CN110292916B

CN110292916B - Phospholipid nanodisk chromatography medium and preparation method and application thereof

Info

Publication number: CN110292916B
Application number: CN201910561133.9A
Authority: CN
Inventors: 马光辉; 赵岚; 黄永东; 魏炜; 朱凯; 陶娇丽; 周炜清; 苏志国
Original assignee: Senhui Microsphere Tech Suzhou Co ltd; Institute of Process Engineering of CAS
Current assignee: Senhui Microsphere Tech Suzhou Co ltd; Institute of Process Engineering of CAS
Priority date: 2019-06-26
Filing date: 2019-06-26
Publication date: 2020-11-03
Anticipated expiration: 2039-06-26
Also published as: CN110292916A

Abstract

The invention relates to a phospholipid nanodisk chromatography medium, and a preparation method and application thereof. The phospholipid nanodisk chromatography medium comprises a charged phospholipid nanodisk and an affinity matrix; the charge type phospholipid nanodiscs comprise phospholipid and membrane scaffold protein with affinity labels; the charged phospholipid nanodiscs are attached to an affinity matrix via an affinity tag. The phospholipid nano-disc chromatography medium has the binding capacity and the protective capacity to membrane protein at the same time, so that the purification and reconstruction of an actual system containing the membrane protein are completed in one step on a chromatographic column; and has reversibility and regeneration performance; the method solves the problems of serious loss of protein activity, tedious and fussy steps and time and labor consuming operation in the traditional membrane protein research technology, is simple and efficient to operate, improves the membrane protein activity recovery rate by more than 20 times, and shortens the processing time by more than 95%.

Description

Phospholipid nanodisk chromatography medium and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a chromatography medium for separating and purifying membrane protein, in particular to a phospholipid nanodisk chromatography medium, and a preparation method and application thereof.

Background

The membrane protein, as a main bearer of the function of the biological membrane, not only provides a stable internal environment for the vital activities of a single cell, but also mediates the connection between cells and a matrix, thereby playing rich and important functions in the aspects of signal transduction, physiological transport, enzyme catalysis, structural connection and the like of a living body. Therefore, membrane proteins have been the focus of research in the fields of biology, medicine and materials. However, membrane proteins are also a recognized difficulty of research. Most of membrane proteins are embedded in a biological membrane layer, the content is very low, the hydrophobicity is very strong, and the separation and purification are very difficult. The conventional method is to dissociate the membrane protein from the membrane layer using a large amount of surfactant and then purify it by a multi-step chromatography technique. However, the use of a large amount of surfactant easily causes inactivation of membrane protein, and the damage to physiological structure and function is serious.

The reconstruction of membrane proteins is currently the main strategy for restoring the structure and function of membrane proteins. The membrane protein after purification is reinserted into the lipid membrane to protect the structure and function of the membrane protein, so that the inactivation phenomenon after the membrane protein is separated from the membrane layer can be effectively avoided. The membrane protein reconstruction system mainly comprises liposome, a double-layer membrane micro-package and a nano disc. Compared with other two systems, the nano-disc is formed by mixing and incubating membrane scaffold protein (MSP for short) and phospholipid dissolved in a surfactant for a period of time, removing the surfactant and then carrying out self-assembly, has the characteristics of controllable structure, no surfactant interference, good stability and the like, and has excellent application effect in the field of membrane protein research.

The traditional research method of firstly purifying membrane protein and then reconstructing the membrane protein in the prior art has the following problems: 1. no matter what reconstruction form, the membrane protein has only a protection function and no binding capacity, so that the purification and reconstruction of the membrane protein are the splitting process, and the whole operation process is complicated and tedious, consumes time and is laborious; 2. the activity loss is large, taking plasma membrane protein as an example, after a series of purification steps, the protein activity is only about 30%, and the activity is only improved to 60% at most in the reconstruction process, namely, the membrane protein activity is still lost nearly half by adopting the step-by-step treatment method of reconstructing after purification; 3. the research surface is narrow, the reconstructed membrane protein is combined on the surface of the matrix and cannot be peeled off, and the reconstructed membrane protein can be researched only by adopting a certain specific research technology aiming at the type of the matrix, so that the information quantity is small.

At present, no chromatography system capable of realizing one-step completion of purification and reconstruction of membrane proteins exists in the prior art, and therefore, a new membrane protein research strategy is urgently needed to be developed so as to achieve the aims of realizing efficient purification and activity maintenance of the membrane proteins at the same time.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a phospholipid nanodisk chromatography medium, a preparation method and an application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the present invention provides a phospholipid nanodisk chromatography medium comprising a charged phospholipid nanodisk and an affinity matrix; the charge type phospholipid nanodiscs comprise phospholipid and membrane scaffold protein with affinity labels; the charged phospholipid nanodiscs are attached to an affinity matrix via an affinity tag.

The invention uses the charged phospholipid nanodisk as ligand to connect with affinity matrix, to obtain a phospholipid nanodisk chromatography medium for one-step completion of membrane protein purification and reconstruction, after the phospholipid nanodisk chromatography medium is filled into a column, the actual system containing membrane protein is loaded, the membrane protein is reconstructed and protected on the chromatography column by the charged phospholipid nanodisk ligand through electrostatic combination, and the membrane protein-nanodisk system can be used for further research after being dissociated.

Preferably, the diameter of the charged phospholipid nanodiscs is 1-100nm, such as 1nm, 5nm, 10nm, 12nm, 20nm, 25nm, 30nm, 40nm, 50nm, 60nm, 80nm or 100nm, etc., preferably 5-50nm, more preferably 10-25 nm.

The diameter of the charge type phospholipid nanodisk is one of key factors influencing the purification effect of the finally prepared phospholipid nanodisk chromatography medium, the value is 1-100nm, so that the effect is better, 5-50nm is better, 10-25nm is the range with the best effect, and if the value is too large, the coupling of the nanodisk is not facilitated due to the steric hindrance effect, so that the coupling density of the nanodisk is low; if too small, it will not bind to the membrane protein due to size effects, thereby reducing the efficiency of membrane protein purification.

Preferably, the charged phospholipid nanodiscs have a density of 0.1-25mg, e.g., 0.1mg, 0.5mg, 1mg, 2mg, 5mg, 10mg, 12mg, 15mg, 20mg or 25mg, etc., preferably 1-10mg, per mL of affinity matrix.

The density of the charge type phospholipid nanodisk is also one of key factors influencing the purification effect of the finally prepared phospholipid nanodisk chromatography medium, the value is 0.1-25mg, the effect is better, 1-10mg is the optimal range of the effect, and if the value is too large, the irreversible damage of the structure of the membrane protein caused by excessive combination is easily caused; if too small, the binding ability of the membrane protein is reduced.

Preferably, the Zeta potential of the charged phospholipid nanodisk at pH7.4 is (-50) eV, such as-50 eV, -40eV, -30eV, -25eV, -20eV, -10eV, 0eV, 10eV, 25eV, or 50eV, preferably (-25) eV.

