Protein molecular weight standard reference substance and preparation method therefor

Abstract

The present invention belongs to the technical field of fusion protein expression and purification. Disclosed are a protein molecular weight standard reference substance and a preparation method therefor. The preparation method comprises: constructing a fusion protein expression vector, inducing expression to generate an inclusion body, recovering the inclusion body and precipitating same, carrying out a renaturation treatment after dissolving same, and purifying same to obtain the reference substance, wherein the fusion protein is formed by combining a rubredoxin as a fusion linker with another heat-resistant protein as a fused party, and the fusion protein does not contain a purification tag. According to the preparation method, purification of the fusion protein by means of Ni metal ion affinity chromatography can be achieved in the absence of a purification tag to avoid the trouble brought about by the use of a protein Marker by a HIS tag, and the use of the easily renatured inclusion body as a purification starting raw material is beneficial to efficiently and economically preparing the protein molecular weight standard reference substance.

Description

A protein molecular weight standard reference substance and preparation method thereof Technical Field

[0001] The invention belongs to the technical field of fusion protein expression and purification, and in particular relates to a protein molecular weight standard reference and a preparation method thereof. Background Art

[0002] Proteins are a class of organic macromolecules that often underlie life's activities. The study of protein structure and function has long been a hot topic in life science research. Before conducting research or applying a protein, it's often necessary to conduct relevant testing.

[0003] Electrophoresis is a commonly used method for protein detection. Polyacrylamide gels have a network structure, allowing small proteins to easily pass through the gel pores with minimal resistance and rapid migration. Larger proteins, however, experience greater resistance and lag behind. Consequently, proteins are separated according to their molecular weight during electrophoresis. Given these characteristics, polyacrylamide gel electrophoresis is often used for protein detection.

[0004] During electrophoresis experiments, protein samples of known molecular weight are often used as references to help researchers quickly determine the molecular weight of the protein being tested. These proteins of known molecular weight are called protein molecular weight standards. Several proteins of varying molecular weights are often mixed together to facilitate testing. These mixed protein molecular weight standards are also called protein markers.

[0005] Currently, most protein marker products sold on the market are formulated using a mixture of natural protein molecules. For example, a widely used protein marker from Thermo Scientific is a mixture of seven natural proteins (14.4 to 116 kDa): lysozyme from egg white (14.4 kDa), β-lactoglobulin from milk (18.4 kDa), restriction endonuclease Bsp98I from Escherichia coli (25.0 kDa), lactate dehydrogenase from porcine muscle (35.0 kDa), ovalbumin from egg white (45.0 kDa), bovine serum albumin from bovine plasma (66.2 kDa), and β-galactosidase from Escherichia coli (116.0 kDa). Because the content of natural proteins in cells is usually low, purification is difficult and the preparation cost is high, resulting in a high price for the final protein marker product.

[0006] The development of modern genetic engineering technology has made it possible to clone and express proteins in large quantities. Recombinant expression offers numerous advantages over natural extraction, such as leveraging the multi-copy nature of plasmids or expressing the target gene under a strong promoter to increase protein expression levels; high-density culture of recombinant bacteria to increase target protein production; and the inclusion of purification tags during expression vector construction to simplify isolation and purification steps. Clearly, using recombinant expression technology to prepare protein molecular weight standards is more efficient.

[0007] Recombinant DNA technology enables the large-scale expression of heterologous proteins, but extracting and purifying the target protein to a high degree of purity is challenging. This is because the target protein is generally expressed intracellularly. After cell disruption, the target protein is released and mixed with the cell's own proteins and other macromolecules. To facilitate the purification of recombinant proteins, various affinity purification tags have been developed, such as the commonly used histidine tag (HIS-Tag), glutathione sulfhydryltransferase tag (GST-Tag), and maltose binding protein tag (MBP-Tag). Of these, the HIS-Tag is the most widely used due to its small size, mild elution conditions, low purification media cost, and the availability of commercially available antibodies against the tag.

[0008] The widespread use of purification tags, particularly HIS tags, has greatly facilitated the purification of recombinant proteins. However, to facilitate the use of protein markers, it is best to use HIS-tag-free protein as a molecular weight standard. This is because, when performing Western blotting experiments, if the primary antibody is His-tagged, the secondary antibody will bind not only to the His-tagged primary antibody but also to the His-tagged protein marker band. Therefore, upon addition of ECL luminescence solution, both the protein marker and the target protein in the sample are exposed simultaneously. Some biotech companies employ the strategy of removing the HIS tag during post-purification to avoid interference, but this undoubtedly complicates the purification step, as the protease involved in the cleavage must be removed later.

[0009] Protein markers are commonly used laboratory consumables. Users require high purity and low cost. Therefore, it is essential to find a simple, efficient, and economical method for preparing protein molecular weight standards.

