A method for preparing high-purity human Orai2 membrane protein based on a mammalian cell expression system

Abstract

The application provides a high-purity human Orai2 membrane protein preparation method based on a mammalian cell expression system. The method obtains a recombinant expression vector containing a human Orai2 gene and an affinity tag sequence; the recombinant expression vector is transfected into a host cell line for expression; a membrane dissolving buffer containing dodecyl-beta-D-maltopyranoside and cholesteryl hemisuccinate is used for lysis extraction of the host cell; after centrifugation to obtain supernatant, the supernatant is sequentially separated and purified by using an affinity chromatography medium and a gel exclusion chromatography medium, and high-purity human Orai2 protein is obtained. The application constructs a thermodynamic equilibrium micellar microenvironment, solves the conformational inactivation problem in the extraction process of multiple transmembrane proteins, and realizes efficient and high-purity enrichment of human Orai2 protein.

Description

Technical Field

[0001] This application relates to the fields of biotechnology and protein engineering technology, and more specifically, to a method for preparing high-purity human Orai2 membrane protein based on a mammalian cell expression system. Background Technology

[0002] In modern molecular biology and drug target research, human Orai2 protein, as a core calcium ion channel protein mediating the storage and manipulation of calcium influx on the cell membrane, relies heavily on the preparation of high-purity, highly stable soluble proteins in vitro for structural analysis and functional verification. However, existing technologies face a fundamental technical contradiction when processing such transmembrane proteins: to achieve efficient extraction, strong detergents are typically introduced to disrupt the hydrophobic field of the lipid bilayer structure; however, the introduction of strong detergents inevitably strips away the boundary lipids that maintain the stability of the transmembrane helical bundles, leading to irreversible conformational collapse and aggregation inactivation of the target protein in an aqueous environment. This theoretical ceiling between extraction efficiency and conformational fidelity means that traditional extraction processes can only obtain products with extremely low expression levels, mostly in a denatured state, severely hindering pharmacological studies of the physiological mechanisms of Orai2 protein in diseases of nerve and muscle smooth muscle. Summary of the Invention

[0003] Firstly, this application provides a method for preparing high-purity human Orai2 membrane protein based on a mammalian cell expression system. This method involves constructing a recombinant vector containing an affinity tag, transiently transfecting it into a eukaryotic expression system, innovatively employing a composite microenvironment containing dodecyl-β-D-maltodextrin and cholesterol succinate for targeted membrane dissolution, and coupling affinity chromatography with gel size exclusion chromatography for two-dimensional chromatographic purification. This technical solution not only solves the fundamental problem of low membrane protein yield but, more importantly, reconstructs the thermodynamic equilibrium system after the transmembrane protein separates from the natural lipid bilayer. Through the synergistic effect of a surfactant and cholesterol derivative with a specific chemical ratio, this application constructs an artificial biomimetic lipid raft in vitro that perfectly adapts to the hydrophobic interface of the Orai2 protein. This converts the energy dissipation during the separation process into a stabilizing force for conformational maintenance, thereby overcoming the conformational inactivation dilemma caused by traditional detergents and endowing the purified protein with extremely high structural uniformity and biological activity at the molecular level.

[0004] Secondly, this application provides a system for purifying human Orai2 protein, as described in claim 9, characterized by comprising a vector construction module, a cell transfection and expression module, a transmembrane protein lysis module, an ultracentrifugation module, an affinity chromatography purification module, and a gel size exclusion chromatography module. This system engineered and modularized the reconstruction process of the aforementioned thermodynamic equilibrium system, ensuring high-fidelity transfer of the entire extraction and purification process in physical and chemical environments, and achieving full-process automation and scalable reproducibility.

[0005] Thirdly, this application provides a computer-readable storage medium, as claimed in claim 10, characterized in that it stores a computer program for executing the aforementioned process control steps.

[0006] Beneficial Effects: The method and system for purifying human Orai2 protein established in this application, through precise control of the lysosomal microenvironment and two-stage fluid dynamics chromatographic sieving, completely breaks the involution paradox of quantitative and qualitative transmembrane protein expression in vitro. It not only significantly improves the recovery rate and absolute purity of the target membrane protein, but also maintains its natural three-dimensional conformation for forming functional polymers, providing a solid material basis for high-throughput screening and precise crystallographic structure determination of drugs targeting the Orai2 channel. Attached Figure Description

[0007] Figure 1 A schematic flowchart illustrating a method for preparing high-purity human Orai2 membrane protein based on a mammalian cell expression system, provided for embodiments of this application; Figure 2 This is a schematic diagram of the recombinant plasmid pcDNA-Flag-Orai2 involved in the embodiments of this application; Figure 3 This is a schematic diagram of SDS-PAGE verification of the affinity chromatography process involved in the embodiments of this application; Figure 4 This is a schematic diagram of Western blot verification of the affinity chromatography process involved in the embodiments of this application; Figure 5 This is a schematic diagram of SDS-PAGE verification of the gel size exclusion chromatography process involved in the embodiments of this application; Figure 6 This is a schematic diagram of Western blot verification of the gel size exclusion chromatography process involved in the embodiments of this application; Figure 7 This is a UV absorption peak diagram of the gel size exclusion chromatography involved in the embodiments of this application. Detailed Implementation

[0008] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0009] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0010] This embodiment provides a method and system for preparing high-purity human Orai2 membrane protein based on a mammalian cell expression system. In one specific implementation, this method utilizes a composite membrane solution buffer containing dodecyl-β-D-maltodextrin and cholesterol succinate, combined with a two-stage chromatography purification mechanism, to obtain human Orai2 membrane protein with high native conformation fidelity. This method solves the technical problems of conformational inactivation and low yield of multi-transmembrane proteins during extraction in existing technologies, achieving the beneficial effect of obtaining high-purity and biologically active Orai2 protein stably and in large quantities in vitro.

[0011] The technical solution provided in this application will be described in detail below, with specific implementation steps and experimental systems.

[0012] S100. Obtain a recombinant expression vector containing the human Orai2 gene and an affinity tag sequence.

