CN116589591B - Application of MBP (MBP) tag, spy tag or MBP-Spy tandem tag in auxiliary recombinant protein expression - Google Patents
Application of MBP (MBP) tag, spy tag or MBP-Spy tandem tag in auxiliary recombinant protein expression Download PDFInfo
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/24—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a MBP (maltose binding protein)-tag
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- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses an application of MBP (MBP) tag, spy tag or MBP-Spy tandem tag in assisting recombinant protein expression. Also discloses a recombinant protein which is formed by connecting the above-mentioned label at the N-terminal of the protein. And corresponding coding genes, expression vectors and host bacteria. The invention uses the Spy label or the MBP label to promote the dissolubility of the recombinant protein, assist the protein to fold correctly, and is helpful for improving the expression quantity of the protein. Wherein the MBP-Spy tandem tag is significantly more effective than either the Spy tag or the MBP tag alone.
Description
Technical Field
The invention relates to an application of an MBP tag, a Spy tag or an MBP-Spy tandem tag in assisting recombinant protein expression, and belongs to the technical field of protein expression.
Background
Collagen (Collagen) is a major component of extracellular matrix, accounting for about 25-30% of total protein in animals, and is widely present in connective tissue of animals to support and protect organisms and organs. At present, recombinant collagen has become a hot spot in the field, and not only can obtain a large amount of gene expression products in a short time, but also the required cost is relatively low. However, the lack of post-translational processing of the prokaryotic-expressed recombinant collagen results in incorrect protein folding to form insoluble inclusion bodies, and the subsequent complex process treatment is required to improve the protein yield and purity, so that the cost is increased and the protein yield loss is increased. In order to express large amounts of soluble foreign proteins, a lytic tag is often used to facilitate soluble expression of the foreign recombinant protein.
Maltose Binding Protein (MBP) labeled recombinant protein can reduce toxicity, improve expression level and solubility, however, MBP has too large molecular weight (42 kDa) and can cause insolubility or aggregation of target protein when MBP tag is removed; the dissolution-promoting label Spy is a bacterial periplasm partner, is a dimer with a cradle-shaped surface, and utilizes the interaction of hydrophobic and electrostatic force with a substrate thereof to inhibit protein aggregation and assist protein folding or refolding, and can promote the solubility of different recombinant proteins and improve the stability and yield of target proteins. Therefore, the MBP label, the Spy label or the MBP-Spy tandem label can not only increase the solubility of the recombinant protein, but also assist the folding of the protein and increase the expression quantity of the protein.
Disclosure of Invention
The present invention aims at utilizing a dissolution promoting tag to enhance the expression and dissolution of recombinant proteins.
The invention adopts the technical scheme that:
use of MBP tags or Spy tags to assist in recombinant protein expression.
The invention also discloses an MBP-Spy serial label which is formed by connecting the MBP label and the Spy label in series.
Preferably, the MBP tag and the Spy tag are connected through a Linker 1.
Preferably, the amino acid sequence of Linker1 is GSGGGS.
The use of the tandem tag described above to assist in recombinant protein expression.
The invention also discloses a recombinant protein, which is formed by connecting a label at the N end of the protein, wherein the label is an MBP label or a Spy label or the MBP-Spy serial label.
Preferably, the protein is collagen or fibronectin.
Preferably, the tag is linked to the protein by Linker2, wherein Linker2 is (EAAAK) 3 。
Preferably, a TEV protease cleavage sequence ENLYFQG is also arranged between the Linker2 and the protein.
The invention also discloses a coding gene of the recombinant protein.
The invention also discloses an expression vector of the recombinant protein.
Preferably, the vector is pSEVA321.
The invention also discloses an expression host bacterium of the recombinant protein.
Preferably, the host bacterium is E.coli.
The invention has the beneficial effects that:
the invention uses the Spy label or the MBP label to promote the dissolubility of the recombinant protein, assist the protein to fold correctly, and is helpful for improving the expression quantity of the protein. Wherein the MBP-Spy tandem tag is significantly more effective than either the Spy tag or the MBP tag alone.