[0010] Summary of the Invention

[0011] To solve the above problems, the present invention provides a method for preparing a protein molecular weight standard reference material, comprising:

[0012] A fusion protein expression vector is constructed, expression is induced to generate inclusion bodies, the inclusion body precipitate is recovered, dissolved and then renatured by a dilution renaturation method, and the protein molecular weight standard reference is obtained after purification; wherein the fusion protein of the fusion protein expression vector is composed of rubredoxin as a fusion linker and another heat-resistant protein as a fused partner, and the fusion protein does not contain a purification tag.

[0013] The method of constructing a fusion protein expression vector, inducing expression to generate inclusion bodies, recovering the inclusion body precipitate, dissolving it, and then performing renaturation treatment by a dilution renaturation method, and obtaining the protein molecular weight standard reference after purification, comprises:

[0014] The fusion junction and another heat-resistant protein are directly connected to construct the protein expression vector and induce expression to generate the inclusion body; or, the fusion junction and another heat-resistant protein are connected via a flexible connecting peptide to construct the protein expression vector and generate the inclusion body;

[0015] performing a renaturation treatment on the inclusion bodies to obtain a renaturation solution;

[0016] Purifying the refolded solution by Ni metal ion affinity chromatography to obtain the protein molecular weight standard reference;

[0017] Preferably, the flexible connecting peptide includes any one or more of GGS, (GGS)2, (GGS)3, (GGS)4, SPGS, GSGSG, (GSGSG)2, (GSGSG)3, GGSGG, (GGSGG)2, (GGSGG)3, GGGGS, (GGGGS)2, (GGGGS)3, GGGGSGGGG and GSAGSASAGSGEF.

[0018] Preferably, the rubrenoxin is a small molecular weight rubrenoxin derived from hyperthermophilic bacteria;

[0019] Preferably, the rubredoxin consists of 53 amino acids;

[0020] Preferably, the amino acid sequence of the rubredoxin is as shown in SEQ ID NO.1.

[0021] Preferably, the method of using the rubrenoxin in the heat-resistant protein as a fusion linker and another heat-resistant protein as a fused partner to construct an inclusion body connected by the fusion linker and the fused partner comprises:

[0022] Construct a heat-resistant protein fusion expression vector;

[0023] According to the heat-resistant protein fusion expression vector, rubredoxin is used as a fusion linker to construct a fusion vector;

[0024] The fusion protein is expressed on the fusion vector based on Escherichia coli to obtain the inclusion body bacteria.

[0025] Preferably, the construction of a heat-resistant protein fusion expression vector comprises:

[0026] The target EGFP plasmid is used as a template and EGFP protein primers are used to perform PCR amplification to obtain the heat-resistant protein fusion expression vector.

[0027] Preferably, the heat-resistant protein fusion expression vector is constructed using rubredoxin as a fusion linker, comprising:

[0028] Using the rubrenoprotein plasmid as a template, PCR amplification of the Rub gene was performed using Rub primers to obtain PCR products;

[0029] The PCR product is digested with enzymes and then combined with the heat-resistant protein fusion expression vector to obtain the fusion vector.

[0030] Preferably, the expression of the fusion protein by the fusion vector based on Escherichia coli comprises:

[0031] Transforming the fused vector into competent Escherichia coli cells and culturing them to obtain seed solution;

[0032] The seed liquid is subjected to self-induction culture, and the culture liquid is collected to obtain the inclusion body bacteria.

[0033] Preferably, the renaturing treatment of the inclusion bodies to obtain a renaturing solution comprises:

[0034] The inclusion body bacteria are prepared into a suspension, crushed and then centrifuged to obtain an inclusion body precipitate, and then the inclusion body is dissolved to obtain a dissolved mixture;

[0035] The dissolved mixture is renatured by adding a buffer dropwise thereto through a dilution renaturation method, and the renatured solution is obtained after centrifugation and removal of the supernatant.

[0036] Preferably, the refolding solution is purified by Ni metal ion affinity chromatography to obtain the protein molecular weight standard reference, comprising:

[0037] Take Ni metal chelating resin and balance it with buffer;

[0038] The renaturation solution is loaded onto a Ni metal chelate resin, and eluted with an imidazole buffer solution. Purity is tracked by gel electrophoresis to obtain the protein molecular weight standard reference.

[0039] In addition, to solve the above problems, the present invention also provides a protein molecular weight standard reference material, which is prepared by the above-mentioned method for preparing a protein molecular weight standard reference material.

[0040] The present invention provides a protein molecular weight standard reference and a preparation method thereof, the preparation method comprising: constructing a fusion protein expression vector, inducing expression to generate inclusion bodies, recovering the inclusion body precipitate, dissolving and renaturing the inclusion body by a dilution renaturation method, and obtaining the protein molecular weight standard reference after purification; wherein, the fusion protein uses rubredoxin as a fusion linker, another heat-resistant protein as a fused party, and the fusion protein does not contain a purification tag. The fusion linker and the other heat-resistant protein can be directly connected; or, the fusion linker and the other heat-resistant protein are connected by a flexible connecting peptide. The preparation method provided by the present invention can realize the purification of the fusion protein by Ni metal ion affinity chromatography without a purification tag, thereby avoiding the trouble caused by the presence of the HIS tag to the use of the protein marker. The inclusion body that is easy to renature is used as the purification starting material, which is conducive to the efficient and economical preparation of the protein molecular weight standard reference, and provides a simpler, more economical and efficient solution for the preparation of the protein molecular weight standard reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG1 is a schematic flow diagram of a method for preparing a protein molecular weight standard reference material according to the present invention;

[0042] FIG2 is a schematic diagram of the SDS-PAGE gel electrophoresis results after Ni metal ion affinity chromatography purification in Example 4 of the present invention.