The Zeta potential of the charge type phospholipid nano disc under the condition of pH7.4 is also one of key factors influencing the purification effect of the finally prepared phospholipid nano disc chromatography medium, the value of the Zeta potential is (-50) eV, which is a better range, the (-25) eV enables the best effect, when the Zeta potential is too high, membrane protein can be excessively combined, and when the Zeta potential is too low, the binding force is insufficient, which can cause that the charge type phospholipid nano disc cannot be effectively combined on the nano disc.

Preferably, the phospholipid comprises any one of or a combination of at least two of phosphatidylcholine (lecithin), Dipalmitoylphosphatidylethanolamine (DPPE), Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), Phosphatidylserine (PS), glycerophospholipid (PG), trimethyl-2, 3-dioleoyloxypropylammonium bromide (DOTAP), or trimethyl-2, 3-dioleoyloxypropylammonium chloride (DOTMA), such as a combination of phosphatidylcholine and dipalmitoylphosphatidylethanolamine, a combination of dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine, a combination of phosphatidylserine and glycerophospholipid, a combination of phosphatidylcholine and trimethyl-2, 3-dioleoyloxypropylammonium bromide, etc., and any other combinations are not necessarily repeated herein.

The membrane scaffold protein is amphiphilic polypeptide obtained by modifying Apoliprotein A-1 protein, the amphiphilic polypeptide has better structural flexibility and can be combined with phospholipid to form a nanodisk, such as MSP1 containing 200 amino acid residues.

Preferably, the affinity tag includes any one or a combination of at least two of histidine (His), Maltose Binding Protein (MBP), streptococcus (Strep) or glutathione mercaptotransferase (GST), such as a combination of histidine and maltose binding protein, streptococcus and glutathione mercaptotransferase, maltose binding protein and streptococcus, and the like, and any combination thereof is not necessarily described herein.

Preferably, the affinity matrix comprises an organic material and/or an inorganic material.

Preferably, the affinity matrix comprises any one or a combination of at least two of agarose, dextran, cellulose, konjac glucomannan, polymethacrylate, polystyrene, polyacrylamide, silica gel or hydroxyapatite, such as a combination of agarose and dextran, a combination of cellulose and konjac glucomannan, a combination of polystyrene and polyacrylamide, and the like, and any combination thereof is not necessarily described herein.

The affinity matrix is prepared by a mechanical stirring method, a membrane emulsification method or a spraying method. Each preparation mode needs to be provided with specific operating parameters, for example, the parameters needing to be set for mechanical stirring comprise geometric parameters of a stirring tank body and a stirrer, the rotating speed of the stirrer and the like; the parameters required to be set by the membrane emulsification method comprise the aperture of a membrane tube, the membrane passing pressure, the membrane passing times and the like; the parameters that the ejection method needs to set include nozzle size, ejection pressure, and the like.

Preferably, the particle size of the affinity matrix is 1-500. mu.m, such as 1. mu.m, 5. mu.m, 10. mu.m, 20. mu.m, 30. mu.m, 50. mu.m, 80. mu.m, 100. mu.m, 200. mu.m, 300. mu.m, 400. mu.m or 500. mu.m, etc., preferably 5-200. mu.m, more preferably 10-100. mu.m.

The particle size of the affinity matrix is one of the key factors influencing the purification efficacy of the finally prepared phospholipid nanodisk chromatography medium, the value is 1-500 mu m, the effect is better, the effect is optimal, the 10-100 mu m enables the effect to be optimal, and if the particle size exceeds the range, the membrane protein purification efficiency is greatly reduced. This is because too large a particle size leads to a decrease in column resolution, and too small a particle size leads to a great decrease in separation speed.

In the present invention, the charged phospholipid nanodiscs are linked to an affinity ligand in an affinity matrix via an affinity tag.

Preferably, the affinity ligand comprises any one or a combination of at least two of metal ions, glutathione, amylose or streptavidin variants, such as a combination of metal ions and glutathione, a combination of amylose and streptavidin variants, a combination of glutathione and amylose, and the like, and any other combination is not repeated herein.

Preferably, the metal ions comprise Ni²⁺、Cu²⁺、Co²⁺、Fe²⁺Or Zn²⁺Any one of or a combination of at least two of, for example, Ni²⁺And Cu²⁺Combination of (2) and Cu²⁺And Co²⁺Combination of (1), Fe²⁺And Zn²⁺The combinations of the above-mentioned components, and other arbitrary combinations are not necessarily described herein.

Preferably, the metal ion is attached to the affinity matrix by chelation with a chelating agent.

Preferably, the chelating agent includes any one or a combination of at least two of iminodiacetic acid (IDA), Tricarboxymethylethylenediamine (TED), nitrilotriacetic acid (NTA), carboxymethylaspartic acid (CM-ASP), Tetraethylenepentamine (TEPA), or carboxymethyl- α, β -diaminosuccinic acid (CM-DASA), such as a combination of iminodiacetic acid and tricarboxymethylethylenediamine, a combination of nitrilotriacetic acid and carboxymethylaspartic acid, a combination of iminodiacetic acid and carboxymethyl- α, β -diaminosuccinic acid, and the like, any combination of which is not necessarily described herein.

In another aspect, the present invention provides a method for preparing the phospholipid nanodisk chromatography medium as described above, comprising the steps of:

(1) mixing and reacting phospholipid membrane assembly liquid and membrane scaffold protein with affinity labels to obtain a charge type phospholipid nanodisk;

(2) and (2) carrying out coupling reaction on the charge type phospholipid nanodisk prepared in the step (1) and an affinity matrix to obtain the phospholipid nanodisk chromatography medium.

In the invention, the preparation method of the phospholipid membrane assembling liquid in the step (1) comprises the following steps: and (2) preparing a phospholipid membrane by using a nitrogen blow-drying membrane preparation method or a rotary evaporation membrane preparation method for the phospholipid solution, and dissolving the phospholipid membrane by using the assembly liquid to obtain the phospholipid membrane assembly liquid.

Preferably, the assembly liquid is a buffer at pH 7.0-7.4 (e.g., pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH7.4, etc.).

Preferably, the assembly liquid comprises phosphate buffer and Na₂HPO₄-citric acid buffer, KH₂PO₄-NaOH buffer, barbiturate sodium-hydrochloric acid buffer or Tris-buffer.

Preferably, the composition contains sodium chloride at a concentration of 0.02-0.1M, such as 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, or 0.1M.

Preferably, the composition contains sodium cholate at a concentration of 0.01-0.5M, such as 0.01M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.1M, 0.2M, or 0.5M.

In the present invention, the mixing time of the phospholipid membrane assembling solution and the membrane scaffold protein with affinity tag in step (1) is 0.5-12h, such as 0.5h, 1h, 2h, 4h, 5h, 6h, 8h, 10h or 12h, etc., preferably 1-8h, and more preferably 2-4 h.