Drawings
FIG. 1 shows pSEVA321-His 6 Plasmid map of MBP-Spy-Col 1.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting. Specific materials and sources thereof used in embodiments of the present invention are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention, as materials that are the same as or similar to the type, model, quality, nature, or function of the reagents and instruments described below may be used in the practice of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1: preparation of single tag fusion proteins
1. Plasmid construction
(1) Construction of pSEVA321-Spy and pSEVA321-MBP expression vectors
Synthesizing Spy and MBP gene sequences according to a protein sequence commission biological company, wherein the amino acid sequence of a dissolution promoting tag Spy is shown as SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 4; the amino acid sequence of the dissolution promoting tag MBP is shown as SEQ ID NO.5, and the nucleotide sequence is shown as SEQ ID NO. 6. Primers are designed through Snapgene software, gene fragments of Spy and MBP are amplified through a PCR technology, PCR products are recovered, and the Spy gene and the MBP sequence are connected to the vector through a Gibson connection technology by utilizing homology arm primers to construct expression vectors pSEVA321-Spy and pSEVA321-MBP. And (3) transforming the connection product into E.coli DH5 alpha competent cells, screening positive transformants by chlormyces resistance, selecting the transformants for agarose gel electrophoresis verification, and selecting the positive transformants for sequencing verification and plasmid extraction.
And (3) PCR amplification:
| system components | Volume of the composition |
| Primer F | 2.5μL |
| Primer R | 2.5μL |
| Q5 High-Fidelity 2x Master Mix | 2μL |
| Template DNA | 25μL |
| ddH 2 O | To 50μL |
After the PCR system is prepared, the mixture is evenly mixed and centrifuged, and the PCR amplification conditions are as follows: the first stage is pre-denaturation at 98 ℃ for 30s; the second stage of denaturation at 98 ℃ for 10s, annealing at 50-72 ℃ for 30s, extension at 72 ℃ for 30s/kb,32 cycles; the third stage extends at 72 deg.C for 2min. The above vectors and gene fragments were recovered using a universal DNA purification kit (Tiangen Biochemical Co., ltd.) and carried out according to the procedure of the product instruction.
Gibson connection:
| system components | Volume of the composition |
| Gibson Assembly Master Mix(2X) | 5μL |
| Ligation fragment | 0.2–1pmols*XμL |
| dd H 2 O | (5-X)μL |
| Total system | 10μL |
Mixing above components on ice, and heating at 37deg.C for 60min to obtain a connection product, and storing on ice or at-20deg.C for subsequent competent transformation.
Transformation of E.coli DH 5. Alpha. Competent cells: slowly thawing competent bacteria on ice (the competent bacteria can be directly used for fresh preparation), adding 3-5ul of the connection product, flicking a centrifuge tube with a light finger to mix the bacteria and the plasmid connection product, standing in an ice bath for 30min, performing heat shock in a water bath at 42 ℃ for 2min after the completion, adding 500ul of LB, performing shaking culture at a constant temperature of 37 ℃ and 220rpm for 1 hour, centrifuging 2000-3000g for 1min after culture to precipitate thalli, leaving about 50-100ul of supernatant to resuspend and precipitate, then coating all on an LB plate containing ampicillin resistance, and performing inversion culture at 37 ℃ for overnight.
(2)pSEVA321-His 6- Spy-Col1 and pSEVA321-His 6- MBP-Col1 expression vector construction
Synthesizing Col1 gene sequence from biological company according to nucleotide sequence, wherein the amino acid sequence of type I collagen Col1 is shown as SEQ ID NO.1, the nucleotide sequence is shown as SEQ ID NO.2, respectively designing primers, amplifying by PCR, recovering PCR product, connecting the gene to pSEVA321-Spy and pSEVA321-MBP vector by Gibson, simultaneously connecting His to N terminal of Spy and MBP respectively 6 The tag is used for facilitating purification, and a primer is respectively designed between the Spy/MBP tag and the Col1 to insert a Tobacco Etching Virus (TEV) protease cleavage sequence (ENLYFQG), so that subsequent cutting and removal of the tag are facilitated. With a linker L between the tag and Col1 2 Ligation to construct pSEVA321-His 6 -Spy-Col1 and pSEVA321-His 6 MBP-Col1. Transforming the connection product into E.coli DH5 alpha competent cells, screening positive transformants by chlormyces resistance, selecting transformants for agarose gel electrophoresis verification, carrying out transfer test on positive clones, determining that each gene is connected correctly if sequencing is correct, and constructing a vector pSEVA321-His 6 Col1 served as control.