[0043] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0045] The present invention provides a method for preparing a protein molecular weight standard reference material, comprising:

[0046] A fusion protein expression vector is constructed, expression is induced to form inclusion bodies, the inclusion body precipitate is recovered, dissolved, and then renatured using a dilution renaturation method. After purification, the protein molecular weight standard reference material is obtained. The fusion protein in the fusion protein expression vector is composed of rubredoxin as the fusion linker and another heat-resistant protein as the fused partner, and the fusion protein does not contain a purification tag. As mentioned above, heat-resistant proteins are proteins that can maintain structural and functional stability in high-temperature environments. These proteins are typically derived from microorganisms that live in extreme environments, such as deep-sea hydrothermal vents and the area around volcanic craters. Because the environmental temperatures in which these microorganisms live are extremely high, their proteins must be heat-resistant to ensure their normal metabolism and survival.

[0047] Further, referring to Figure 1,

[0048] The method of constructing a fusion protein expression vector, inducing expression to generate inclusion bodies, recovering the inclusion body precipitate, dissolving it, and then performing renaturation treatment by a dilution renaturation method, and obtaining the protein molecular weight standard reference after purification, comprises:

[0049] Step S1, the fusion linker and another heat-resistant protein are directly connected to construct the protein expression vector and induce expression to generate the inclusion body; alternatively, the fusion linker and another heat-resistant protein are connected via a flexible connecting peptide to construct the protein expression vector and generate the inclusion body;

[0050] Step S2, performing renaturation treatment on the inclusion bodies to obtain a renaturation solution;

[0051] Step S3, purifying the refolding solution using Ni metal ion affinity chromatography to obtain the protein molecular weight standard reference;

[0052] Furthermore, the flexible connecting peptide includes any one or more of GGS, (GGS)2, (GGS)3, (GGS)4, SPGS, GSGSG, (GSGSG)2, (GSGSG)3, GGSGG, (GGSGG)2, (GGSGG)3, GGGGS, (GGGGS)2, (GGGGS)3, GGGGSGGGG and GSAGSASAGSGEF.

[0053] As mentioned above, rubredoxin (Rub) is a small iron-containing protein widely found in many organisms, including bacteria, archaea, and plants. It plays a vital role in electron transport within organisms, participating in redox reactions during cellular metabolism. Rubredoxin typically contains a single iron ion, which is linked to a thiol group to achieve electron transport. These properties make rubredoxin an important mediator of redox reactions in organisms.

[0054] In the present invention, rubredoxin is used as the fusion linker because it has the following characteristics and advantages:

[0055] (1) Small molecular weight: Rubredoxin is a small molecular weight protein composed of 53 amino acids with a relatively simple structure. This means that it will not significantly affect the structure and function of the target protein when expressed in fusion.

[0056] (2) Good stability: Rubrenodoxin has high thermal stability and chemical resistance, and can maintain its integrity and activity under high temperature and acid-base conditions. This enables it to take on the role of fusion linker while protecting the fused target protein from the influence of the external environment.

[0057] (3) Affinity chromatography purification capability: Rubredoxin can bind to Ni metal ions with appropriate strength, so the fusion protein can be purified using Ni metal ion affinity chromatography without a purification tag. This method is simple and efficient, avoiding the problems caused by the HIS tag used in traditional purification methods.

[0058] In summary, the use of rubredoxin as a fusion linker has the advantages of small molecular weight, stability and affinity purification ability, and is suitable for use in the preparation of protein molecular weight standard reference materials.

[0059] The different forms of the amino acid sequence of the flexible connecting peptide provided in the present invention can be specifically shown in the following table:

[0060] Table 1. Different amino acid representations of flexible linker peptides

[0061] In the present invention, the purpose of providing a flexible linker is to connect rubredoxin and other heat-resistant proteins, slightly increasing the molecular weight of the fusion protein to reach the commonly used molecular weight standards such as 35.0 kDa, 45.0 kDa, etc.

[0062] It should be noted that, in the method for preparing the protein molecular weight standard reference provided by the present invention, when rubredoxin is used as the fusion linker and another heat-resistant protein is used as the fused partner, there are two schemes as follows:

[0063] Specifically, the two proteins (rubredoxin and another heat-resistant protein) can be directly connected, or a flexible connecting peptide as described above can be added between the two proteins, and the fusion protein molecule does not contain a purification tag.

[0064] As mentioned above, refolding refers to the process of refolding a protein that has been expressed in the form of inclusion bodies in a prokaryotic or eukaryotic expression system into an active protein with correct structure and function through appropriate conditions and methods.

