin solution comprises the following steps:
transforming the linearized plasmid pPIC9-hfbI into Pichia pastoris GS 115;
selecting positive transformants for shake flask fermentation and induced expression;
purifying hydrophobin by hollow fiber column ultrafiltration and acetonitrile extraction two-step method;
lyophilizing hydrophobin to hydrophobin powder;
dissolving hydrophobin powder in a solvent and carrying out ultrasonic oscillation to prepare a hydrophobin solution;
and diluting the hydrophobin solution and carrying out ultrasonic oscillation to obtain the hydrophobin solution with the concentration of 1-1000 mug/mL.
7. The method for preparing an electron microscope mesh using a hydrophobin film as a support film according to claim 6, wherein: the solvent is ultrapure water, PBS buffer, Tris buffer or HEPES buffer.
8. The method for preparing an electron microscope mesh using a hydrophobin film as a support film according to claim 1, wherein: the volume of the hydrophobin solution dropped onto the substrate with hydrophobic properties is 10-500. mu.L.
9. The method for preparing an electron microscope mesh using a hydrophobin film as a support film according to claim 8, wherein: the substrate is a sealing film or a glass slide.
10. The method for preparing an electron microscope mesh using a hydrophobin film as a support film according to claim 1, wherein: the incubation time after the electron microscope carrier mesh is contacted with the hydrophobin film is more than 5 min.
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| ANNELOES S. OUDE VRIELINK ET.AL: "Suspended crystalline films of protein hydrophobin I (HFBI)", 《JOURNAL OF COLLOID AND INTERFACE SCIENCE》 * |
| 牛宝龙: "《中国博士学位论文全文数据库 (基础科学辑)》", 15 July 2013 * |
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