2. Expression of fusion proteins
pSEVA321-His 6 -Col1、pSEVA321-His 6 -Spy-Col1 and pSEVA321-His 6 The MBP-Col1 plasmid was transformed into competent cells of E.coli BL21 (DE 3) to obtain three recombinant E.coli BL21 (DE 3). Three recombinant E.coli BL21 (DE 3) cultured overnight were picked and inoculated into 5mL of LB liquid medium with ampicillin, and shake-cultured at a constant temperature of 37℃and 220rpm for 10-12 hours as seed solution. Inoculating the obtained seed solution into 20mL culture medium at a volume ratio of 1:100, placing at 37deg.C, and shake culturing at 220rpm until bacterial liquid OD 600 When the value of (2) is 0.6-0.8, 1mM IPTG (isopropyl-beta-D-thiogalactoside) is added to induce the expression of collagen, and the culture is continued for 5-6 hours.
3. Fusion protein solubility and expression level detection
After induction was completed, the cell particles were collected by centrifugation at 12000 rpm for 3min at 4℃and resuspended in 100. Mu.L of lysis buffer (50 mM Tris-HCl, 1mM EDTA,pH 7.5), 100 Xprotease inhibitor cocktail was added, the sample was incubated at 4℃for 5min, then after disruption of the cells by sonication using a 300W sonicator for 30min (sonication 3s gap 3 s), the supernatant and pellet were collected by centrifugation at 4℃and 12000 rpm for 20min, the pellet was resuspended with 100. Mu.L of 2% SDS, 10. Mu.L of each of the supernatant and pellet was mixed with 6 Xprotein loading buffer, SDS-PAGE was performed after boiling for 10 min, and the expression of the soluble fraction and insoluble fraction was compared. Western blot was performed according to standard protocols, proteins were transferred to polyvinylidene fluoride (PVDF) membranes and then detected with anti-His antibodies.
The results are shown in Table 1, and compared with the control group pSEVA321-His 6 The foreign proteins in the E.coli strain containing the pro-lytic tags MBP and Spy produce a relatively higher amount of soluble proteins than Col1. The MBP-tagged strain had a 32% increase in soluble protein and the Spy-tagged recombinant strain had a 30% increase in soluble expression of its foreign protein compared to the control group, indicating that both tags promote soluble expression of the foreign protein in e.
TABLE 1 control group and soluble protein expression containing the pro-lytic tag MBP, spy
| Level of solubility% | Expression level% | |
| pSEVA321-His 6 -Col1 | 18％ | 31％ |
| pSEVA321-His 6 -Spy-Col1 | 30％ | 60％ |
| pSEVA321-His 6 -MBP-Col1 | 32％ | 58％ |
Example 2: preparation of serial label MBP-Spy fusion type I collagen
1. Carrier preparation
(1) Construction of pSEVA321-MBP-Spy expression vector
Using homology arm primers, the expression vector pSEVA321-MBP-Spy was constructed by selecting Gibson ligation to ligate the Spy tandem gene to the vector. With L between MBP-Spy 1 Joint connection, L 1 For GSGGGS, the ligation product is transformed into E.coli DH5 alpha competent cells, positive transformants are screened by chlormyces resistance, the transformants are selected for agarose gel electrophoresis verification, and the positive transformants are selected for sequencing verification and plasmid extraction.
(2)pSEVA321-His 6 Construction of the expression vector of MBP-Spy-Col1
The Col1 gene was ligated to pSEVA321-MBP-Spy vector by Gibson, and the linker L was used between Col1 and the tag 2 Connection, L 2 For (EAAAK) 3 Simultaneously, the primers are designed to be respectively connected with His at the N end and the C end of the Spy of the MBP 6 Tag and TEV protease cleavage sequence, construction of vector pSEVA321-His 6 MBP-Spy-Col1, as shown in particular in FIG. 1. In addition, the vector pSEVA321-His constructed in example 1 6- Col1 served as a control.
2. Fusion protein induced expression
The two recombinant plasmids are respectively transformed into competent cells of escherichia coli BL21 (DE 3) to be cultured overnight, single colonies are selected and inoculated into 5mL of LB liquid medium with ampicillin, and the culture is carried out for 10-12 hours at a constant temperature of 37 ℃ and 220rpm to serve as seed liquid. Inoculating the obtained seed solution into 20mL culture medium at a volume ratio of 1:100, and shake culturing at constant temperature of 37deg.C and 220rpm until bacterial liquid OD 600 When the value of (2) is 0.6-0.8, adding 1mM IPTG to induce collagen expression, and culturing for 5-6h.