[0065] In bacterial expression systems such as E. coli, overexpression of exogenous proteins or the inherent properties of the expressed proteins often results in the formation of inclusion bodies, which fail to fold properly. These inclusion bodies typically exist as aggregates or insoluble granules and lose their native biological activity. To restore the protein's biological activity, renaturation treatment is necessary.

[0066] The fundamental principle of renaturation is to provide an environment suitable for protein folding under appropriate conditions to promote proper protein folding and the formation of a functional structure. The conditions and methods of renaturation can be determined based on the characteristics and needs of the target protein, including temperature, pH, the addition of reducing agents, oxidizing agents, buffers, auxiliary proteins, and gradual dilution.

[0067] The renaturation process usually includes the following steps:

[0068] (1) Dissolution of inclusion bodies: The inclusion bodies are extracted from bacteria and dissolved in an appropriate buffer to destroy the granular structure of the inclusion bodies.

[0069] (2) Addition of folding buffer: Add appropriate buffers, reducing agents, auxiliary proteins, etc. to the protein solution to provide a suitable folding environment.

[0070] (3) Slow dilution: By gradually diluting the protein solution, the protein is gradually exposed to suitable folding conditions, avoiding aggregation caused by rapid folding.

[0071] (4) Monitoring and optimization of the folding process: By monitoring the biological activity, solubility, structure and other indicators of the protein, the folding conditions and methods are adjusted to achieve the best folding effect.

[0072] Renaturation treatment is one of the technical means in the field of protein engineering and biotechnology, which can purify the target protein expressed in inclusion bodies and restore its functionality.

[0073] As mentioned above, nickel-affinity chromatography is a commonly used protein purification technology that uses Ni 2+ The ions are separated and purified based on their affinity for the His tag in the protein.

[0074] In Ni metal ion affinity chromatography purification, the target protein sample containing His tag is usually added to a Ni-containing 2+ ion in the resin solid phase material, so that it can interact with the Ni 2+ The ions react with each other and are separated from other impurity proteins. Then, by gradually adjusting the buffer conditions, the target protein is eluted from the resin, and the purified target protein is finally obtained.

[0075] The principle of this technology is based on the affinity of His tag with Ni 2+ Strong interactions between ions, Ni 2+ The ions can form coordination bonds with the nitrogen and oxygen atoms in the His tag, thereby allowing the protein to bind to the Ni 2+ The method is simple, efficient and reproducible.

[0076] Furthermore, the rubrenoxin is a small molecular weight rubrenoxin derived from hyperthermophilic bacteria.

[0077] Hyperthermophilic bacteria, as mentioned above, are a type of bacteria that thrive in extremely high-temperature environments, capable of surviving and multiplying in temperatures approaching or exceeding 100°C. These bacteria typically inhabit high-temperature environments such as deep-sea hydrothermal vents and volcanic eruptions, demonstrating remarkable adaptability to these temperatures.

[0078] As mentioned above, cytochrome oxidoreductases are a class of proteins involved in electron transport in the cellular respiratory chain, transferring electrons from one molecule to another. Cytochrome oxidoreductases in hyperthermophilic bacteria possess a specific structure and sequence that allows them to remain stable and function normally in high-temperature environments.

[0079] The term "low molecular weight rubrenoxin" refers to its low relative molecular weight. In hyperthermophiles, to adapt to high temperatures, intracellular proteins typically have smaller molecular weights. A smaller molecular weight can improve the thermal stability and structural compactness of proteins, helping them maintain their structural and functional stability at high temperatures. Therefore, rubrenoxin proteins in hyperthermophiles typically have smaller molecular weights to accommodate the extreme high temperatures.

[0080] Furthermore, the rubredoxin is composed of 53 amino acids;

[0081] Furthermore, the amino acid sequence of the rubrenoxin is as shown in SEQ ID NO.1.

[0082] Furthermore, the step S1a, using the rubrenoxin in the heat-resistant protein as a fusion linker and another heat-resistant protein as a fused partner, constructing an inclusion body connected by the fusion linker and the fused partner, comprises:

[0083] Step S11, constructing a heat-resistant protein fusion expression vector;

[0084] This step, described above, involves combining the heat-resistant protein used to construct a protein molecular weight standard with other components to form a new protein molecule. Specifically, under laboratory conditions, the heat-resistant protein is chemically reacted or biologically synthesized with the other desired components, linking them together to form a protein fusion construct. This method allows the heat-resistant protein to be combined with other functional components, improving the protein's functionality and applicability.

[0085] Step S12, constructing a fusion vector based on the heat-resistant protein fusion expression vector using rubredoxin as a fusion linker;

[0086] As described above, the constructed thermostable protein fusion expression vector was used to link rubrenoxin to it via a flexible linker peptide to form a new post-fusion vector. Specifically, based on the designed molecular structure, the flexible linker peptide and rubrenoxin were specifically linked to the thermostable protein fusion expression vector to produce a post-fusion vector protein containing rubrenoxin.