[0013] The vector construction module within the system performs the preparation of recombinant DNA fragments and the fusion of the vector backbone. The module obtains the sequence-optimized human Orai2 gene and seamlessly splices an affinity tag sequence for subsequent affinity capture into the gene's reading frame, ultimately inserting it into a eukaryotic expression vector backbone to construct a circular plasmid molecule for transient transfection.

[0014] S110. Design forward and reverse amplification primers based on the human Orai2 gene, and integrate a first restriction endonuclease site sequence and a nucleotide sequence corresponding to the affinity tag sequence into the forward amplification primer, and integrate a second restriction endonuclease site sequence into the reverse amplification primer.

[0015] The vector construction module employs a primer design algorithm to perform thermodynamic analysis on the sequences at both ends of the human Orai2 gene to generate primer pairs with specific annealing temperatures. To achieve efficient binding without disrupting the transmembrane topology of the target protein, an affinity tag is designed and added to the N-terminus. The vector construction module sequentially tandem a first restriction endonuclease site, an affinity tag coding sequence, and a homologous binding region corresponding to the Orai2 gene start codon at the 5' end of the forward amplification primer. Simultaneously, a second restriction endonuclease site and a complementary region corresponding to the Orai2 gene stop codon sequence are tandemly tandem at the 5' end of the reverse amplification primer. The restriction endonuclease sites are selected only from enzyme sequences that do not contain the same recognition site within the Orai2 gene.

[0016] Exemplarily, the vector construction module performs a primer design process using the human Orai2 gene as a template. The designed forward amplification primer sequence integrates a BamHI restriction site (…). ) and the encoded Flag tag ( nucleotide sequence of ) The complete sequence of the forward amplification primers is as follows: The reverse amplification primers integrate the XhoI restriction site ( ), its sequence is: The calculated annealing temperatures of the two primer segments. The temperatures were set at 65.2 degrees Celsius and 64.8 degrees Celsius, respectively, to ensure thermodynamic synchronization during the polymerase chain reaction cycle.

[0017] S120. The human Orai2 gene is amplified by polymerase chain reaction using the forward amplification primer and the reverse amplification primer to obtain the target gene fragment.

[0018] The vector construction module mixes the synthesized primer pairs, template DNA, deoxyribonucleoside triphosphate substrate, and high-fidelity DNA polymerase in a buffer system, performing periodic denaturation, annealing, and extension operations. The high-fidelity DNA polymerase utilizes its 3'-5' exonuclease activity to proofread mismatched bases, ensuring the absolute accuracy of the amplified sequence. The extension time of the polymerase chain reaction is set to the quotient of the target gene length and the synthesis rate of the high-fidelity polymerase.

[0019] For example, the vector construction module prepares a PCR reaction system with a total volume of 25 μL, containing 1.0 μL of forward primer at a concentration of 10 μmol / L, 1.0 μL of reverse primer at a concentration of 10 μmol / L, 0.5 μL of template DNA at a concentration of 100 ng / μL, 0.5 μL of dNTP mixture at a concentration of 10 mmol / L, 0.5 μL of magnesium sulfate at a concentration of 0.1 mol / L, 2.5 μL of 10×Phusion reaction buffer, 0.6 μL of Phusion high-fidelity DNA polymerase, and 18.4 μL of sterile double-distilled water. The reaction program is defined as follows: pre-denaturation at 95°C for 5 minutes; followed by 30 cycles, each cycle consisting of denaturation at 95°C for 45 seconds, annealing (here set to 60°C) for 45 seconds, and extension at 72°C for 1 minute and 15 seconds (based on...). kilobase pair length and per minute (The amplification rate of 1000 base pairs was calculated); a final extension was performed at 72 degrees Celsius for 3 minutes and 15 seconds. After amplification, the product appeared on a 1% agarose gel. A bright, specific band at kb.

[0020] S130. The target gene fragment and an expression vector backbone are subjected to double restriction enzyme digestion using a first restriction endonuclease and a second restriction endonuclease, respectively.

[0021] The vector construction module exposes the purified target gene fragment and the empty expression plasmid to the catalytic environments of the first restriction endonuclease and the second restriction endonuclease, respectively. The endonuclease recognizes specific short nucleotide sequences and hydrolyzes phosphodiester bonds, generating complementary sticky ends at both ends of the target gene fragment and at corresponding positions on the expression vector backbone, providing a molecular interface with minimized steric hindrance for subsequent ligation conformations.

[0022] Exemplarily, the vector construction module is used to prepare a double enzyme digestion reaction system. Take 1 μg of the target gene recovery product or pcDNA4.0 vector plasmid, add 1 μL of BamHI enzyme, 1 μL of XhoI enzyme, 2 μL of 10×Fast Digest Green Buffer, and add sterile double-distilled water to a total volume of 20 μL. Incubate the system at 37°C for 1 hour. After enzyme digestion, the target gene fragment will expose a 4-base fragment. and The pcDNA4.0 vector was linearized with sticky ends, exposing complementary end sequences. High-purity linearized fragments were obtained by gel recovery.

[0023] S140. The target gene fragment that has undergone the double enzyme digestion process is ligated to the expression vector backbone to obtain the recombinant expression vector.

[0024] The vector construction module, catalyzed by T4 DNA ligase, covalently condenses the target gene fragment with complementary sticky ends to the expression vector backbone via phosphodiester bonds. In the ligation system, the molar ratio of the target fragment to the vector is strictly controlled within a specific range to maximize the probability of bimolecular collisions and suppress single-molecule self-ligation. The ligation product is then introduced into competent *E. coli* for cloning amplification and single-clone screening. Finally, after sequence verification, a high concentration of the recombinant expression vector *pcDNA-Flag-Orai2* is extracted.

[0025] For example, the carrier construction module performs a linkage reaction. Based on the amount of substance... The proportion of the target gene fragment volume is used to calculate the target gene fragment volume. With carrier volume In a connection system with a total volume of 10 microliters, add Target gene, The vector, 1 μL of T4 DNA ligase, and 1 μL of T4 DNA ligase buffer were used. After incubation at room temperature for 30 minutes, the entire system was added to Trans1-T1 competent cells, and transformation was completed by heat shock at 42°C for 1.5 minutes (then 1 mL of LB was added, and the cells were incubated at 37°C, 220 rpm for 1 hour, centrifuged at 3600 rpm for 2 minutes, the supernatant was discarded, and the cells were resuspended in 100 μL of fresh culture medium before plating). Resistant monoclonal antibodies were then screened on LB agar plates coated with 50 μg / mL ampicillin. The purity ratio of the extracted plasmid was measured. for (Dimensionless) This proves that the plasmid purity meets the requirements.