3. Fusion protein solubility and expression level detection
After induction was completed, cells were collected, sonicated using an sonicator after adding lysis buffer, and then supernatant and pellet were collected by centrifugation, pellet was resuspended with 100 μl of 2% SDS, protein samples were mixed with 6X protein loading buffer, boiled for 10 minutes, and subjected to SDS-PAGE. Western blot was performed according to standard protocols.
TEV protease cleavage fusion proteins
Cells collected after induction were resuspended in lysis buffer (20 mM HEPES-NaOH, pH 7.5,400mM NaCl,0.5mM EDTA,0.15. Mu.M aprotinin, 1.3mM benzamidine hydrochloride, 4.2. Mu.M leupeptin, 1. Mu.M pepstatin, 1mM phenylmethylsulfonyl fluoride) and disrupted using a cell homogenizer and centrifuged at 9500 Xg for 50min at 4 ℃. The supernatant was loaded onto a 3mL Ni-NTA agarose column and washed with lysis buffer. The fusion proteins were eluted with 300mM imidazole in lysis buffer (pH 7.5), dialyzed into rTEV buffer (25 mM Tris-HCl,150mM NaCl,pH 8.0,0.1mM DTT) overnight before TEV protease digestion, and finally centrifuged at 18000rpm for 20min at 4 ℃. According to the manufacturer's guidelines (recombinant tobacco etch virus protease, china Biotechnology Co.) TEV protease (1U/3. Mu.g recombinant protein) was added to the supernatant and the reaction was performed at 30℃for 1h (or overnight digestion at 4 ℃). After digestion, the samples were centrifuged at 18000rpm for 20min at 4℃and the supernatant and precipitate were separated. SDS-PAGE was performed on the two fractions and protein expression was observed after tag removal.
The results are shown in Table 2, and the above-mentioned composition contains pSEVA321-His 6 -Col1、pSEVA321-His 6 -Spy-Col1 and pSEVA321-His 6 Compared with the escherichia coli strain of the MBP-Col1 plasmid, the escherichia coli strain with the MBP-Spy tandem tag has the advantage that the expression level of soluble protein is 62%, which indicates that the use of the tandem tag has better effect than that of a single tag.
TABLE 2 control group and soluble type I collagen expression containing the pro-lytic tags MBP, spy and MBP-Spy tandem tags
| Level of solubility% | Expression level% | |
| pSEVA321-His 6 -Col1 | 18％ | 31％ |
| pSEVA321-His 6 -Spy-Col1 | 30％ | 60％ |
| pSEVA321-His 6 -MBP-Col1 | 32％ | 58％ |
| pSEVA321-His 6 -MBP-Spy-Col1 | 62％ | 72％ |
Example 3: preparation of serial label MBP-Spy fusion III type collagen
1.pSEVA321-His 6 Construction of the expression vector of MBP-Spy-Col3
The Col3 gene was ligated to the pSEVA321-MBP-Spy vector constructed in the above example by Gibson, the amino acid sequence of human type III collagen Col3 was shown in SEQ ID NO.7, the nucleotide sequence was shown in SEQ ID NO.8, and a linker L was used between Col3 and the tag 2 Connecting, simultaneously respectively introducing His at N end and C end of Spy of MBP 6 Tag and TEV protease cleavage sequence, construction of vector pSEVA321-His 6 MBP-Spy-Col3, while constructing the vector pSEVA321-His 6- Col3 served as control.
2. Fusion protein induced expression
The two plasmids which are successfully constructed are respectively transformed into competent cells of escherichia coli BL21 (DE 3) to be cultured overnight, single colonies are selected and inoculated into 5mL of LB liquid medium with ampicillin, and the culture is carried out for 10-12 hours at a constant temperature of 37 ℃ and 220rpm to be used as seed liquid. Inoculating the obtained seed solution into 20mL culture medium at a volume ratio of 1:100, and shake culturing at constant temperature of 37deg.C and 220rpm until bacterial liquid OD 600 When the value of (2) is 0.6-0.8, adding 1mM IPTG to induce collagen expression, and culturing for 5-6h.
3. Fusion protein solubility and expression level detection
After induction was completed, cells were collected, disrupted by sonication using an sonicator after addition of lysis buffer, supernatant and pellet were collected by centrifugation, pellet was resuspended in 100 μl of 2% SDS, protein samples were mixed with 6X protein loading buffer, boiled for 10 minutes and subjected to SDS-PAGE. Western blot was performed according to standard protocols.