[0087] Step S13, expressing the fusion protein based on Escherichia coli using the fusion vector to obtain the inclusion body bacteria.

[0088] In this step, E. coli is used as a host cell, allowing the fusion vector to be expressed within the cells to produce the target inclusion bodies. Specifically, the fusion vector is introduced into E. coli, allowing the cells' own biological mechanisms to express the fusion vector, thereby producing inclusion bodies. This step efficiently produces the target inclusion bodies using the E. coli expression system, laying the foundation for the subsequent preparation of protein molecular weight standards.

[0089] Furthermore, the step S11, constructing a heat-resistant protein fusion expression vector, comprises:

[0090] Step S111, using the target EGFP plasmid as a template and using EGFP protein primers to perform PCR amplification to obtain the heat-resistant protein fusion expression vector.

[0091] In step S111, the target EGFP plasmid is used as a template and specific EGFP protein primers are used to perform PCR amplification to ultimately obtain the desired heat-resistant protein fusion expression vector.

[0092] Specifically, PCR (polymerase chain reaction) is a commonly used molecular biology technique for amplifying DNA fragments in vitro. In this step, PCR technology is used to amplify the target sequence from the target EGFP plasmid to achieve subsequent protein fusion.

[0093] EGFP stands for Enhanced Green Fluorescent Protein, a fluorescent labeling tool widely used in biological research. Here, the target EGFP plasmid was selected as a template for PCR amplification to obtain the gene sequence of the EGFP protein.

[0094] EGFP protein primers are specific primers designed to recognize and amplify the EGFP gene sequence. Through PCR amplification, the EGFP gene sequence can be amplified from the target EGFP plasmid to obtain the desired heat-resistant protein fusion expression vector.

[0095] In summary, in the process of constructing the heat-resistant protein fusion expression vector, step S111 uses PCR technology to amplify the EGFP gene sequence from the target EGFP plasmid to provide the required protein gene fragment for subsequent steps.

[0096] Furthermore, the step S12, based on the heat-resistant protein fusion expression vector, uses rubredoxin as a fusion linker to construct a fusion vector, including:

[0097] Step S121, using the rubrenoprotein plasmid as a template, and using Rub primers to perform PCR amplification of the Rub gene to obtain a PCR product;

[0098] Step S122 , performing enzyme digestion on the PCR product, and then combining it with the heat-resistant protein fusion expression vector to obtain the fusion vector.

[0099] In step S12, the following two steps are specifically included:

[0100] The first step is step S121, in which the Rub gene is amplified by PCR using a rubrenoprotein plasmid as a template and specific Rub primers to obtain the rubrenoprotein gene sequence. Rub is a bifunctional protein with important biological functions and can serve as a fusion linker for the fusion vector. The PCR product is a DNA fragment amplified from the target DNA.

[0101] The second step, step S122, involves enzymatically digesting the PCR product and ligating it to a thermostable protein fusion expression vector to form a new protein molecule. Specifically, a specific enzyme can be used to ligate the PCR product and thermostable protein fusion expression vector, thereby forming a link with the rubrenoxin. Ultimately, the desired fusion vector is obtained.

[0102] In addition, in step S122, the Rub primer can be edited to add a fragment of the flexible connecting peptide so that it contains the amino acid sequence of the flexible connecting peptide, so that when the post-fusion vector is constructed, a post-fusion vector is prepared in which a flexible connecting peptide is added between the two proteins and the fusion protein molecule does not contain a purification tag.

[0103] In summary, in step S12, the rubrenoprotein gene sequence is obtained by PCR amplification and used as a fusion linker to connect with the heat-resistant protein fusion expression vector to construct the fusion vector. This step is one of the important steps in preparing protein molecular weight standard reference materials.

[0104] Furthermore, the step S13, expressing the fusion protein in Escherichia coli using the fusion vector, comprises:

[0105] Step S131, transforming the fused vector into competent Escherichia coli cells and culturing them to obtain a seed solution;

[0106] In this step, the fused vector is introduced into competent E. coli cells, and the bacteria continue to reproduce by utilizing the self-replication mechanism of the bacteria, and eventually an E. coli seed solution containing the fused vector is obtained.

[0107] Specifically, the fused vector can be treated together with E. coli competent cells, and after transformation, the E. coli seed solution containing the fused vector can be obtained through culture and screening.

[0108] Step S132, subjecting the seed solution to self-induction culture, collecting the culture solution to obtain the inclusion body bacteria.

[0109] In this step, the E. coli seed solution containing the fusion vector obtained in the previous step is cultured, and the protein expression is promoted by using an auto-induction expression system. Finally, the culture solution is collected and the inclusion body bacteria are extracted from it.

[0110] For this step, an autoinducible expression system can be used, such as one driven by a T7 promoter. During the culture process, E. coli will automatically induce expression of the target protein based on the culture conditions. Finally, inclusion bodies can be extracted from the culture medium.

