CN111662388A

CN111662388A - Protein expression and purification method

Info

Publication number: CN111662388A
Application number: CN202010506686.7A
Authority: CN
Inventors: 陈郑; 郑兰花; 洪礼清; 蒋奎胜; 刘仁源
Original assignee: Dongguan Dongyangguang Diagnostic Products Co ltd
Current assignee: Dongguan Dongyangguang Diagnostic Products Co ltd
Priority date: 2020-06-05
Filing date: 2020-06-05
Publication date: 2020-09-15

Abstract

The invention relates to a method for protein expression and purification, which comprises the following steps of firstly constructing an expression vector of a fusion protein: one end of the target protein is sequentially connected with a first enzyme cutting site, a report mark and a first purification label, and the other end of the target protein is sequentially connected with a second enzyme cutting site and a second purification label; transferring the expression vector into an expression strain for expression culture, and collecting a culture product; purifying the product according to the second purification tag, and then carrying out enzyme digestion on the first enzyme digestion site and the second enzyme digestion site; and purifying the enzyme digestion product according to the first purification label and the second purification label respectively to obtain the target protein. The expression purification method increases the solubility and yield of the protein, so that the expression of the protein is visualized; the purification steps are simple, and the purity of the target protein is obviously improved under the condition of not increasing the purification process.

Description

Protein expression and purification method

Technical Field

The invention relates to the technical field of biology, in particular to a protein expression and purification method.

Background

The general strategy for expressing the foreign protein is to connect a target gene to a proper vector, then transfer the vector into an expression strain to express the target protein, and finally, crush the expression strain, enrich and purify the required target protein.

The process needs to solve two problems of expression and purification of target protein: expressing a sufficient amount of protein in the expression strain by the target gene; 2. and selecting a proper means to purify the target protein. In addition, sufficient expression is also advantageous for protein purification. For the convenience of subsequent purification, the skilled artisan often uses tags such as 6 × His, MBP, etc. to purify the protein of interest by affinity chromatography. However, these tags often cause new problems, for example, 6 × His tag often causes a problem of low purification efficiency due to its poor specificity; however, MBP-tag is too bulky and may affect the structural function of the protein. In addition, it is also a problem how to identify whether a gene of interest is expressed in an expression strain.

The general method is to use a proper magnetic bead to adsorb and enrich the crushed expression strain, and then judge whether the target gene is expressed or not through SDS-PAGE gel, and the construction of one expression vector is not always done at once and needs to be continuously optimized, which causes much time waste and reagent loss. In order to solve this problem, the skilled person proposes to link a gfp gene at the end of the target gene, so that the target gene and gfp gene are expressed in a fusion manner, and when the expression strain has green fluorescence during the culture process, it can be determined that the target gene has been expressed.

Patent document CN110628801A provides a method for inserting GFP gene into the end of target gene for fusion expression, which comprises constructing a target gene-GFP-his expression vector, and then determining whether the target protein is expressed by using fluorescence emitted by GFP protein expressed by GFP gene; purifying the target protein by using a Ni ion affinity chromatography column purification method. However, due to the nonspecific adsorption of Ni ion affinity chromatography column, there are often more foreign proteins after affinity chromatography treatment, and more complicated purification means such as molecular sieves and other means are required to obtain a protein with higher purity, and 6 XHis-tag is not cleaved.

Patent documents CN103819563A and CN108508210A provide a method for fusion expression and then purification of MBP-tag and target protein, respectively, and the introduction of MBP-tag significantly improves the solubility of fusion protein, but when detecting whether target protein is expressed, escherichia coli liquid still needs to be collected, after being crushed and enriched, MBP-beads are used for adsorption, and SDS-PAFE gel is used for judgment, which increases many complicated procedures.

In summary, it is desirable for those skilled in the art to develop a novel protein expression and purification method, which can not only intuitively determine the expression of the target protein, but also purify and enrich the target protein with high efficiency and high purity.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a protein expression and purification method, which promotes the soluble expression of target protein, can visually judge the expression condition of the target protein and carry out high-efficiency and high-purity purification and enrichment on the target protein.