[0026] S200. The recombinant expression vector is transfected into a host cell line for expression to obtain host cells expressing the human Orai2 protein.

[0027] The cell transfection and expression module mediates the transperfusion of exogenous DNA across the cell membrane and nuclear membrane. This step uses a cationic polymer as a carrier to concentrate plasmid DNA into nanoparticles through electrostatic adsorption, which are then absorbed into the cytoplasm via endocytosis.

[0028] S210. The recombinant expression vector is mixed with polyethyleneimine at a preset mass ratio to obtain a transfection complex with a preset volume.

[0029] The cell transfection and expression module is formulated with a transfection solution containing the recombinant expression vector and polyethyleneimine. The high density of protonated secondary and tertiary amine groups in polyethyleneimine imparts a strong positive charge. When mixed with negatively charged phosphate backbone DNA molecules, a spontaneous polymeric complexation reaction occurs at a specific mass ratio, compressing the DNA molecule volume and forming electrically neutral or slightly positively charged complex particles. This positively charged shell facilitates the adsorption of the complex to the negatively charged cell membrane surface.

[0030] For example, for a volume of 100 ml and a cell density of Each milliliter of HEK293F suspension cell line, the cell transfection and expression module calculates the required substances. (Per...) The ratio of 1 microgram of plasmid to 1 cell requires a plasmid mass of 120 micrograms. Since the plasmid stock solution concentration is 500 nanograms per microliter, a plasmid volume of 240 microliters is required. The mass ratio of plasmid to polyethyleneimine (PEI) is set as follows: This requires 360 micrograms of PEI, corresponding to a volume of 360 microliters (PEI stock solution concentration set at 1 mg / mL). The module is described in volume ratio... Both were diluted separately in SMM 293-TII medium, i.e., 3.36 mL of medium was used to dilute the plasmid and 5.04 mL of medium was used to dilute the PEI. The two were then mixed uniformly and incubated at room temperature for 20 minutes to form transfection complex particles with a diameter in the range of 100 to 200 nm.

[0031] S220. The transfection complex is added to a human embryonic kidney cell derivative in suspension culture for co-culture.

[0032] The cell transfection and expression module adds the matured transfection complex to a suspension of host cells in the logarithmic growth phase. Under constant temperature and rotation speed in a shaker, the host cells take the complex into endosomes via endocytosis. The proton sponge effect of polyethyleneimine causes an increase in osmotic pressure in the endosomes, eventually leading to rupture and release of the plasmid into the cytoplasm. The plasmid then enters the nucleus and, utilizing the host's transcription and translation mechanisms, synthesizes a large amount of the human Orai2 protein on the endoplasmic reticulum membrane, ultimately embedding it into the cell membrane surface. After a certain expression cycle, host cell bodies enriched with the target protein are collected by centrifugation.

[0033] Exemplarily, the cell transfection and expression module adds the prepared complex solution dropwise to 100 mL of HEK293F cell suspension and incubates in a shaker at 37°C and 120 rpm. 24 hours post-transfection, SMS 293-SUPI supplement is added at a ratio of 35 mL per liter of culture medium to maintain nutrition. 48 hours post-transfection, the cell suspension is transferred to centrifuge tubes and subjected to… Centrifuge for 10 minutes and discard the supernatant culture medium. Resuspend the precipitated cell pellet in 1×TBS solution, wash again, and centrifuge to collect the purified cell pellet containing Orai2 membrane protein. Transfer the pellet to a -80°C freezer for storage or proceed directly to the next lysis step.

[0034] S300. The host cells are lysed using a membrane lysis buffer containing a first surfactant and a second surfactant to obtain a cell lysate containing the human Orai2 protein, wherein the first surfactant is dodecyl-β-D-maltopyranoside and the second surfactant is cholesterol succinate.

[0035] The transmembrane protein dissolution module performs physicochemical deconstruction of the host cell membrane and soluble extraction of transmembrane proteins. Because the Orai2 protein contains four transmembrane helices, its hydrophobic region is tightly bound by a lipid bilayer. Simple mechanical disruption cannot dissociate it from the membrane lipids. An amphiphilic surfactant molecule must be introduced to disrupt the lipid bilayer while simultaneously replacing natural phospholipids to encapsulate the hydrophobic transmembrane region of Orai2, forming a water-soluble protein-detergent complex.

[0036] S310. Construct the film-dissolving buffer based on a preset buffer matrix, wherein the buffer matrix contains tris(hydroxymethyl)aminomethane hydrochloride at a concentration of 20 mmol / L and sodium chloride at a concentration of 200 mmol / L.

[0037] The transmembrane protein lysis module is first prepared with a basic isotonic and acid-base buffer system. Tris(hydroxymethyl)aminomethane hydrochloride acts as a proton acceptor and donor pair, maintaining the solution pH within a physiologically neutral-to-alkaline range with minimal fluctuations, thus protecting the ionic and hydrogen bond networks within the protein. Sodium chloride provides the necessary ionic strength, shielding the protein surface from non-specific electrostatic interactions and preventing the flocculation and precipitation of large amounts of nucleic acids and proteins during lysis.

[0038] For example, the transmembrane protein dissolution module is prepared using basic Buffer A. 2.4228 g of Trizma base powder (equivalent to 121.14 g / mol) and 11.688 g of sodium chloride powder (equivalent to 58.44 g / mol) are weighed and dissolved in approximately 800 mL of sterile double-distilled water. Subsequently, dilute hydrochloric acid with a molar concentration of 1 mol / L is slowly added dropwise, and the pH of the solution is precisely adjusted to the specified value using a pH meter for real-time monitoring. Finally, adjust the volume to the required total volume. The resulting Buffer A has a Tris buffer capacity of 20 mmol / L and an ionic strength of 200 mmol / L.