[0111] Furthermore, the step S2 of renaturing the inclusion bodies to obtain a renaturation solution comprises:

[0112] Step S21, preparing the inclusion body cells into a suspension, crushing them and then centrifuging them to obtain inclusion body precipitates, and then dissolving the inclusion bodies to obtain a dissolved mixture;

[0113] In this step, the inclusion bodies are first collected and prepared into a suspension. Then, the cell membranes are disrupted through a cell disruption process, releasing the inclusion bodies into the lysis mixture.

[0114] Step S22, the dissolved mixture is subjected to a regeneration treatment by adding a buffer dropwise thereto through a dilution and regeneration method, and the regeneration solution is obtained after the supernatant is removed by centrifugation.

[0115] In this step, an appropriate amount of buffer is added dropwise to the dissolved mixture for renaturation via the dilution renaturation method. This method is a commonly used technique that manipulates temperature, pH, and ion concentration to restore the protein from its unfolded state to its correct three-dimensional structure. The treated mixture is centrifuged and the supernatant removed to obtain the renaturation solution.

[0116] As mentioned above, renaturation treatment can refold the proteins in the inclusion bodies into the correct conformation and restore their native functionality.

[0117] As mentioned above, the dilution renaturation method is a simple and effective renaturation treatment method that can promote protein renaturation under controlled conditions.

[0118] In the dilution renaturation method, the protein concentration in the solution can be reduced by gradually adding buffer, thereby reducing the interaction between proteins and providing a more favorable environment for their correct folding. At the same time, the renaturation process can be optimized by adjusting parameters such as temperature, pH and ion concentration.

[0119] Furthermore, the step S3, purifying the refolding solution by Ni metal ion affinity chromatography to obtain the protein molecular weight standard reference, comprises:

[0120] Step S31, taking Ni metal chelate resin and balancing it with a buffer solution;

[0121] Step S32, taking the renaturation solution and loading it on Ni metal chelate resin, and eluting it with imidazole buffer, and tracking the purity by gel electrophoresis to obtain the protein molecular weight standard reference.

[0122] Ni metal chelate resin is an affinity chromatography material that can be used to purify and enrich recombinant proteins containing HIS-Tag. This resin is usually obtained by cross-linking and functionalizing polypropylene microspheres or agarose gel particles. Its surface has Ni 2+ Ionically coordinated amino or carboxyl functional groups.

[0123] In the present invention, the Ni metal chelating resin used may be Ni-IDA 6FF agarose purification resin.

[0124] The reason for using imidazole buffer for elution is because imidazole has the ability to compete with Ni ions for coordination. In the Ni metal chelate resin, the target protein is captured by the affinity between the HIS-Tag and the Ni ions. When the target protein needs to be eluted, a buffer containing imidazole can be used to dissociate the target protein from the resin through the competitive coordination between imidazole and Ni ions.

[0125] In addition, the present invention also provides a protein molecular weight standard reference material, which is prepared by the above-mentioned method for preparing a protein molecular weight standard reference material.

[0126] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0127] Example 1: Construction of expression vector for fusion protein.

[0128] In this example, an expression vector for a fusion protein was constructed.

[0129] Experimental methods:

[0130] 1. Construction of heat-resistant protein fusion expression vector (24a-EGFP vector): Using EGFP-F as upstream primer, EGFP-R as downstream primer, and commercial plasmid pEGFP-N1 as template, PCR amplified the EGFP gene.

[0131] The nucleotide sequences of EGFP-F and EGFP-R are as follows:

[0132] (1) EGFP-F upstream primer: 5′-cgcGGATCCgtgagcaagggcgagg-3′, where the underline indicates the BamH I restriction site, and the sequence is shown in SEQ ID NO. 2;

[0133] (2) EGFP-R downstream primer: 5′-ccgCTCGAGttagtggtgatggtgatggtg-3′, where the underline indicates the Xho I restriction enzyme site, and the sequence is shown in SEQ ID NO. 3;

[0134] The PCR product was recovered and double-digested with BamH I and Xho I. The double-digested product was recovered and ligated into the commercial plasmid pET-24a that had also been double-digested with BamH I to obtain the 24a-EGFP vector.

[0135] 2. Construction of the post-fusion vector (24a-Rub-EGFP vector): Using Rub-F as the upstream primer, Rub-R as the downstream primer, and the constructed plasmid 28a-Rub as the template, PCR amplified the Rub gene.

[0136] The PCR product was recovered and double-digested with Nde I and BamH I. The double-digested product was recovered and ligated into the plasmid 24a-EGFP that had also been double-digested to obtain the 24a-Rub-EGFP vector.

[0137] The nucleotide sequences of Rub-F and Rub-R are as follows:

[0138] (1) Rub-F upstream primer: 5'-cggcagcCATATGaagaaataccgttgcaaactgtg-3', where the underline indicates the Nde I restriction site, and the sequence is shown in SEQ ID NO. 4;

[0139] (2) Rub-R downstream primer: 5′-cgcGGATCCttctaccggttcgaagtcttc-3′, in which the BamHI restriction site is underlined and the sequence is shown in SEQ ID NO.5.

[0140] Example 2: Fusion protein expression.