To this end, the first aspect of the present invention provides a fusion protein for expressing a purified protein of interest, comprising: the kit comprises a target protein, a first enzyme cutting site, a report mark and a first purification label which are sequentially connected with one end of the target protein, and a second enzyme cutting site and a second purification label which are sequentially connected with the other end of the target protein;

wherein the target protein does not contain the first enzyme cutting site or the second enzyme cutting site.

As is known to those skilled in the art, proteins have an amino terminus (N-terminus) and a carboxy terminus (C-terminus). When one end of the target protein is an N end, the other end of the target protein is a C end; when one end of the target protein is a C end, the other end of the target protein is an N end.

Further, the first enzyme cutting site and the second enzyme cutting site are the same, and are selected from one of Thrombin (Thrombin) cutting site, HRV3C (human rhinovirus 3C) cutting site, tev (tobaco etch virus) cutting site, enterokinase (enterokinase) cutting site and SUMO protease (SUMOProtease) cutting site.

Further, the first purification tag is selected from one of histidine tag (His-tag), Flag tag (Flag-tag), HA tag (HA-tag) and SUMO tag (SUMO-tag).

Further, the second purification tag has a function of increasing the expression level and/or solubility of the fusion protein.

Further, the second purification tag is a fusion tag formed by connecting one or two of a maltose binding protein tag (MBP-tag) and a glutathione mercaptotransferase tag (GST-tag).

Further, the report mark is selected from one of Chloramphenicol Acetyl Transferase (CAT), beta-galactosyltransferase, horseradish peroxidase, luciferase, alkaline phosphatase and fluorescent protein. Further, the fluorescent protein is selected from one of Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), Yellow Fluorescent Protein (YFP), mCherry, mTAGBFP2, mCLOVER3, EGFP, Venus, mTAGBFP2, mffp and TagRFP 657.

In a specific embodiment, the protein of interest is a 2019-nCov nucleocapsid protein having an amino acid sequence as set forth in seq id NO: 1, the N end of the probe is sequentially connected with an HRV3C enzyme cutting site and an MBP-tag, and the C end of the probe is sequentially connected with the HRV3C enzyme cutting site, GFP and His-tag; the amino acid sequence of the fusion protein is SEQ ID NO: 8.

in a second aspect of the invention, a gene encoding the fusion protein is provided.

In a specific embodiment, the amino acid sequence of the fusion protein is SEQ ID NO: 8, the nucleotide sequence of the gene for coding the fusion protein is SEQ ID NO: 7.

in a third aspect of the invention, an expression vector or cassette containing the gene is provided.

In a fourth aspect of the present invention, there is provided a method for purifying a protein by expression, comprising:

s1, constructing an expression vector for expressing the fusion protein, and transferring the expression vector into an expression strain; culturing the expression strain under the condition suitable for expressing the fusion protein, collecting and culturing to obtain thalli, and performing cell disruption to obtain a crude product I;

s2, purifying the crude product I in a mode of specifically sorting the second purification tag to obtain a crude product II;

s3, adding enzymes which are specifically identified and cut the first enzyme cutting site and the second enzyme cutting site into the crude product II, and carrying out enzyme cutting reaction to obtain a crude product III;

s4, purifying the crude product III respectively in a mode of specifically sorting the second purification tag and a mode of specifically sorting the first purification tag to obtain the target protein.

Further, the construction step of the expression vector comprises: the nucleotide sequence encoding the protein of interest is codon optimized for expression in the expression strain.

Codon optimization can employ strategies common in the art, such as optimization based on codon preference of the expression strain, optimization based on predicted mRNA secondary structure, optimization based on GC content or thermostability, and the like.

The expression vector is a molecule or entity, such as a nucleic acid, plasmid, phage or virus, suitable for transformation into the expression strain and which, upon transformation into an expression strain, can express the fusion protein under conditions; preferably a plasmid. Unless otherwise stated, the specific procedures for constructing the expression vector should not be construed as limiting the present invention, and the construction of the expression vector can be performed by methods generally used in the art, such as enzymatic ligation, Gibson assembly, Golden Gate, etc.

Further, the expression strain is a prokaryotic expression strain or a eukaryotic expression strain.

Further, the expression strain is selected from escherichia coli, pichia pastoris, saccharomyces cerevisiae or schizosaccharomyces; preferably E.coli.