Preferably, the mixing temperature of the phospholipid membrane assembling solution and the membrane scaffold protein with affinity tag in the step (1) is 4-60 ℃, such as 4 ℃, 10 ℃, 20 ℃, 25 ℃, 30 ℃, 40 ℃, 50 ℃ or 60 ℃, preferably 20-60 ℃, and more preferably 30-50 ℃.

Preferably, the molar ratio of the membrane scaffold protein to the phospholipid in the phospholipid membrane assembling solution in the step (1) is 1 (10-100), such as 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100, etc., preferably 1 (20-60).

Preferably, the coupling in step (2) is performed by metal chelation or covalent interaction.

Preferably, the coupling reaction time in step (2) is 10-30h, such as 10h, 12h, 18h, 20h, 22h, 24h, 25h, 26h, 28h or 30h, etc.

Preferably, the temperature of the coupling reaction in step (2) is 20-40 deg.C, such as 20 deg.C, 22 deg.C, 25 deg.C, 30 deg.C, 32 deg.C, 35 deg.C, 38 deg.C or 40 deg.C.

Preferably, the charged phospholipid nanodiscs are dialyzed to equilibrium in a coupling buffer prior to performing the coupling reaction as described in step (2).

The time for dialysis of the charged phospholipid nanodiscs to equilibrium in the coupling buffer is typically 10-15h, and different affinity matrices correspond to different coupling conditions. For example, the coupling conditions for metal chelation are generally that the charged phospholipid nanodisk solution is dialyzed to equilibrium by neutral/weak alkaline (pH 7-8) buffer solution, wherein a phosphate buffer system is most commonly used, and 0.15-1.0M NaCl or other neutral salt can be added to inhibit nonspecific adsorption, and the balanced phospholipid nanodisk buffer solution is mixed with the metal chelation medium at room temperature for 12 h.

As for the coupling conditions of covalent interactions, in particular, for glutathione media, phospholipid nanodisk solutions are typically dialyzed to equilibrium in neutral/weakly basic (pH 7-8) buffers, with Tris-HCl buffer systems being most commonly used, such as 50mM Tris-HCl, pH 8.0, 0.1-0.2M NaCl being added to suppress nonspecific adsorption; for amylose, phospholipid nanodisk solutions are typically dialyzed to equilibrium in neutral/slightly alkaline (pH 7-8) buffers, with Tris-HCl buffer systems being most commonly used, such as 20mM Tris-HCl, 0.2M NaCl, 1mM EDTA, 1mM DTT, pH 7.4; for streptavidin variants, phospholipid nanodisk solutions are typically dialyzed to equilibrium in neutral/slightly alkaline (pH 7-8) buffers, with Tris-HCl buffer systems being most commonly used, such as 100mM Tris-HCl, 0.15M NaCl, 1mM EDTA, pH 8.0.

As a preferred technical scheme of the invention, the preparation method specifically comprises the following steps:

(1) preparing a phospholipid membrane by a phospholipid solution through a nitrogen blow-drying membrane preparation method or a rotary evaporation membrane preparation method, and dissolving the phospholipid membrane by an assembly liquid to obtain the phospholipid membrane assembly liquid;

(2) mixing the phospholipid membrane assembling liquid prepared in the step (1) and membrane scaffold protein with affinity labels at 4-60 ℃ for reaction for 0.5-12h, wherein the molar ratio of the membrane scaffold protein to phospholipid in the phospholipid membrane assembling liquid is 1 (10-100), so as to obtain a charge type phospholipid nanodisk;

(3) and (3) dialyzing the charge type phospholipid nanodisk prepared in the step (2) in a coupling buffer solution until the charge type phospholipid nanodisk is balanced, and performing coupling reaction with an affinity matrix at the temperature of 20-40 ℃ for 18-30h to obtain the phospholipid nanodisk chromatography medium.

In a further aspect, the invention provides a use of the phospholipid nanodisk chromatography media as described above for membrane protein purification and reconstitution.

Preferably, the method of application is: and sequentially carrying out column packing, balancing, protein loading, leaching, eluting and cleaning on the phospholipid nano-disc chromatography medium to realize the purification and reconstruction of the membrane protein.

The balance conditions are as follows: after the medium is loaded into the column, the chromatographic column is equilibrated with 5-10 column volumes of equilibration buffer solution until the effluent conductance and pH are unchanged (consistent with the equilibration solution), and the equilibration buffer solution is screened and optimized according to the stability and isoelectric point of the target protein and the type of ion exchange medium.

The protein loading conditions are as follows: the buffer solution of the sample is consistent with the balance buffer solution, the solid sample is dissolved and prepared by the balance solution, the low-concentration sample solution is dialyzed by the balance solution or is added with salt with corresponding amount, and the high-concentration sample solution is diluted by the balance solution.

The leaching conditions are as follows: the elution was performed with equilibration buffer to baseline.

The elution conditions were: eluting with elution buffer solution, dissociating membrane protein-nanodisc system bound to affinity matrix by metal chelation and covalent interaction, and collecting eluate. For the nano-disc medium prepared by metal chelation, a buffer solution containing imidazole is adopted for elution; for nanodisk media prepared by covalent interactions, e.g. glutathione type, elution is performed with glutathione containing buffers, also amylose type, with maltose containing buffers, and further streptavidin type, with desthiobiotin containing buffers.

The cleaning conditions are as follows: washing buffer solution is adopted to wash out the residual precipitate, denatured protein and non-specific adsorption protein on the medium. For example, it is washed with 70% ethanol, 30% isopropanol or 0.05-1.0M NaOH solution, and then rinsed with pure water.

Compared with the prior art, the invention has the following beneficial effects:

(1) firstly, the phospholipid nanodisk chromatography medium related by the invention has binding capacity and protective capacity to membrane protein simultaneously, so that the purification and reconstruction of an actual system containing the membrane protein are completed in one step on a chromatography column, the protective function of the traditional nanodisk is maintained, and the nanodisk has a certain electric charge amount and can be combined with the membrane protein under the electrostatic action to play a role in purifying the protein;

(2) secondly, the phospholipid nanodisk chromatography medium is applied to the technical field of protein, solves the problems of serious loss of protein activity, tedious and fussy steps and time and labor consuming operation in the traditional membrane protein research technology, is simple and efficient to operate, improves the membrane protein activity recovery rate by more than 20 times, and shortens the processing time by more than 95%;

(3) thirdly, the phospholipid nano-disc chromatography medium has reversibility and regeneration performance. The combination of the phospholipid nano disc and the affinity matrix has reversibility, after the nano disc is combined with the membrane protein, the nano disc loaded with the membrane protein is dissociated from the affinity matrix by adopting a certain elution condition, on one hand, the membrane protein-nano disc system which simultaneously realizes the membrane protein purification and activity protection functions can be directly used for the research on the structure and the function of the membrane protein and is suitable for a plurality of membrane protein research technologies such as Surface Plasma Resonance (SPR), Nuclear Magnetic Resonance (NMR), Electrochemical Impedance Spectroscopy (EIS) and Atomic Force Microscope (AFM); on the other hand, the substrate after the dissociation of the nano disc can be continuously coupled with the nano disc and can be reused after regeneration, so that the service life of the medium is greatly prolonged, and the application field is wider.