[0141] In this example, the expression of the fusion protein was induced to obtain bacteria that produced inclusion bodies.

[0142] Experimental methods:

[0143] (1) The fusion vector (24a-Rub-EGFP) was transformed into Escherichia coli BL21 (DE3) competent cells, coated on LB plates (containing 50 mg / L kanamycin), and cultured in a 37°C incubator overnight.

[0144] (2) Pick a single colony from the overnight cultured LB plate and place it in a 250 mL shake flask containing 50 mL of LB medium. Place it in a constant temperature shaker at 37°C, 200 rpm, and culture for 20 hours as a seed solution.

[0145] (3) The cultured seed liquid was inoculated into 1000 mL shake flasks containing 100 mL of autoinduction medium. A total of 8 flasks were inoculated, with an inoculum volume of 5% (v / v) per flask. The flasks were placed in a constant temperature shaker at 200 rpm and 37°C for 20 h.

[0146] The formula of the autoinduction medium is: lactose 2 g / L; peptone 10 g / L; yeast powder 5 g / L; NaCl 10 g / L; glycerol 8 mL / L.

[0147] (4) Collect all the culture medium, centrifuge and remove the supernatant to obtain the bacterial bodies, which are the inclusion bodies.

[0148] Example 3: Renaturation treatment of inclusion bodies.

[0149] In this example, the inclusion bodies were dissolved and renatured to obtain a renatured solution.

[0150] Experimental methods:

[0151] (1) Add 200 mL of 1× PBS buffer containing 300 mmol / L NaCl to the centrifuge bottle where the bacteria were collected to suspend the bacteria, and then use a high-pressure homogenizer to break the cells.

[0152] (2) Centrifuge the cell disruption solution (8000 rpm, 10 min), discard the supernatant, and use a small steel spoon to break up the white precipitate (inclusion body) at the bottom of the centrifuge bottle. Add 50 mL of urea (2 mol / L) solution and place it on a seesaw decolorization shaker for 2-3 hours. After the precipitate is fully dispersed, pour the suspension into a 50 mL centrifuge tube (it can be observed that the solution is turbid, which indicates that the inclusion body precipitate cannot be dissolved by 2 mol / L urea). Place the centrifuge tube containing the suspension in a -20°C freezer. The next day, take out the centrifuge tube and thaw it at room temperature. It can be observed that after the freeze-thaw treatment, the inclusion body precipitate suspension becomes clear, which indicates that the inclusion body has been dissolved in the urea (2 mol / L) solution.

[0153] (3) 50 mL of urea solution was slowly added dropwise to a beaker containing 450 mL of 1× PBS buffer by dilution and refolding. The beaker was placed on a magnetic stirrer and stirred slowly by a magnetic levitation rotor. The addition rate was controlled to be 5 mL / h. After the addition was completed, the solution was stirred slowly for 2-3 hours, and the solution in the beaker was centrifuged (8000 rpm, 10 min). The supernatant was taken and the immobilized metal ion (Ni 2+ ) chromatography purification.

[0154] Example 4: Ni metal ion affinity chromatography purification.

[0155] In this example, the refolded solution was purified by Ni metal ion affinity chromatography.

[0156] Experimental methods:

[0157] (1) The purification column containing 16 mL of Ni-IDA 6FF agarose purification resin was equilibrated with 1× PBS buffer, and then the refolding supernatant was loaded. The column was then washed with 1× PBS buffer and then gradient eluted with 1× PBS buffer containing different concentrations of imidazole, wherein the imidazole concentrations were 50 mmol / L, 100 mmol / L, 200 mmol / L, and 500 mmol / L, respectively.

[0158] (2) Collect the washed components, add loading buffer to prepare loading samples, and perform SDS-PAGE gel electrophoresis.

[0159] Experimental results:

[0160] The experimental results are shown in Figure 2. M represents protein marker; 1 represents the loading buffer; 2 represents the flowthrough; 3 represents 50 mmol / L imidazole elution; 4 represents 100 mmol / L imidazole elution; 5 represents 200 mmol / L NaCl elution; and 6 represents 500 mmol / L imidazole elution. Arrows indicate the locations of the Rub-EGFP fusion protein bands.

[0161] As can be seen from Figure 2, the purity of the target protein in the elution samples of 100mmol / L imidazole, 200mmol / L imidazole and 500mmol / L imidazole is already high, but the elution sample of 50mmol / L imidazole still contains more impurities.

[0162] Considering that the refolding solution is not suitable for heating due to the presence of urea, the purity can be further improved through other purification methods such as heat treatment and ion exchange chromatography.

[0163] In summary, the preparation method provided by the present invention can realize Ni metal ion affinity chromatography purification of fusion proteins without a purification tag, thereby avoiding the problem of the presence of the His tag in the use of protein markers. The inclusion bodies that are easy to renature are used as the purification starting material, which is conducive to the efficient and economical preparation of protein molecular weight standard reference materials, and provides a simpler, more economical and efficient solution for the preparation of protein molecular weight standard reference materials.