Further, when the purification tag is His-tag, the specific sorting of the His-tag is performed by eluting with a Ni ion affinity chromatography column; when the purification tag is Flag-tag, the specific sorting of Flag-tag is performed by elution through an anti-Flag affinity column; when the purification label is HA-tag, the mode of specifically sorting HA-tag is Anti-HA immunomagnetic bead affinity column elution; when the purification tag is SUMO-tag and Hist-ag combined, the tag can be specifically sorted by eluting with Ni ion affinity chromatography column; when the purification tag is MBP-tag, the specific MBP-tag sorting mode is MBP-magnetic bead adsorption; when the purification tag is GST-tag, the GST-tag can be specifically sorted by GST-magnetic bead adsorption.

In a specific embodiment, the step of constructing an expression vector expressing the fusion protein comprises:

(1) codon optimizing a nucleotide sequence encoding a protein of interest for expression in said expression strain;

(2) connecting the optimized nucleotide sequence for coding the target protein and the nucleotide sequence for coding the report marker through PCR reaction to obtain a nucleic acid fragment I; a first enzyme cutting site is arranged between the optimized nucleotide sequence for coding the target protein and the nucleotide sequence for coding the report marker, and the 5 'end and the 3' end of the nucleic acid fragment I are respectively provided with an enzyme cutting site I and an enzyme cutting site II;

(3) ligating the nucleic acid fragment I into a plasmid vector by enzyme digestion connection, wherein the plasmid vector comprises the following components in sequence from 5 'to 3': and preparing the expression vector of the fusion protein by using a second purification marker, a second enzyme cutting site, an enzyme cutting site I, an enzyme cutting site II and a first purification marker.

Taking the preferred specific purification tags (the first purification tag is His-tag, the second purification tag is MBP-tag) and the report tag (GFP) as examples, the invention concept of the invention is explained as follows:

his-tag is generally small, but the specificity is poor in the purification process, and other impurity proteins are often left; MBP-tag can increase the expression level of a target protein, and the purification efficiency is high, but the MBP-tag itself is bulky and needs to be cleaved by a protease after affinity purification. In addition, His-tag and MBP-tag have the common disadvantage that whether the protein is expressed or not can not be intuitively known, and whether the target protein is expressed or not can be generally known only by carrying out SDS-PAGE analysis after cells are crushed, enriched and purified. In the prior art, GFP is fused at one end of a target protein, so that whether the fusion protein is expressed or not can be intuitively known by using a fluorescence microscope, but His-tag carried by GFP is poor in specificity during purification, and is complicated in subsequent purification.

In the expression purification method provided by the invention, in the aspect of expression, the MBP-tag can improve the expression yield and solubility of the fusion protein, and the fused GFP can provide a visual judgment basis; in the aspect of purification, an MBP-magnetic bead one-step purification mode is firstly used, then a purification tag and a report tag on the upstream and downstream of the target protein are cut off by one-step enzyme digestion, and the cut product is sequentially purified by a Ni ion affinity chromatography column (to remove GFP-His-tag) and an MBP-beads purification column (to remove MBP), namely the target protein with high concentration and high purity is obtained by purification.

Compared with the prior art, the technical scheme of the invention has the following advantages:

(1) the protein expression and purification method provided by the invention increases the solubility and yield of the protein, so that the expression of the protein is visualized; the purification steps are simple, and the purity of the target protein is obviously improved under the condition of not increasing the purification process.

(2) The protein expression and purification method provided by the invention has good universality and is particularly suitable for expressing foreign proteins in escherichia coli.

(3) The preparation method provided by the invention is convenient to operate, can visually obtain the protein expression condition, does not need repeated screening and verification, saves the time cost, and has good application prospect.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:

FIG. 1 is a schematic representation of a fusion protein for expression of a purified protein of interest; 1-a first purification tag, 2-a report tag, 3-a first enzyme cutting site, 4-a target protein, 5-a second enzyme cutting site, and 6-a second purification tag;

FIG. 2 is a plasmid map of recombinant vector pET 26-MBP-x-gfp-his;

FIG. 3 shows the results of fluorescence expression of the expression strain before induction culture;

FIG. 4 shows the results of fluorescence expression of the expression strain after induction culture;

FIG. 5 is an SDS-PAGE electrophoresis of the first MBP-beads column purification;

FIG. 6 is an SDS-PAGE of 2019-nCov nucleocapsid protein purified from example 2;