[0039] S320. Add the dodecyl-β-D-maltopyranoside to the buffer matrix to achieve a final concentration of one percent, and add the succinate cholesterol ester to achieve a final concentration of one-thousandth.

[0040] The transmembrane protein dissolution module constructs the detergent formulation in this step. The nonionic detergent, dodecyl-β-D-maltopyranoside (DDM), possesses a long dodecyl hydrophobic tail and a large maltose hydrophilic head. Its gentle detergency effectively dissolves polyester membranes without easily disrupting the internal hydrogen bonds of membrane proteins. To compensate for the shortcomings of detergent micelles in lateral pressure and rigidity, the transmembrane protein dissolution module also introduces cholesterol succinate (CHS). CHS molecules are embedded in the hydrophobic core and hydrophilic shell interface layer of the DDM micelles, mimicking the cholesterol environment in natural membranes and significantly improving the structural rigidity and thermal stability of the protein-detergent complex.

[0041] For example, the transmembrane protein dissolution module is formulated with a mass-volume fraction DDM and CHS 10× high-concentration stock solution. Weigh 5 g of DDM powder into a 50 mL centrifuge tube, add 10 mL of 1 mol / L Tris (pH 8.0) buffer, and then add approximately 35 mL of double-distilled water to completely dissolve it. Separately weigh 0.5 g of CHS powder and add it to the above solution. Use an ultrasonic cell disruptor to apply intermittent ultrasonic energy until the powder particles are completely dispersed and dissolved in the microphase. Make up to 50 mL and filter through a 0.22 μm filter membrane. In actual membrane dissolution operations, use this 10× stock solution according to the volume ratio. Diluted into Buffer A, which resuspends the cells, to lock the final detergent concentration in the lysis system to [value missing]. DDM and CHS (volume fraction).

[0042] S330. The host cells are suspended in the membrane lysis buffer and incubated under a preset low temperature condition for a first preset time span to obtain the cell lysate.

[0043] The transmembrane protein dissolution module mixes a buffer system containing a high concentration of detergent with the cell clusters. Under the dual effects of mechanical resuspension and the physicochemical penetration of the detergent, the cell membrane undergoes a phase transition, breaking down from a lamellar phase and reorganizing into a mixed micelle phase. Nucleic acids and organelles within the cell are released, while the Orai2 protein on the cell membrane is encapsulated by DDM / CHS mixed micelles, completing the transfer from the solid phase to the liquid phase. The preset low-temperature conditions reduce the catalytic activity of endogenous proteases and the kinetic rate of protein thermal denaturation.

[0044] For example, the transmembrane protein lysis module takes the cell pellet frozen at -80 degrees Celsius in step S220. It then adds a pre-cooled solution containing... DDM and A mixed membrane lysis buffer containing CHS and a 1×cocktail broad-spectrum protease inhibitor was used. The suspension was repeatedly pipetted until no visible cell clots remained. The suspension was then placed on a rotary shaker in a 4°C room and incubated slowly at 30 rpm for the specified incubation period. This process ensures that transmembrane proteins have sufficient thermodynamic time to overcome the dissolution energy barrier, generating a homogeneous suspension of the cell lysate.

[0045] S400. The cell lysate is centrifuged to separate insoluble matter and obtain a supernatant containing the human Orai2 protein.

[0046] The ultracentrifugation module performs density gradient and mass exclusion sedimentation operations. After chemiluminescence, the lysis system exhibits a multiphase suspension mixture containing water-soluble protein micelles, incompletely disintegrated cytoskeleton networks, insoluble polynucleotide clusters, and unsolvated lipid raft microregions. To prevent column bed pore blockage and improve the phase partition coefficient during subsequent chromatographic purification, extremely high centrifugal forces must be applied to force high sedimentation coefficient non-target macromolecular particles to overcome Brownian motion and undergo forced precipitation.

[0047] S410. Place the cell lysate in a centrifuge and run it for a second preset time span at an ambient temperature of 4 degrees Celsius with a relative centrifugal force of 120,000 times the acceleration due to gravity.

[0048] The ultracentrifugation module evenly injects the lysate containing a large amount of particulate suspension into the ultracentrifuge tube and precisely balances it to prevent dynamic imbalance during high-speed rotor rotation. As the rotational speed increases exponentially along the time axis, the discrete phase particles in the mixture are subjected to the outward radial vector of the centrifugal field. According to the sedimentation velocity formula, the sedimentation rate of a particle is proportional to the square of its equivalent radius and the density difference between it and the medium. The ultra-high field strength provided by the acceleration of gravity 120,000 times is sufficient to cause large, or much denser, unsolvated membrane fragments and nucleoprotein complexes to rapidly migrate and deposit towards the bottom of the tube.

[0049] For example, the ultracentrifugation module dispenses 10 mL of the incubated cell lysis buffer (for example, 10 mL of cell lysis buffer is reasonable for 100 mL of cells) into polycarbonate ultracentrifuge tubes. The centrifuge tubes are then placed in a Ti-70 titanium alloy rotor pre-cooled to 4 degrees Celsius and loaded into an ultracentrifuge. Centrifugation begins by evacuating to below 300 micrometers of mercury and setting the relative centrifugal force. After accelerating to the set speed, maintain stable operation for the specified time span. Minutes. Under this extreme centrifugal field, even tiny organelle fragments with a sedimentation coefficient of tens of Svedberg units will be compacted to the bottom of the tube.

[0050] S420. Separate and extract the liquid phase component above the cell debris precipitate at the bottom, and use it as the supernatant.

[0051] After the ultracentrifugation module slows down and stops, it immediately and smoothly removes the centrifuge tubes from the rotor, avoiding resuspension of the precipitate layer caused by liquid phase oscillation. The clear liquid phase at the top is enriched with the human Orai2 protein complex, which has been successfully encapsulated and completely dissolved by detergent micelles, as well as other water-soluble host proteins. The liquid phase components are carefully aspirated and transferred to a new sterile container.