TEV protease cleavage fusion proteins
Recombinant type III collagen was subjected to tag removal as described in example 2, and then subjected to SDS-PAGE to determine protein expression. The results are shown in Table 3, and compared with the control group pSEVA321-His 6 pSEVA321-His possessing the MBP-Spy tandem tag compared to Col3 6 The E.coli strain MBP-Spy-Col3 had an increase in soluble protein expression of about 40%, indicating that the use of tandem tags is not limited to type I collagen, but is equally applicable to the soluble solubilization of type III collagen.
TABLE 3 control group and soluble III-type collagen expression containing the tandem osteolytic tag MBP-Spy
| Level of solubility% | Expression level% | |
| pSEVA321-His 6 -Col3 | 20％ | 30％ |
| pSEVA321-His 6 -MBP-Spy-Col3 | 63％ | 68％ |
Example 4: preparation of tandem tag MBP-Spy fusion fibronectin
1.pSEVA321-His 6 Construction of MBP-Spy-fibro expression vector
Construction of vector pSEVA321-His as in example 2 6 MBP-Spy-fibro, while constructing the vector pSEVA321-His 6 As a control, fibronectin fibrio has the amino acid sequence shown in SEQ ID NO.9 and the nucleotide sequence shown in SEQ ID NO. 10.
2. Fusion protein induced expression
The two plasmids which are successfully constructed are respectively transformed into competent cells of escherichia coli BL21 (DE 3) to be cultured overnight, single colonies are selected and inoculated into 5mL of LB liquid medium with ampicillin, and the culture is carried out for 10-12 hours at the constant temperature of 37 ℃ and 220rpm to be used as seed liquid. Inoculating 1% seed solution into 20mL culture medium, shake culturing at 37deg.C and 220rpm until bacterial solution OD 600 When the value of (2) is 0.6-0.8, adding 1mM IPTG to induce collagen expression, and culturing for 5-6h.
3. Fusion protein solubility and expression level detection
After induction was completed, cells were collected, disrupted by sonication using an sonicator after addition of lysis buffer, supernatant and pellet were collected by centrifugation, pellet was resuspended in 100 μl of 2% SDS, protein samples were mixed with 6X protein loading buffer, boiled for 10 minutes and subjected to SDS-PAGE. Western blot was performed according to standard protocols.
TEV protease cleavage fusion proteins
After removal of the recombinant fibronectin by the tag as described in example 2, SDS-PAGE was used to determine protein expression. The results are shown in Table 4, which shows that the cells have pSEVA321-His compared with the control group 6 The expression level of the soluble protein of the MBP-Spy-fibrino escherichia coli strain is increased by 45 percent, and the expression level of the MBP-Spy-fibrino escherichia coli strain is doubled, which shows that the dissolution promoting effect of the MBP-Spy tandem tag not only acts on different types of collagen, but also acts on fibronectin.
TABLE 4 control group and soluble fibronectin expression with tandem promotion tag MBP-Spy
The calculation mode of the percentage of the soluble protein and the expression level is as follows:
Claims (13)
1. the MBP-Spy tandem tag is characterized by being formed by connecting an MBP tag and a Spy tag in series, wherein the sequence of the MBP tag is shown as SEQ ID No.5, and the sequence of the Spy tag is shown as SEQ ID No. 3.
2. MBP-Spy tandem tag according to claim 1, characterised in that the MBP tag and Spy tag are linked by Linker 1.
3. MBP-Spy tandem tag according to claim 2, characterised in that the amino acid sequence of Linker1 is GSGGGS.
4. Use of the MBP-Spy tandem tag of any one of claims 1-3 to assist in recombinant protein expression.
5. A recombinant protein, characterized in that: the protein comprises a label connected to the N-terminal of the protein, wherein the label is the MBP-Spy tandem label as claimed in any one of claims 1 to 3.
6. The recombinant protein according to claim 5, wherein: the protein is collagen or fibronectin.
7. The recombinant protein according to claim 5 or 6, wherein: the tag is connected with the protein through a Linker2, and the Linker2 is (EAAAK) 3 。
8. The recombinant protein according to claim 7, wherein: a TEV protease cleavage sequence ENLYFQG is also arranged between the Linker2 and the protein.
9. A gene encoding the recombinant protein of any one of claims 5 to 8.
10. An expression vector for a recombinant protein according to any one of claims 5-8.
11. The expression vector according to claim 10, characterized in that said vector is pSEVA321.
12. An expression host bacterium for a recombinant protein according to any one of claims 5 to 8.
13. The expression host bacterium according to claim 12, characterized in that the host bacterium is escherichia coli.
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