(4) Meanwhile, the phospholipid nanodisk chromatography medium has the advantages of controllable structure and performance. The membrane proteins are various in types and complex in structure, and the activity characteristics of different membrane proteins are greatly different from the purification requirements. By adjusting the preparation conditions of the nano-disc chromatography medium, the controllability of the structure and performance parameters such as the size and distribution, the charge property, the density and the like of the nano-disc is realized, so that the method is favorable for selecting the chromatography medium type which is beneficial to the efficient combination and activity maintenance of the membrane protein according to the actual system characteristics of the membrane protein. The controllable preparation of the size and the distribution of the nano-disc is beneficial to the combination of membrane proteins with different sizes, the controllable preparation of the charged property and the density is beneficial to the combination of membrane proteins with different isoelectric points, and the affinity matrix with wide variety is beneficial to the satisfaction of chromatography systems of different types, so that the efficient protection and purification of the chromatography media of the nano-disc on different membrane protein systems are finally realized.

Drawings

FIG. 1 is a schematic diagram of the application of the phospholipid nanodisk chromatography media in membrane protein purification and reconstitution in accordance with the present invention;

FIG. 2 is a particle size distribution and Zeta potential diagram of nanodiscs in the phospholipid nanodisc chromatography medium prepared in example 1;

FIG. 3 is a transmission electron micrograph of a nanodisk in a phospholipid nanodisk chromatography medium made in example 1;

FIG. 4 is a Zeta potential diagram and a particle size distribution of nanodiscs in the phospholipid nanodisc chromatography medium prepared in example 2;

FIG. 5 is a transmission electron micrograph of a nanodisk in a phospholipid nanodisk chromatography medium made in example 2;

FIG. 6 is a Zeta potential diagram and a particle size distribution of nanodiscs in the phospholipid nanodisc chromatography medium prepared in example 3;

FIG. 7 is a transmission electron micrograph of a nanodisk in a phospholipid nanodisk chromatography medium made in example 3;

FIG. 8 is a chromatogram of example 9;

FIG. 9 is a CO difference spectrum in example 9;

FIG. 10 is a diagram showing the result of electrophoretic analysis (SDS-PAGE) in example 9;

FIG. 11 is a gel filtration chromatogram of example 17;

FIG. 12 is a diagram showing the results of SDS-PAGE analysis in example 17;

FIG. 13 is a Native-PAGE analysis result chart in example 17;

FIG. 14 is a transmission electron micrograph of example 17 (a shows a cell membrane disruption solution, and b shows an eluate after passing through a chromatography medium).

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The following examples relate to assays that include the following:

(1) and (3) measuring the particle size and distribution of the nano-discs and the Zeta potential: measured by a dynamic light scattering instrument.

(2) And (3) nano-disc shape analysis: and (5) observing the appearance of the nano disc collected in stages by adopting a transmission electron microscope.

(3) Density measurement of the nanodisk: and (3) measuring the protein concentration of the supernatant before and after the coupling of the nano-disc solution and the affinity matrix by adopting a Coomassie brilliant blue method, and calculating the density of the nano-disc according to the following formula.

Density of nanodisk ═ C₁V₁-C₀V₀)/V

In the formula, C₀、C₁The concentrations of the protein (mg/mL) before and after the reaction of the supernatant of the coupling solution, V₀、V₁Respectively, the volume (mL) of the supernatant of the coupling solution before and after the reaction, and V is the affinityMatrix volume (mL).

(4) Determination of the average particle size of the affinity matrix: and measuring by using a laser particle size analyzer.

(5) CO differential spectroscopy experiment: adding sodium dithionite into the collected components by gel filtration chromatography, and standing for 5 s. After the system is stabilized, baseline scanning is carried out from 400nm to 500nm by using an enzyme-labeling instrument, then carbon monoxide is introduced into the system for 60s (bubbles are continuous and solution does not overflow), and scanning is carried out from 400nm to 500nm by using the enzyme-labeling instrument continuously. The experiment uses liver microsome broken liquid as blank control. The CYP450 content was calculated according to the following formula:

wherein Δ OD is the difference in OD values at wavelengths of 450nm and 490nm, and 91 is the extinction coefficient (mmol/L).

(6) Electrophoretic analysis (SDS-PAGE): SDS-PAGE experiments are carried out according to the steps of separating gel preparation, gel concentration, sample loading, electrophoresis, dyeing, decoloring and the like. The separation gel concentration was 13.5%. Purity was quantified by scanning densitometer.

The amino acid sequence of the membrane scaffold protein involved in the following examples is: GHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ are provided.

Example 1

This example prepares a phospholipid nanodisk chromatography medium, named nanodisk-Ni-IDA agarose chromatography medium, whose preparation method is as follows:

(1) preparing a membrane scaffold protein (MSP protein for short) connected with a His tag: connecting the MSP protein sequence gene monomer with His label with pET-21a to obtain expression vector, and converting to E.coli BL21 to obtain corresponding gene engineering bacteria. Inoculating the strain to LB solid culture medium by plate-drawing method, selecting single colony, transferring to LB liquid culture medium, shaking overnight at 30 deg.C, and continuously culturing to obtain first-stage seed and second-stage seed. When the OD600 of the secondary seed is between 0.8 and 1.0, IPTG induction is carried out, bacterial suspension is collected, and centrifugation is carried out at 4 ℃. And (3) carrying out Ni-IDA column chromatography on the supernatant, carrying out loading conditions (20mM PB, 0.15M NaCl, 20mM imidazole, pH7.4) and elution conditions (20mM PB, 0.15M NaCl, 200mM imidazole, pH7.4), collecting elution peaks, concentrating, and storing at 4 ℃ to obtain the MSP protein connected with the His tag.

(2) Preparing a charged phospholipid nanodisk: accurately measuring 1mL of 100mM lecithin solution, adding into a centrifuge tube, and drying by nitrogen. To the resulting phospholipid membrane, 5mL of an assembly solution (20mM phosphate, 0.15M sodium chloride, 100mM sodium cholate at pH7.4) was added, and the mixture was subjected to a water bath at 50 ℃. After the phospholipid membrane was completely dissolved in the assembly liquid, 15mg/mL of His-tagged membrane scaffold protein (MSP protein to phospholipid molar ratio 1:60) was added thereto and mixed for 2 h. The resulting system was dialyzed overnight at 4 ℃.