[0052] For example, the ultracentrifugation module uses a long-needle syringe to slowly aspirate from approximately 0.5 cm below the liquid surface in the centrifuge tube, maintaining relative stillness between the tube wall and the needle throughout the process. The resulting pale yellow, clear liquid is the supernatant containing the target protein, with a volume of approximately 9 mL. The tightly compacted, translucent and grayish-white gelatinous precipitate at the bottom of the tube (mainly unly lysed membrane material and nucleic acid-protein complexes) is discarded.

[0053] S500. The supernatant is specifically bound and eluted using an affinity chromatography medium to obtain the first-stage purified protein solution.

[0054] The affinity chromatography purification module performs affinity adsorption and desorption operations based on molecular recognition. This module utilizes the Flag affinity tag located at the N-terminus of the human Orai2 protein (sequence: The antigen-antibody interaction with the immobilized anti-Flag monoclonal antibody exhibits spatial complementarity and non-covalent binding properties. In a highly complex host protein background pool, this highly specific antigen-antibody interaction enriches extremely low concentrations of the target protein on the solid-phase carrier surface, achieving crude purification of the target molecule.

[0055] S510. The magnetic microspheres containing the monoclonal antibody corresponding to the affinity tag sequence are equilibrated using an equilibration buffer.

[0056] The affinity chromatography purification module is formulated with an affinity chromatography washing and equilibration system (Buffer B). This system needs to maintain not only the matrix buffering capacity and ionic strength, but also a specific detergent concentration. If the detergent is removed, the Orai2 protein will immediately expose its hydrophobic transmembrane region and undergo irreversible precipitation; if the detergent concentration is too high, it will disrupt the antibody conformation or interfere with antigen-antibody binding. The detergent concentration in the equilibration buffer is precisely adjusted to slightly above its critical micelle concentration (CMC) to achieve a dynamic balance between maintaining the dissolved state and ensuring affinity binding titer.

[0057] For example, the affinity chromatography purification module is prepared with Buffer B. Based on Buffer A (20 mmol / L Tris-HCl, 200 mmol / L NaCl, pH 7.5), trace amounts of DDM and CHS are added respectively, to ensure the final concentration is strictly controlled. DDM and CHS (volume fraction). Subsequently, 100 μL of commercial anti-FLAG magnetic beads (FLAG-beads) were measured and washed with 5 column volumes of pure water to remove the ethanol preservative in the storage solution. Then, 10 column volumes of the aforementioned Buffer B were added for three resuspension centrifugation washes to completely transition the ligand microenvironment on the bead surface to the buffer state containing an appropriate amount of detergent.

[0058] S520. The supernatant is mixed with the equilibrated magnetic microspheres and incubated in a horizontal rotation state for a third preset time span to obtain a magnetic microsphere complex bound to the human Orai2 protein.

[0059] The affinity chromatography purification module directly pours the filtered, sterilized, highly clarified supernatant into a reactor containing equilibrium magnetic beads. To ensure sufficient spatial freedom for the antibody ligands to capture large protein micelle complexes with extremely low diffusion coefficients, the reactor is placed on a gently rotating three-dimensional pedestal. During continuous liquid-phase mixing, Orai2 micelles carrying the Flag sequence follow their binding constants. The antibody continuously adheres to the surface of the magnetic bead into the active groove until the binding and dissociation reach thermodynamic dynamic equilibrium.

[0060] For example, the affinity chromatography purification module uses a 0.45-micron pore size filter membrane to perform dead-end filtration on 9 mL of centrifuged supernatant to thoroughly remove large nucleic acid aggregates precipitated due to temperature fluctuations. The filtered supernatant is then added to a 15 mL suspension culture tube containing 100 μL of FLAG-beads. The tube is sealed and placed on a horizontal incubator in a 4°C freezer at 15 rpm. The system is continuously incubated under this three-dimensional convection state for a specified time span. Within hours, the FLAG terminus of the Orai2 protein molecule in the supernatant is firmly held by the antibody, forming an immobilized bead-antibody-antigen complex.

[0061] S530. The magnetic microsphere complex is eluted with a washing buffer to remove impurities and proteins.

[0062] The affinity chromatography purification module precipitates and separates the magnetic microsphere complex by low-speed centrifugation, discarding the flow-through fluid containing a large amount of unbound host proteins. To remove background protein clusters weakly bound to the microsphere surface by van der Waals forces or nonspecific electrostatic interactions, the module repeatedly flushes the microspheres with a large amount of washing solution with the same composition as the equilibration buffer.

[0063] For example, the affinity chromatography purification module centrifuges the culture tube containing magnetic beads at low speed at approximately 1000g for 2 minutes, and aspirates the supernatant as the breakthrough fraction. The remaining liquid containing magnetic beads in the culture tube is collected into a 1.5ml EP tube, washed with 1ml Buffer B, centrifuged at low speed, and the supernatant is discarded. This process is repeated several times. Remove the culture tube and add 10 ml (equivalent to 10 columns). Volume) The aforementioned Buffer B ( DDM, (CHS) Complete resuspension of the magnetic bead complex. Place it back in the flip position. After washing for 5 minutes on the instrument, sedimentation is carried out using a magnetic rack. This washing cycle is repeated 5 times. A high volume ratio of Buffer B continuously dilutes the free impurity phase, gradually expelling endogenous ribosomes, organelle skeletal proteins, etc., that have not specifically bound to the FLAG antibody from the magnetic bead pores and surface.

[0064] S540. The magnetic microsphere complex is competitively eluted using an elution buffer containing a high concentration of affinity-tagged peptides to obtain the first-stage purified protein solution.

[0065] The affinity chromatography purification module performs target molecule desorption based on a similar ligand competitive substitution mechanism. The elution buffer contains a high molar concentration of free affinity peptides (such as Flag peptides). Based on Le Chatelier's principle and the law of mass action, the exponentially excessive free peptide molecules in the system possess extremely high diffusion penetration, continuously competing for antibody binding sites. As a large amount of free peptides are incorporated into the antibody groove structure, the originally anchored Orai2 macromolecular complex is forced to dissociate from the solid phase surface and re-release into the liquid phase.