[0164] The above are preferred embodiments and corresponding examples of the present invention. It should be noted that, for those skilled in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, including but not limited to adjustments in proportions, processes, dosages and reaction vessels, such as the use of a continuous flow reactor, all of which fall within the scope of protection of the present invention.

Claims

1. A method for preparing a protein molecular weight standard reference, characterized in that: include: A fusion protein expression vector is constructed, expression is induced to generate inclusion bodies, the inclusion body precipitate is recovered, and after dissolution, a dilution renaturation method is used for renaturation treatment, and after purification, the protein molecular weight standard reference is obtained; wherein the fusion protein of the fusion protein expression vector is formed by combining rubredoxin as a fusion linker and another heat-resistant protein as a fused party, and the fusion protein does not contain a purification tag.

2. The method for preparing a protein molecular weight standard reference material according to claim 1, characterized in that: The method of constructing a fusion protein expression vector, inducing expression to generate inclusion bodies, recovering the inclusion body precipitate, dissolving it, and then performing renaturation treatment by a dilution renaturation method, and obtaining the protein molecular weight standard reference after purification, comprises: The fusion linker and another heat-resistant protein are directly connected to construct the protein expression vector and induce expression to generate the inclusion body; or, the fusion linker and another heat-resistant protein are connected via a flexible connecting peptide to construct the protein expression vector and generate the inclusion body; Performing a renaturation treatment on the inclusion body to obtain a renaturation solution; Purifying the refolded solution by Ni metal ion affinity chromatography to obtain the protein molecular weight standard reference; Preferably, the flexible connecting peptide includes any one or more of GGS, (GGS)2, (GGS)3, (GGS)4, SPGS, GSGSG, (GSGSG)2, (GSGSG)3, GGSGG, (GGSGG)2, (GGSGG)3, GGGGS, (GGGGS)2, (GGGGS)3, GGGGSGGGG and GSAGSASAGSGEF.

3. The method for preparing a protein molecular weight standard reference material according to claim 2, characterized in that: The rubrenoxin is a small molecular weight rubrenoxin derived from hyperthermophilic bacteria; Preferably, the rubrenoxin consists of 53 amino acids; Preferably, the amino acid sequence of the rubrenoxin is as shown in SEQ ID NO.

1.

4. The method for preparing a protein molecular weight standard reference material according to claim 2, characterized in that: The fusion linker and another heat-resistant protein are directly connected to construct the protein expression vector and induce expression to generate the inclusion body; or, the fusion linker and another heat-resistant protein are connected via a flexible connecting peptide to construct the protein expression vector and generate the inclusion body, comprising: Constructing a heat-resistant protein fusion expression vector; According to the heat-resistant protein fusion expression vector, rubrenoxin is used as a fusion linker to construct a fusion vector; The fusion protein is expressed on the fusion vector based on Escherichia coli to obtain the inclusion body.

5. The method for preparing a protein molecular weight standard reference material according to claim 4, characterized in that: The method for constructing a heat-resistant protein fusion expression vector comprises: The target EGFP plasmid is used as a template and EGFP protein primers are used for PCR amplification to obtain the heat-resistant protein fusion expression vector.

6. The method for preparing a protein molecular weight standard reference material according to claim 4, characterized in that: The method of constructing a fusion vector based on the heat-resistant protein fusion expression vector and using rubrenoxin as a fusion linker comprises: Using the rubrenoprotein plasmid as a template, PCR was performed to amplify the Rub gene using Rub primers to obtain PCR products; The PCR product is digested with enzymes and then combined with the heat-resistant protein fusion expression vector to obtain the fusion vector.

7. The method for preparing a protein molecular weight standard reference material according to claim 4, characterized in that: The method of expressing the fusion protein by using the fusion vector in Escherichia coli comprises: Transforming the fused vector into competent Escherichia coli cells and obtaining seed solution after culturing; The seed solution is subjected to self-induction culture, and the culture solution is collected to obtain the inclusion body bacteria.

8. The method for preparing a protein molecular weight standard reference material according to claim 2, characterized in that: The step of performing a renaturation treatment on the inclusion bodies to obtain a renaturation solution comprises: The inclusion body bacteria are prepared into a suspension, crushed and then centrifuged to obtain an inclusion body precipitate, and then the inclusion body is dissolved to obtain a dissolved mixture; The dissolved mixture is rejuvenated by adding a buffer dropwise through a dilution refolding method, and the refolding solution is obtained after centrifugation and removal of the supernatant.

9. The method for preparing a protein molecular weight standard reference material according to claim 2, characterized in that: The refolding solution is purified by Ni metal ion affinity chromatography to obtain the protein molecular weight standard reference material, including: Take Ni metal chelating resin and balance it with buffer; The renaturation solution is loaded onto a Ni metal chelate resin, and eluted with an imidazole buffer solution. Purity is tracked by gel electrophoresis to obtain the protein molecular weight standard reference.

10. A protein molecular weight standard reference, characterized in that: The protein molecular weight standard reference material is prepared by the method for preparing the protein molecular weight standard reference material according to any one of claims 1 to 9.

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