The average diameter of the resulting nanodisk, and the Zeta potential at pH7.4 were measured and are shown in FIG. 2: the diameter of the nano disc is 10nm, and the Zeta potential value under the pH value of 7.4 is-8 eV; the transmission electron microscopy analysis was performed on the prepared nanoplate, and the results are shown in fig. 3: the nano-discs are approximately round in shape, and are regularly arranged without aggregation.

(3) Preparing a phospholipid nanodisk chromatography medium: 5g of agarose medium was weighed out, washed, drained and charged with Ni-IDA as affinity ligand, and placed in a flask, and 5mL of the above nanodisk solution was added thereto, followed by shaking overnight at room temperature. After the reaction is finished, washing the medium by using a large amount of deionized water to prepare the nanodisk-Ni-IDA agarose chromatography medium.

The coupling density of the prepared nanodiscs was determined to be 12mg/mL and the average particle size of the affinity media was determined to be 90 μm.

Example 2

This example prepares a phospholipid nanodisk chromatography medium, named nanodisk-glutathione-dextran chromatography medium, and its preparation method is as follows:

(1) preparing a membrane scaffold protein (MSP protein for short) connected with a GST tag: the procedure was as described in example 1, and glutathione column chromatography was used for purification.

(2) Preparing a charged phospholipid nanodisk: 1mL of 100mM lecithin and DPPC compounded solution (compounded according to the mol ratio of 1: 1) is accurately measured, added into a centrifuge tube and dried by nitrogen. To the resulting phospholipid membrane, 5mL of an assembly solution (20mM phosphate, 0.15M sodium chloride, 100mM sodium cholate at pH7.4) was added, and the mixture was subjected to a water bath at 50 ℃. After the phospholipid membrane was completely dissolved in the assembly liquid, 1mg/mL of GST tag membrane scaffold protein (molar ratio of MSP protein to phospholipid was 1:10) was added thereto and mixed for 2 hours. The resulting system was dialyzed overnight at 4 ℃.

The average diameter of the resulting nanodisk, and the Zeta potential at pH7.4 were measured and are shown in FIG. 4: the diameter of the nano disc is 1nm, and the Zeta potential value under the pH value of 7.4 is-1 eV; the transmission electron microscopy analysis was performed on the prepared nanoplate, and the results are shown in fig. 5: the nano-discs are approximately round in shape, and are regularly arranged without aggregation.

(3) Preparing a phospholipid nanodisk chromatography medium: weighing 5g of dextran medium which is washed and drained and takes glutathione as affinity ligand, placing the dextran medium into a triangular flask, adding 5mL of the nano-disc solution, and shaking the solution at room temperature overnight. After the reaction is finished, washing the medium by using a large amount of deionized water to prepare the nanodisk-glutathione-glucan chromatography medium.

The coupling density of the prepared nanodisk was determined to be 0.1mg/mL and the average particle size of the affinity medium was determined to be 60 μm.

Example 3

In this embodiment, a phospholipid nanodisk chromatography medium named nanodisk-amylose-konjac glucomannan chromatography medium is prepared by the following preparation method:

(1) preparing membrane scaffold protein (MSP protein for short) connected with Maltose Binding Protein (MBP) label: the procedure was as in example 1, and amylopectin column chromatography was used for purification.

(2) Preparing a charged phospholipid nanodisk: accurately measuring 1mL of a compound solution (compounded according to a mol ratio of 1: 5) of lecithin and Phosphatidylserine (PS) with the total molar weight of 100mM, adding the compound solution into a centrifugal tube, and drying by nitrogen. To the resulting phospholipid membrane, 5mL of an assembly solution (20mM phosphate, 0.15M sodium chloride, 100mM sodium cholate at pH7.4) was added, and the mixture was subjected to a water bath at 50 ℃. After the phospholipid membrane was completely dissolved in the assembly solution, 30mg/mL of MBP-tagged membrane scaffold protein (MSP protein to phospholipid molar ratio 1:100) was added thereto and mixed for 2 h. The resulting system was dialyzed overnight at 4 ℃.

The average diameter of the resulting nanodisk, and the Zeta potential at pH7.4 were measured and are shown in FIG. 6: the diameter of the nano disc is 100nm, and the Zeta potential value under the pH value of 7.4 is-50 eV; the transmission electron microscopy analysis was performed on the prepared nanoplate, and the results are shown in fig. 7: the nano-discs are approximately round in shape, and are regularly arranged without aggregation.

(3) Preparing a phospholipid nanodisk chromatography medium: weighing 5g of konjac glucomannan medium which is cleaned and drained and takes amylopectin as affinity ligand, placing the konjac glucomannan medium into a triangular flask, adding 5mL of the nano-disc solution, and shaking the mixture at room temperature overnight. After the reaction is finished, cleaning the medium by using a large amount of deionized water to prepare the nanodisk-amylose-konjac glucomannan chromatography medium.

The coupling density of the prepared nanodiscs was determined to be 25mg/mL and the average particle size of the affinity media was determined to be 120 μm.

Example 4

This example prepares a phospholipid nanodisk chromatography medium, named nanodisk-streptavidin variant (Strep-Tactin) -polymethacrylate chromatography medium, by the following preparation method:

(1) preparing membrane scaffold protein (MSP protein for short) connected with Strep label: the method is as described in example 1, and purification is carried out by column chromatography using a streptavidin variant.

(2) Preparing a charged phospholipid nanodisk: accurately weighing 1mL of a compound solution (compounded according to a molar ratio of 1:10) of lecithin and DOTAP, wherein the total molar amount of the lecithin and the DOTAP are 100mM, adding the compound solution into a centrifugal tube, and drying the solution by nitrogen. To the resulting phospholipid membrane, 5mL of an assembly solution (20mM phosphate, 0.15M sodium chloride, 100mM sodium cholate at pH7.4) was added, and the mixture was subjected to a water bath at 50 ℃. After the phospholipid membrane was completely dissolved in the assembly solution, 12mg/mL of MBP-tagged membrane scaffold protein (MSP protein to phospholipid molar ratio 1:70) was added thereto and mixed for 2 h. The resulting system was dialyzed overnight at 4 ℃.

The average diameter of the prepared nano disc and the Zeta potential value under the pH value of 7.4 are measured, the diameter of the nano disc is 12nm, and the Zeta potential value under the pH value of 7.4 is 50 eV; and (3) carrying out transmission electron microscope analysis on the prepared nano disc: the nano-discs are approximately round in shape, and are regularly arranged without aggregation.

(3) Preparing a phospholipid nanodisk chromatography medium: weighing 5g of polymethacrylate medium which is washed and drained and takes streptavidin variant as affinity ligand, placing the polymethacrylate medium in a triangular flask, adding 5mL of the nanodisk solution, and shaking the nanodisk solution at room temperature overnight. After the reaction is finished, the medium is washed by a large amount of deionized water, and a nanodisk-streptavidin variant (Strep-Tactin) -polymethacrylate chromatography medium is prepared.