[0066] For example, after thoroughly washing away impurities and discarding the supernatant, the affinity chromatography purification module adds 500 μL of [a specific substance] to the tube. Elution buffer for FLAG peptide (its base component remains Buffer B). Free FLAG peptide (sequence) was eluent at a final concentration of 150 μg / mL at an extremely high concentration. This creates an overwhelming molar advantage competition for the binding site. The suspension tubes were then placed on a 4°C horizontal inverter and subjected to slow overnight shaking for up to 12 hours. The next day, 2 mL of the supernatant containing the displaced high concentration of the Orai2 protein-micelle complex was magnetically separated to obtain the first-stage purified protein solution (1000 g, 2 min low-speed centrifugation to obtain supernatant, 200 μL of Buffer B added for elution, repeated 5-6 times, and all supernatants were combined as the first-stage protein solution). SDS-PAGE gel electrophoresis revealed a highly significant coarse band of the target protein at approximately 30 kDa (30 kDa).

[0067] S600. The first-stage purified protein solution is subjected to molecular weight sieving using a gel size exclusion chromatography medium to obtain a final eluent containing high-purity human Orai2 protein.

[0068] The gel size exclusion chromatography module performs physical volumetric chromatography separation based on differences in hydrodynamic radius. Although the first-stage affinity purification removes most of the flag-free impurities, antibody fragments, some non-specifically bound impurities, and aggregates or degradation byproducts of Orai2 protein during elution may still be present in the system. Gel size exclusion chromatography (SEC) utilizes a chromatographic column packed with microporous gel particles, causing micelles of different molecular weights flowing through the matrix to experience different path lengths in the microporous network based on their hydrodynamic volume, thereby achieving high-resolution sieving along the time axis.

[0069] S610. The first-stage purified protein solution is subjected to ultrafiltration concentration treatment to obtain concentrated protein solution.

[0070] The gel size exclusion chromatography module uses ultrafiltration centrifuge tubes with a molecular weight cutoff smaller than the target micelle molecular weight to force concentration of the large volume of eluent by high-speed centrifugation. The core purpose of this step is to minimize the sample loading volume, because in gel filtration chromatography, an extremely narrow loading band width is a prerequisite geometric condition for ensuring baseline separation of each molecular weight peak and maintaining extremely high resolution.

[0071] Exemplarily, the gel size exclusion chromatography module transfers 2 mL of the first-stage purified protein solution obtained in step S540 into an ultrafiltration tube with a molecular weight cutoff (MWCO) of 100 kDa. It is then placed in a benchtop refrigerated centrifuge at 4 degrees Celsius for further processing. Concentration was performed by centrifugation at high speed. After every 10 minutes of centrifugation, the mixture was paused and gently resuspended by pipetting to prevent localized precipitation and polarization of extremely high-concentration proteins on the filter membrane surface. After several centrifugations, the total protein solution volume was precisely concentrated to 1.0 mL, which is exactly equal to the nominal volume of the hard sample loop of the chromatographic system selected for subsequent experiments. Ultracentrifugation for 5 minutes was then performed to eliminate any air bubbles, obtaining a homogeneous concentrated protein solution.

[0072] S620. Inject the concentrated protein solution into a molecular sieve chromatography column that has been pre-equilibrated.

[0073] The gel size exclusion chromatography module executes the injection sequence on an automated protein liquid chromatography (FPLC) system. To prevent air bubbles from entering the column and damaging the dense structure of the gel bed, the module strictly adheres to a pre-cleaning procedure, using pure water and buffer to replace residual liquid in the system pump and tubing dead volume, and utilizes cross-linked agarose matrix molecular sieves with a specific pore size distribution for separation.

[0074] For example, the gel size exclusion chromatography module uses a pre-packed molecular sieve column loaded with Superose 6 Increase 10 / 300 GL (rated column bed volume). milliliters, maximum tolerable back pressure The Bio-Rad automated protein purification chromatograph (MPA) was used. Using the connection port, the 1 mL sample loop and infusion pump assembly were first rinsed with pre-degassed purified water to remove internal crystalline salt particles. The detergent buffer B equilibration system was then connected using the same procedure. Subsequently, the system was switched to injection mode, and 1.0 mL of the prepared concentrate was non-destructively injected into the rigid metal sample loop inside the system using a blunt-tipped syringe, awaiting the column flow path switching command.

[0075] S630, drive the mobile phase through the molecular sieve chromatography column at a constant volumetric flow rate, and collect the eluted components sequentially according to a preset elution volume interval.

[0076] The gel size exclusion chromatography module activates a high-pressure constant-flow dual-plunger pump, forcing the mobile phase to carry the sample zone through the column matrix. Polymeric proteins or ultra-large aggregated micelles with large hydrodynamic radii cannot penetrate the deep capillary pores inside the gel microspheres; they can only flow rapidly along the gaps between gel particles, forming the initial dead volume peak (external water volume component). However, the target human Orai2 protein-complex micelles, due to their medium to large molecular weight, moderately penetrate the particle pores, forming a peak with a specific retardation coefficient. ) and outflow volume ( The main peak of the FLAG peptide is observed; extremely small free FLAG peptide fragments and salt ions permeate every pore and are eluted at the end. The system continuously retains the eluted fraction according to fixed volume cutting units.

[0077] For example, with the parameter configuration as described in claim 8, the gel size exclusion chromatography module locks the infusion pump flow rate to a constant volumetric flow rate. The chromatographic mobile phase was strictly prepared using Filter Buffer B solution (containing 20 mmol / L Tris-HCl, 200 mmol / L NaCl, and 0.03% DDM and 0.03% CHS). The run command was initiated, and the loading loop was connected to the main flow path. During a total elution run of 1.2 column volumes (approximately 32 mL), the UV detector at the system's tail end (set to a detection wavelength of 280 nm) acquired real-time data on changes in optical density and plotted chromatograms. The fraction collector was started simultaneously, operating in unit volume increments. Milliliters per tube, the effluent from the chromatographic tubing is continuously and sequentially dispensed into the collection tube array.

[0078] S640. Based on the ultraviolet absorption spectroscopy analysis and polyacrylamide gel electrophoresis analysis performed on each of the eluted components, the elution tubes corresponding to the target molecular weight peak of the human Orai2 protein are identified and merged to obtain the final eluent.