FIG. 7 is an SDS-PAGE of 2019-nCov nucleocapsid protein purified from comparative example, wherein lane 1 is the whole cell lysate and

lanes

2 and 3 are the proteins purified by Ni ion affinity column.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

EXAMPLE 1 construction of expression vectors

(1) Obtaining a gene sequence of 2019-nCov nucleocapsid protein (hereinafter referred to as X) from 2019-nCov genome DNA, and then calculating that the amino acid sequence of the target protein X is SEQ ID NO: 1, obtaining the sequence of the target gene x (SEQ ID NO: 2) by a codon optimization mode, and obtaining the target gene x by gene synthesis. By plasmid PBBR1-gfp (OuahraniBettache S, Porte F, Teyssier J, L)iautard JP,&

S. (1999), Pbbr1-gfp, a broad-host-range vector for prokarstic promoter, biotechniques,26(4),620-2, amplifying to obtain gfp gene, and obtaining x-gfp fragment by two-round PCR, wherein HRV3C enzyme cutting site is arranged between x and gfp. Primers F1, R1, F2 and R2 used in the amplification process are shown in Table 1, wherein the tail end of F1 is designed with a Nco1 enzyme cutting site, and the tail end of R2 is designed with an Xho1 enzyme cutting site;

TABLE 1

(2) T7 promoter and lacI in the vector pET26-MBP (purchased from Addgene) constitute inducible expression elements; the end of HRV3C enzyme cutting site at the downstream of MBP-tag is provided with Nco1 enzyme cutting site, and XHo1 enzyme cutting site is arranged between Nco1 enzyme cutting site and 6 XHis;

and carrying out double enzyme digestion on the vector pET26-MBP by Nco1 and Xho1, connecting the x-gfp fragment to the vector subjected to double enzyme digestion, and obtaining the recombinant vector pET26-MBP-x-gfp-his after successful connection and sequencing verification. Wherein the nucleotide sequence of the fusion protein MBP-x-gfp-his is shown as SEQ ID NO: shown at 7.

As shown in figure 2, in an expression vector pET26-MBP-x-gfp-His, the terminal 6 XHis of the gfp gene can make the recombinant protein carry histidine tag, and the MBP-tag can promote the expression and dissolution of the recombinant protein, improve the protein expression yield and facilitate the protein purification; HRV3C enzyme cutting sites are arranged between the MBP-tag and the target protein X and between the target protein X and the GFP, and the MBP-tag and the GFP-6 XHis can be cut off by using the enzyme cutting sites without influencing the structure of the target protein X.

Example 2 expression purification of protein of interest

(1) The recombinant vector pET26MBP-x-gfp-his is introduced into an escherichia coli strain BL21(DE3) through heat shock transformation, after the culture is carried out until the logarithmic phase, 0.5mM IPTG is added under the condition of 25 ℃ for induced expression for 10 hours, bacterial suspensions are respectively taken before and after the induced expression and are observed under a fluorescence microscope, the fluorescence expression condition of the strain before the induction is shown in figure 3, the fluorescence expression of the strain after the induction expression is shown in figure 4, and the strain can emit green fluorescence according to figure 4, so that the recombinant protein is judged to be expressed.

(2) Collecting the induced and expressed bacterial liquid, breaking cells, purifying by using MBP-beads chromatographic column, collecting the purified product, and the SDS-PAGE result is shown in figure 5; adding a proper amount of HRV3C protease into the purified product, incubating for 16h at 4 ℃, eluting the enzyme digestion product by MBP-beads and Ni ion affinity chromatography columns respectively, adsorbing MBP and GFP-6 XHis by the purification columns respectively, collecting effluent liquid to obtain pure target protein X, and finally further verifying and purifying by an SDS-PAGE method to obtain the target protein X, wherein the result is shown in figure 6.

Comparative example

This comparative example used methods commonly used in the art for 2019-nCov nucleocapsid protein expression. The method comprises the following specific steps:

pET26b was used as an expression vector, and the sequence shown in SEQ ID NO: 2 was ligated into pET26b for inducible expression. Escherichia coli strain BL21(DE3) was used as an expression vector, and after culturing to logarithmic phase, 0.5mM IPTG was added at 25 ℃ to induce expression for 10 hours.