The coupling density of the prepared nanodisks was determined to be 10mg/mL and the average particle size of the affinity media was determined to be 50 μm.

Example 5

This example prepares a phospholipid nanodisk chromatography medium, named nanodisk Cu-CM-ASP silica gel chromatography medium, and the preparation method is as follows:

(1) preparing a membrane scaffold protein (MSP protein for short) connected with a His tag: the process was as in example 1.

(2) Preparing a charged phospholipid nanodisk: the process was as in example 1.

The average diameter of the prepared nano disc and the Zeta potential value under the pH value of 7.4 are measured, the diameter of the nano disc is 10nm, and the Zeta potential value under the pH value of 7.4 is-8 eV; and (3) carrying out transmission electron microscope analysis on the prepared nano disc: the nano-discs are approximately round in shape, and are regularly arranged without aggregation.

(3) Preparing a phospholipid nanodisk chromatography medium: 5g of the silica gel medium is weighed, washed, drained and filled in a triangular flask with Cu-CM-Asp as affinity ligand, 5mL of the above nanodisk solution is added, and the mixture is shaken overnight at room temperature. After the reaction is finished, washing the medium by using a large amount of deionized water to prepare the nanodisk Cu-CM-ASP silica gel chromatography medium.

The coupling density of the prepared nanodiscs was determined to be 15mg/mL and the average particle size of the affinity media was determined to be 5 μm.

Example 6

This example prepares a phospholipid nanodisk chromatography medium, named nanodisk-Ni-IDA agarose chromatography medium-2, whose preparation method differs from example 1 only in that: the molar ratio of MSP protein to phospholipid was 1: 100.

The average diameter of the prepared nano disc and the Zeta potential value under the pH value of 7.4 are measured, the diameter of the nano disc is 100nm, and the Zeta potential value under the pH value of 7.4 is-10 eV; and (3) carrying out transmission electron microscope analysis on the prepared nano disc: the nano-discs are approximately round in shape, and are regularly arranged without aggregation.

Example 7

This example prepares a phospholipid nanodisk chromatography medium, named nanodisk-Ni-IDA sepharose chromatography medium-3, which differs from example 1 only in that: accurately measuring 1mL of a compound solution (compounded according to a molar ratio of 1: 4) of lecithin and Phosphatidylserine (PS) with the total molar amount of 100 mM.

The average diameter of the prepared nano disc and the Zeta potential value under the pH value of 7.4 are measured, the diameter of the nano disc is 15nm, and the Zeta potential value under the pH value of 7.4 is-30 eV; and (3) carrying out transmission electron microscope analysis on the prepared nano disc: the nano-discs are approximately round in shape, and are regularly arranged without aggregation.

Example 8

This example prepares a phospholipid nanodisk chromatography medium, named nanodisk-Ni-IDA sepharose chromatography medium-4, which differs from example 1 only in that: the average particle size of the affinity medium was 200. mu.m, the others remained unchanged.

Example 9

This example used the nanodisk-Ni-IDA agarose chromatography media prepared in example 1 for the purification of protein liver microsome CYP450, and was performed as follows:

the nanodisk-Ni-IDA agarose chromatography media prepared in example 1 was packed into a column (column volume 0.5mL) and connected to a PPS protein chromatography system. After the column was equilibrated with Buffer A (20mM PB, pH7.4), the liver microsome disruption solution was applied at a flow rate of 0.2mL/min, and elution was continued with Buffer A and finally with Buffer B (20mM PB, containing 200mM imidazole, pH 7.4).

The chromatogram was recorded, and the results are shown in FIG. 8 (P)₁And P₁' penetration peak, P, of coupled and uncoupled nanodisk chromatography media sets, respectively₂Elution peaks for coupled nanodisk chromatography media set), as can be seen from the figure: the chromatographic medium which is not coupled with the nano disc has no obvious elution peak after being loaded with the sample and eluted by imidazole, which indicates that the chromatographic medium is not combined with protein, and the chromatographic medium coupled with the nano disc generates an obvious elution peak after being eluted by imidazole solution.

The eluted peak was collected and analyzed by CO differential spectroscopy, as shown in FIG. 9, from which it can be seen that: elution Peak P of nanodisk chromatography Medium₂Has obvious absorption at 450nm, and the penetration peak P of coupled nano-disc chromatographic medium group and uncoupled nano-disc chromatographic medium group₁And P₁There is no absorption.

The results of the electrophoretic analysis are shown in fig. 10 (1 is the supernatant of the liver microsome disruption solution, 2 is the membrane scaffold protein solution, 3 is the solution after the liver microsome disruption solution is combined with the nanodisk chromatography medium, 4 is the elution peak of the nanodisk chromatography medium, and 5 is the penetration peak of the unconjugated nanodisk chromatography medium): the nano disc medium elution peak has an obvious band around 5 ten thousand molecular weight, which shows that the medium can achieve the effect of purifying CYP 450. The content of CYP450 of an elution peak is 0.04nmol/mg, and the electrophoresis purity is more than 90 percent. The results show that the medium is capable of purifying CYP450 while at the same time acting as a maintenance of activity.

Example 10

This example uses the nanodisk-glutathione-dextran chromatography media prepared in example 2 for the purification of protein liver microsomal CYP450, which was performed as follows:

the nanodisk-glutathione-dextran chromatography media prepared in example 2 was packed into a column, and the procedure was the same as in example 9. When in elution, 20mM reduced glutathione is added into the loading buffer solution, and the elution peak is collected. CO differential spectroscopy and electrophoretic analysis were performed separately. The determination shows that the content of CYP450 of an elution peak is 0.02nmol/mg, and the electrophoresis purity is more than 90 percent. The results show that the medium is capable of purifying CYP450 while at the same time acting as a maintenance of activity.

Example 11

In this example, the nanodisk-amylose-konjac glucomannan chromatography medium prepared in example 3 was used for the purification of protein liver microsome CYP450, and the operation method thereof was as follows:

the nanodisc-amylose-konjac glucomannan chromatography medium prepared in example 3 was packed into a column, and the procedure was the same as in example 9. During elution, 10mM maltose was added to the loading buffer, and the elution peak was collected. CO differential spectroscopy and electrophoretic analysis were performed separately. The determination shows that the content of the CYP450 of the elution peak is 0.03nmol/mg, and the electrophoresis purity is more than 90 percent. The results show that the medium is capable of purifying CYP450 while at the same time acting as a maintenance of activity.

Example 12

This example uses the nanodisk-streptavidin variant (Strep-Tactin) -polymethacrylate chromatography medium prepared in example 4 for the purification of protein liver microsome CYP450, as follows:

the nanodisk-streptavidin variant (Strep-Tactin) -polymethacrylate chromatography medium prepared in example 4 was pillared in the same procedure as in example 9, wherein the buffer pH was adjusted to pH 11.0(25mM NaHCO)₃). When elution is carried out, 2.5mM desthiobiotin is added into the loading buffer solution, and elution peaks are collected. CO differential spectroscopy and electrophoretic analysis were performed separately. The determination shows that the content of CYP450 of the elution peak is 0.03nmol/mg, and the electrophoresis purity is>90 percent. The results show that the medium is capable of purifying CYP450 while at the same time acting as a maintenance of activity.