[0079] The gel size exclusion chromatography module locates the effective signal range using a multidimensional cross-validation strategy. Asymmetric peaks on the UV absorption chromatographic baseline represent the enrichment of macromolecules at specific elution points. Fractions from several consecutive tubes at this point are extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based on a dual charge-mass difference separation mechanism. Based on the band thickness distribution indicated by Coomassie Brilliant Blue dye and the results of Western blot imaging with antibody-specific binding, an absolutely homogeneous core elution tube array free of macromolecular polymer tails and small molecule impurity degradation segments is identified and merged to obtain the final product.

[0080] For example, the gel size exclusion chromatography module reads the gel size exclusion ultraviolet absorption chromatogram output by the system terminal (such as...). Figure 7 (As shown). The image shows the elution effluent volume span. A tall, narrow peak with extremely high symmetry (Orai2 main peak) appears at the x-axis position in milliliters, preceded by a small number of broad polymer peaks, followed by a large number of sharp peaks of very low molecular weight impurities (such as the Flag peptide signal peak). Trace fractions were extracted from tubes 44 to 54, corresponding to the effluent volumes. Each fraction was then mixed with loading buffer containing or without the reducing agent dithiothreitol (DTT), and a concentration distribution was performed. The SDS-PAGE electrophoresis was performed for verification. The colloid was stained and developed using an enzyme assay kit (e.g., ...). Figure 5 and Figure 6As shown in the diagram, in lanes labeled L' (with DTT added) and each collection tube, a dense, sharp, and completely free band appears at the 30 kDa mass mark indicated by the marker. This band can be verified by high-intensity luminescence response from the anti-FLAG secondary antibody. Homogenizing all the liquids in tubes 44 to 54 yields the final elution buffer of the human Orai2, which is structurally extremely homogeneous and of extremely high purity, perfectly achieving the goal of in vitro separation of transmembrane proteins maintained in their native quaternary conformation.

[0081] In some embodiments, to address complex membrane protein systems with varying depths of intercalation and hydrophobic exposure areas, this application may employ an adaptive regulation mechanism for the dissolution microenvironment in a dependent configuration to maintain the thermodynamic stability of lipid substitution. This mechanism aims to address the three-dimensional structural imbalance arising from the tendency of pure detergents to peel off and the fragility of the internal salt bond and hydrogen bond networks of membrane proteins.

[0082] The adaptive regulation mechanism of the membrane microenvironment is based on the dynamic adjustment of the buffer system by molecular docking affinity and surface tension gradient feedback. This mechanism obtains the extraction rate of different candidate detergents (such as Triton X-100, CHAPS, and DDM) when treating cell suspensions of specific concentrations, as well as the ratio of the integral of the main peak height to the integral of the polymer tail volume in the subsequent gel size exclusion chromatography (conformation homogeneity index). According to Le Chatelier's chemical equilibrium principle, if a certain formulation results in a high extraction rate but a significant increase in polymer tailing (indicating that although the protein is dissolved, hydrophobic exposure polymerization occurs), the system control module determines that the critical micelle concentration of the detergent system is too low or the solubilizing particles are too small to shield the transmembrane region, triggering a repair action that increases the proportion of rigid lipids (such as cholesterol succinate CHS) incorporated.

[0083] For example, addressing the issue of abundant oligomeric degradation products encountered during the initial purification of human Orai2 protein, the mechanism first sets the input vector to use a single 2% CHAPS solution for lysis. In the resulting UV absorbance spectrum, the polymer peak area is 3.5 times that of the monomer target peak, indicating extremely poor conformational uniformity. This is used as a negative feedback bias signal input to the model, which determines that this ionic detergent severely interferes with the electrostatic network of the Orai2 membrane segment. The mechanism then re-outputs a set of... A high concentration of nonionic long-chain detergent, dodecyl-β-D-maltopyranoside (DDM), and cholesterol succinate (CHS) incorporated at a 1 / 10 mass ratio served as the production instructions for optimizing the microenvironment. Under this mixed micellar environment, CHS molecules containing a rigid cyclopentane-polyhydrophenanthrene backbone were extensively embedded in the hydrophobic carbon chains of DDM, reducing the electrostatic repulsion of the detergent head and enhancing the lateral interfacial tension limit of the entire solubilized micelle layer, perfectly meeting the van der Waals force requirements of Orai2's four transmembrane helical interfaces. Verification of the modified extraction process output showed a sharp drop in the peak area of ​​polymeric impurities in the product, while the target peak remained high and prominent, indicating the successful construction of the thermodynamically simulated microenvironment.

[0084] This application involves biochemical operations and mechanical parameters in several places, requiring a specific definition of the unit system for core parameters to avoid confusion. The dimension of centrifugation intensity is not only expressed as rotational angular velocity (e.g., To eliminate the acceleration difference bias caused by different shaft radii, this paper must and mandates the use of relative centrifugal force. ) is the only valid operating dimension, expressed as a multiple of gravitational acceleration (symbol: Absolute numerical values ​​must be defined. In describing component concentrations, when detergent powders such as DDM and CHS are dissolved in a liquid buffer system, this paper must and mandates the use of mass-volume fractions (CLC). ) as an absolute unit of measurement, that is, grams per 100 milliliters ( This accurately characterizes the proportion of solute interface mass displaced within the three-dimensional solvent volume, eliminating ambiguities in molecular weight determination caused by the formation of polydisperse micelles in molar concentration systems. For chromatographic elution records, this paper does not use uncertain elution time as the x-axis measure, but must and mandates the use of the absolute elution volume at a constant pump rate ( ) or by multiples of column bed volume ( Geometric scaling is performed to make the liquid phase separation conclusion independent of the hardware dimensions of the specific pumping equipment.

[0085] Furthermore, the functional units in the various embodiments of this application can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit described above can be implemented in hardware.