When protein expression was carried out by the same or similar method as in comparative example, it was judged after cell disruption, enrichment, purification, and SDS-PAGE for the purpose of determining the yield of the target protein; the protein yield of the method depends on the solubility of the target protein, when the solubility of the target protein is poor, the yield is low, and the prepared protein product is easy to contain impurities. In this comparative example, after purification using a Ni ion affinity column, a protein product was obtained, and the SDS-PAGE electrophoresis pattern of the protein product is shown in FIG. 7, and it can be seen from FIG. 7 that the product contained a certain amount of protein as an impurity.

Compared with the conventional method commonly used in the field, the preparation method provided by the invention can know whether the target protein is expressed or not through a fluorescence microscope, and the yield of the target protein is higher; comparing fig. 6 and fig. 7, it can be seen that, although the present invention adds an enzyme digestion step, the purity of the prepared target protein is significantly higher due to the use of the multi-tag protein and the higher specificity of MBP than His tag, so that the preparation method of the present invention has a higher benefit effect.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. A fusion protein for expression and purification of a protein of interest, said fusion protein comprising: the kit comprises a target protein, a first enzyme cutting site, a report mark and a first purification label which are sequentially connected with one end of the target protein, and a second enzyme cutting site and a second purification label which are sequentially connected with the other end of the target protein;

2. The fusion protein of claim 1, wherein the first and second cleavage sites are the same and are selected from one of a thrombin cleavage site, a HRV3C cleavage site, a TEV cleavage site, an enterokinase cleavage site, and a SUMO protease cleavage site.

3. The fusion protein of claim 1, wherein the first purification tag is selected from one of a histidine tag, a Flag tag, an HA tag, and a SUMO tag.

4. The fusion protein of claim 1, wherein the second purification tag has a function of increasing the expression level and/or solubility of the fusion protein; and/or the presence of a gas in the gas,

the second purification tag is a fusion tag formed by connecting one or two of a maltose-binding protein tag and a glutathione mercaptotransferase tag.

5. The fusion protein of claim 1, wherein the reporter label is selected from the group consisting of chloramphenicol-acetyltransferase, β -galactosyltransferase, horseradish peroxidase, luciferase, alkaline phosphatase, and fluorescent protein;

preferably, the fluorescent protein is selected from one of green fluorescent protein, red fluorescent protein, yellow fluorescent protein, mCherry, mTAGBFP2, mCLOVER3, EGFP, Venus, mTAGBFP2, mffp and TagRFP 657.

6. A gene encoding the fusion protein of any one of claims 1 to 5.

7. An expression vector or cassette comprising the gene of claim 6.

8. A method for expressing and purifying a protein, comprising the steps of:

s1, constructing an expression vector for expressing the fusion protein of any one of claims 1-5, and transferring the expression vector into an expression strain; culturing the expression strain under the condition suitable for expressing the fusion protein, collecting and culturing to obtain thalli, and performing cell disruption to obtain a crude product I;

s4, purifying the crude product III respectively in a mode of specifically sorting the second purification label and a mode of specifically sorting the first purification label to obtain the target protein.

9. The method of claim 8, wherein the step of constructing the expression vector comprises:

codon optimizing a nucleotide sequence encoding said protein of interest for expression in said expression strain;

preferably, the method further comprises the following steps:

connecting the optimized nucleotide sequence for coding the target protein and the nucleotide sequence for coding the report mark through PCR reaction to obtain a nucleic acid fragment I; a first enzyme cutting site is arranged between the optimized nucleotide sequence for coding the target protein and the nucleotide sequence for coding the report marker, and the 5 'end and the 3' end of the nucleic acid fragment I are respectively provided with an enzyme cutting site I and an enzyme cutting site II;

ligating the nucleic acid fragment I into a plasmid vector by enzyme digestion connection, wherein the plasmid vector comprises the following components in sequence from 5 'to 3': and preparing a second purification marker, a second enzyme cutting site, an enzyme cutting site I, an enzyme cutting site II and a first purification marker to obtain the expression vector.

10. The method of claim 8 or 9, wherein the expression strain is a prokaryotic expression strain or a eukaryotic expression strain; and/or the presence of a gas in the gas,

the expression strain is selected from escherichia coli, pichia pastoris, saccharomyces cerevisiae or schizosaccharomyces.