Example 13

This example used the nanodisk Cu-CM-ASP silica gel chromatography media prepared in example 5 for the purification of protein liver microsome CYP450, which was performed as follows:

the nanodisk Cu-CM-ASP silica gel chromatography media prepared in example 5 was loaded onto the column in the same manner as in example 9. When elution is performed, 200mM imidazole is added into loading buffer, and elution peaks are collected. CO differential spectroscopy and electrophoretic analysis were performed separately. The determination shows that the content of CYP450 of an elution peak is 0.04nmol/mg, and the electrophoresis purity is more than 90%. The results show that the medium is capable of purifying CYP450 while at the same time acting as a maintenance of activity.

Example 14

This example used nanodisk-Ni-IDA sepharose chromatography media-2 prepared in example 6 for the purification of protein liver microsome CYP450, and was performed as follows:

the nanodisk-Ni-IDA agarose chromatography media prepared in example 6 was packed into a column, using the same procedure as in example 9. And collecting elution peaks, and performing CO differential spectrum and electrophoretic analysis respectively. The determination shows that the content of CYP450 of an elution peak is 0.02nmol/mg, and the electrophoresis purity is more than 90 percent.

Example 15

This example used nanodisk-Ni-IDA sepharose chromatography media-3 prepared in example 7 for the purification of protein liver microsome CYP450, and was performed as follows:

the nanodisk-Ni-IDA agarose chromatography media prepared in example 7 was packed into a column, using the same procedure as in example 9. And collecting elution peaks, and performing CO differential spectrum and electrophoretic analysis respectively. The determination shows that the content of CYP450 of an elution peak is 0.02nmol/mg, and the electrophoresis purity is more than 80%.

Example 16

This example used nanodisk-Ni-IDA sepharose chromatography media-4 prepared in example 8 for the purification of protein liver microsome CYP450, and was performed as follows:

the nanodisk-Ni-IDA agarose chromatography media prepared in example 8 was packed into a column, using the same procedure as in example 9. And collecting elution peaks, and performing CO differential spectrum and electrophoretic analysis respectively. The determination shows that the content of CYP450 of an elution peak is 0.02nmol/mg, and the electrophoresis purity is more than 90 percent.

Comparative example 1

The comparative example adopts a conventional method to treat the liver microsome CYP450, and the operation method comprises the following steps:

membrane protein is processed by conventional method including purification and reconstitution, membrane protein CYP450 purified from liver microsomes is mixed with amphoteric surfactant CHAPS (3- [3- (cholamidopropyl) dimethylamino ] propanesulfonate inner salt) at a ratio of 1:3, and stirred at 0 deg.C overnight. Centrifuging and taking supernatant. Molecular sieve prepacked column, Superdex200(10mM I.D.. times.300 mM), was connected to the PPS protein chromatography system and equilibrated with Buffer A (20mM PB, 0.03% Chaps, pH 6). After equilibration of the column, the sample was loaded at a flow rate of 0.2 mL/min. The peaks were collected. The nanoplate was added to the collected peak and incubated for 0.5 h. The incubation was analyzed by CO differential spectroscopy. The purified indices of examples 9-16 and comparative example 1 are summarized in Table 1, and it can be seen from Table 1 that: the protein amount, the purity and the processing time limit of CYP450 purified by adopting the phospholipid nanodisk chromatography medium are far better than those of comparative example 1.

TABLE 1

Example 17

This example used the nanodisk-Ni-IDA agarose chromatography media prepared in example 1 for the purification of cell membrane proteins, as follows:

first, the cell membrane disruption solution was subjected to gel filtration chromatography as shown in FIG. 11: the chromatogram retention volume shows a plurality of peaks from 7.0-24.0mL of the broken solution, the peaks respectively correspond to membrane components with different sizes, the components are very complex, and the cell membrane protein is difficult to separate by adopting a conventional chromatographic method.

The nanodisk-Ni-IDA agarose chromatography media prepared in example 1 was packed into a column (column volume 0.5mL) and connected to a PPS protein chromatography system. After a Buffer A (20mM PB, pH7.4) is used for balancing the column, a mouse somatic cell membrane breaking solution is adopted for loading, the flow rate is 0.2mL/min, the Buffer A is continuously used for elution, finally, Buffer B (20mM PB, containing 200mM imidazole, pH7.4) is used for elution, and the extraction condition of cell membrane protein is analyzed through two electrophoresis methods, namely SDS-PAGE, Native-PAGE and Transmission Electron Microscopy (TEM).

The SDS-PAGE analysis results are shown in FIG. 12 (1 is the nanodisk prepared in example 1, 2 is the cell membrane disruption solution, and 3 is the elution peak), and it is evident from the figure that: the cell membrane disruption solution contains a plurality of strips, the components are very complex, and after the cell membrane disruption solution is treated by a nano-disc chromatography medium, an elution peak consists of a nano-disc and membrane protein, which indicates that the cell membrane protein is effectively purified.

The results of Native-PAGE analysis are shown in FIG. 13 (1 is membrane scaffold protein, 2,3, 4, and 7 are nanodiscs prepared in example 1, 5 is cell membrane disruption solution, and 6 is elution peak), and it can be seen from the figure that: the cell membrane disruption solution contains a plurality of strips, the components are very complex, and after the cell membrane disruption solution is treated by a nano-disc chromatography medium, an elution peak consists of a nano-disc and membrane protein, which indicates that the cell membrane protein is effectively purified.

The TEM results are shown in FIG. 14 (a is the cell membrane disruption solution, b is the eluate after passing through the chromatography medium), and it is clear from the figure that: the cell membrane disruption solution contains a large amount of irregularities, which are presumed to be various complex components on the cell membrane, and after the cell membrane disruption solution is treated by a nano-disc chromatography medium, the components are single, and cell membrane protein is combined on the nano-disc to realize purification.