[0086] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing high-purity human Orai2 membrane protein based on a mammalian cell expression system, characterized in that, The method includes: Obtain a recombinant expression vector containing the human Orai2 gene and an affinity tag sequence; The recombinant expression vector was transfected into a host cell line for expression to obtain host cells expressing the human Orai2 protein; The host cells were lysed using a membrane lysis buffer containing a first surfactant and a second surfactant to obtain a cell lysate containing the human Orai2 protein, wherein the first surfactant was dodecyl-β-D-maltopyranoside and the second surfactant was cholesterol succinate. The cell lysate was centrifuged to separate insoluble matter, and a supernatant containing the human Orai2 protein was obtained. The supernatant was specifically bound and eluted using an affinity chromatography medium to obtain a first-stage purified protein solution. The first-stage purified protein solution was molecular weight sieved using a gel size exclusion chromatography medium to obtain a final eluent containing high-purity human Orai2 protein.

2. The method according to claim 1, characterized in that, The affinity tag sequence comprises an aspartic acid-tyrosine-lysine-aspartic acid-aspartic acid-aspartic acid-lysine polypeptide sequence consisting of eight amino acid residues. The steps for obtaining the recombinant expression vector containing the human Orai2 gene and the affinity tag sequence include: Forward and reverse amplification primers were designed based on the human Orai2 gene, and a first restriction endonuclease site sequence and a nucleotide sequence corresponding to the affinity tag sequence were integrated into the forward amplification primer, and a second restriction endonuclease site sequence was integrated into the reverse amplification primer. The human Orai2 gene was amplified by polymerase chain reaction using the forward amplification primers and the reverse amplification primers to obtain the target gene fragment; The target gene fragment and an expression vector backbone were subjected to double restriction enzyme digestion using a first restriction endonuclease and a second restriction endonuclease, respectively. The target gene fragment, which has undergone the double enzyme digestion process, is ligated to the expression vector backbone to obtain the recombinant expression vector.

3. The method according to claim 1, characterized in that, The step of transfecting the recombinant expression vector into a host cell line for expression includes: The recombinant expression vector is mixed with polyethyleneimine at a preset mass ratio to obtain a transfection complex with a preset volume. The transfection complex was added to a human embryonic kidney cell derivative in suspension culture for co-culture.

4. The method according to claim 1, characterized in that, The step of lysing the host cells using a membrane-dissolving buffer containing a first surfactant and a second surfactant includes: The membrane solution buffer is constructed based on a pre-defined buffer matrix, which contains tris(hydroxymethyl)aminomethane hydrochloride at a concentration of 20 mmol / L and sodium chloride at a concentration of 200 mmol / L. The dodecyl-β-D-maltopyranoside was added to the buffer matrix to achieve a final concentration of 1 percent, and the cholesterol succinate was added to achieve a final concentration of 1 part per thousand. The host cells are suspended in the membrane lysis buffer and incubated at a preset low temperature for a first preset time span to obtain the cell lysate.

5. The method according to claim 1, characterized in that, The step of centrifuging the cell lysate to separate insoluble matter includes: The cell lysate was placed in a centrifuge and run for a second preset time span under an ambient temperature of 4 degrees Celsius and a relative centrifugal force of 120,000 times the acceleration due to gravity. The liquid phase component above the cell debris precipitate at the bottom is separated and extracted, and used as the supernatant.

6. The method according to claim 2, characterized in that, The step of specifically binding and eluting the supernatant using an affinity chromatography medium includes: The magnetic microspheres containing monoclonal antibodies corresponding to the affinity tag sequence were equilibrated using a equilibration buffer. The supernatant was mixed with the equilibrated magnetic microspheres and incubated in a horizontal rotation state for a third preset time span to obtain a magnetic microsphere complex bound to the human Orai2 protein. The magnetic microsphere complex was eluted with a washing buffer to remove impurities and proteins. The magnetic microsphere complex was competitively eluted using an elution buffer containing a high concentration of affinity-tagged peptides to obtain the first-stage purified protein solution.

7. The method according to claim 1, characterized in that, The step of molecular weight sieving of the first-stage purified protein solution using a gel size exclusion chromatography medium includes: The first-stage purified protein solution is subjected to ultrafiltration concentration to obtain concentrated protein solution; The concentrated protein solution was injected into a molecular sieve chromatography column that had been pre-equilibrated. The mobile phase is driven through the molecular sieve chromatography column at a constant volumetric flow rate, and the eluted components are collected sequentially according to a preset elution volume interval. Based on the UV absorption spectroscopy and polyacrylamide gel electrophoresis analysis performed on each of the eluted components, the elution tubes corresponding to the target molecular weight peak of the human Orai2 protein were identified and merged to obtain the final eluent.

8. The method according to claim 7, characterized in that, The step of driving the mobile phase through the molecular sieve chromatography column at a constant volumetric flow rate includes: The mobile phase consisted of a solution containing 20 mmol / L of tris(hydroxymethyl)aminomethane hydrochloride, 200 mmol / L of sodium chloride, 0.03% dodecyl-β-D-maltopyranoside, and 0.03% succinate cholesterol ester. The fluid-driven pump is configured to deliver the mobile phase at a constant volumetric flow rate of 0.3 ml per minute.

9. A system for purifying human Orai2 protein, characterized in that, The system includes: The vector construction module is configured to obtain a recombinant expression vector containing the human Orai2 gene and an affinity tag sequence; The cell transfection and expression module is configured to transfect the recombinant expression vector into a host cell line for expression, thereby obtaining host cells expressing the human Orai2 protein; The transmembrane protein lysis module is configured to lyse the host cells using a lysis buffer containing a first surfactant and a second surfactant to obtain a cell lysate containing the human Orai2 protein, wherein the first surfactant is dodecyl-β-D-maltopyranoside and the second surfactant is cholesterol succinate. The ultracentrifugation separation module is configured to centrifuge the cell lysate to separate insoluble matter and obtain a supernatant containing the human Orai2 protein. The affinity chromatography purification module is configured to use an affinity chromatography medium to specifically bind and elute the supernatant to obtain a first-stage purified protein solution. The gel size exclusion chromatography module is configured to use a gel size exclusion chromatography medium to perform molecular weight sieving on the first-stage purified protein solution to obtain a final eluent containing high-purity human Orai2 protein.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by the processor, it implements the process control steps of the method for purifying human Orai2 protein according to any one of claims 1 to 8.

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