The applicant states that the present invention is illustrated by the above examples to the phospholipid nanodisk chromatography media of the present invention, and the preparation method and application thereof, but the present invention is not limited to the above examples, i.e., it is not meant that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

SEQUENCE LISTING

<110> institute of Process engineering of Chinese academy of sciences

<120> phospholipid nanodisk chromatography medium and preparation method and application thereof

<130>2019

<160>1

<170>PatentIn version 3.3

<210>1

<211>211

<212>PRT

<213> artificially synthesized sequence

<400>1

Gly His His His His His His Ile GluGly Arg Leu Lys Leu Leu Asp

1 5 10 15

Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu

20 25 30

Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu

35 40 45

Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys

50 55 60

Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met

65 70 75 80

Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu

85 90 95

Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu

100 105 110

Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg

115 120 125

Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala

130 135 140

Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr

145 150 155 160

His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys

165 170 175

Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser

180 185 190

Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu

195 200 205

Asn Thr Gln

210

Claims

1. A phospholipid nanodisk chromatography media comprising a charged phospholipid nanodisk and an affinity matrix; the charge type phospholipid nanodiscs comprise phospholipid and membrane scaffold protein with affinity labels; the charge type phospholipid nanodisk is connected with an affinity ligand in an affinity matrix through an affinity label;

the diameter of the charge type phospholipid nanodisk is 1-100 nm;

the density of the charged phospholipid nanodiscs is 0.1-25mg per mL of affinity matrix;

the Zeta potential of the charge type phospholipid nanodisk is (-50) eV under the condition of pH 7.4;

the particle size of the affinity matrix is 10-100 μm;

the affinity tag comprises any one or a combination of at least two of histidine, maltose binding protein, streptococcus or glutathione mercaptotransferase;

the affinity matrix comprises any one or the combination of at least two of agarose, dextran, cellulose, konjac glucomannan, polymethacrylate, polystyrene, polyacrylamide, silica gel or hydroxyapatite;

the affinity ligand comprises any one of metal ions, glutathione, amylose or a streptavidin variant or a combination of at least two of the foregoing.

2. The phospholipid nanodisk chromatography media of claim 1, wherein the charged phospholipid nanodisk has a diameter of 5-50 nm.

3. The phospholipid nanodisk chromatography media of claim 1, wherein the charged phospholipid nanodisk has a diameter of 10-25 nm.

4. The phospholipid nanodisk chromatography media of claim 1, wherein the charged phospholipid nanodisk has a density of 1-10mg per mL affinity matrix.

5. The phospholipid nanodisk chromatography medium of claim 1, wherein the charged phospholipid nanodisk has a Zeta potential at ph7.4 of preferably (-25) eV.

6. The phospholipid nanodisc chromatography medium of claim 1, wherein the phospholipid comprises any one of or a combination of at least two of phosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, phosphatidylserine, trimethyl-2, 3-dioleoyloxypropylammonium bromide or trimethyl-2, 3-dioleoyloxypropylammonium chloride.

7. The phospholipid nanodisk chromatography medium of claim 1, wherein the metal ion comprises Ni²⁺、Cu²⁺、Co²⁺、Fe²⁺Or Zn²⁺Any one or a combination of at least two of them.

8. The phospholipid nanodisk chromatography medium of claim 1, wherein the metal ion is associated with the affinity matrix by chelation with a chelating agent.

9. The phospholipid nanodisk chromatography media of claim 8, wherein the chelating agent comprises any one or a combination of at least two of iminodiacetic acid, tricarboxymethylethylenediamine, nitrilotriacetic acid, carboxymethylaspartic acid, tetraethylenepentamine, or carboxymethyl-alpha, beta-diaminosuccinic acid.

10. The method of preparing the phospholipid nanodisk chromatography medium of any one of claims 1-9, wherein the method comprises the steps of:

11. The method for preparing the phospholipid nanodisk chromatography medium of claim 10, wherein the method for preparing the phospholipid membrane assembling solution of step (1) comprises: and (2) preparing a phospholipid membrane by using a nitrogen blow-drying membrane preparation method or a rotary evaporation membrane preparation method for the phospholipid solution, and dissolving the phospholipid membrane by using the assembly liquid to obtain the phospholipid membrane assembly liquid.

12. The method of claim 11, wherein the assembly liquid is a buffer solution with a pH of 7.0-7.4.

13. The method of claim 11, wherein the assembly liquid comprises phosphate buffer, Na₂HPO₄-citric acid buffer, KH₂PO₄-NaOH buffer, barbiturate sodium-hydrochloric acid buffer or Tris-buffer.

14. The method of claim 11, wherein the assembly solution contains sodium chloride at a concentration of 0.02-0.1M.

15. The method of claim 11, wherein the composition comprises sodium cholate at a concentration of 0.01-0.5M.

16. The method for preparing the phospholipid nanodisk chromatography medium of claim 10, wherein the mixing time of the phospholipid membrane assembly liquid and the affinity-tagged membrane scaffold protein of step (1) is 0.5-12 h.

17. The method for preparing the phospholipid nanodisk chromatography medium of claim 10, wherein the mixing time of the phospholipid membrane assembly liquid and the affinity-tagged membrane scaffold protein of step (1) is 1-8 h.

18. The method for preparing the phospholipid nanodisk chromatography medium of claim 10, wherein the mixing time of the phospholipid membrane assembly liquid and the affinity-tagged membrane scaffold protein of step (1) is 2-4 h.

19. The method for preparing the phospholipid nanodisk chromatography medium of claim 10, wherein the mixing temperature of the phospholipid membrane assembly liquid and the membrane scaffold protein with affinity tag in step (1) is 4-60 ℃.

20. The method for preparing the phospholipid nanodisk chromatography medium of claim 10, wherein the mixing temperature of the phospholipid membrane assembly liquid and the membrane scaffold protein with affinity tag in step (1) is 20-60 ℃.

21. The method for preparing the phospholipid nanodisk chromatography medium of claim 10, wherein the mixing temperature of the phospholipid membrane assembly liquid and the membrane scaffold protein with affinity tag in step (1) is 30-50 ℃.

22. The method for preparing the phospholipid nanodisk chromatography medium of claim 10, wherein the molar ratio of the membrane scaffold protein to the phospholipid in the phospholipid membrane assembly liquid of step (1) is 1 (10-100).

23. The method for preparing the phospholipid nanodisk chromatography medium of claim 10, wherein the molar ratio of the membrane scaffold protein to the phospholipid in the phospholipid membrane assembly liquid of step (1) is 1 (20-60).

24. The method for preparing a phospholipid nanodisk chromatography medium as claimed in claim 10, wherein the coupling in step (2) is via metal chelation or covalent bonding.

25. The method for preparing the phospholipid nanodisk chromatography medium of claim 10, wherein the coupling reaction time of step (2) is 10-30 h.

26. The method for preparing a phospholipid nanodisk chromatography medium as claimed in claim 10, wherein the temperature of the coupling reaction in step (2) is 20-40 ℃.

27. The method of claim 10, wherein the charged phospholipid nanodisk is dialyzed to equilibrium in the coupling buffer prior to the coupling reaction in step (2).

28. The method for preparing the phospholipid nanodisk chromatography medium of claim 10, wherein the method specifically comprises the steps of:

29. Use of the phospholipid nanodisk chromatography medium of any one of claims 1-9 for membrane protein purification and reconstitution.

30. The application of claim 29, wherein the method of applying is: and sequentially carrying out column packing, balancing, protein loading, leaching, eluting and cleaning on the phospholipid nano-disc chromatography medium to realize the purification and reconstruction of the membrane protein.