Isopeptide bond-based surface display system and construction method and application thereof

Abstract

The invention relates to a surface display system based on an isopeptide bond and a construction method and application of the surface display system, two conformational proteins including an anchor protein containing the isopeptide bond and a target protein are designed through a modularization method, and through covalent coupling formed between lysine and asparagine or aspartic acid residues in a molecular adhesive system based on the isopeptide bond, the surface display system based on the isopeptide bond is obtained. And displaying the target protein on the surface of escherichia coli. According to the surface display system based on the isopeptide bond, high-efficiency display of complex enzyme and macromolecular protein on the cell surface is realized, and the influence of direct fusion with anchoring protein on the activity of target protein is avoided. According to the constructed surface display whole-cell catalyst, the tedious enzyme purification process is omitted, the stability of the enzyme is improved, the production cost is lower, and the surface display whole-cell catalyst shows a good application prospect in biological catalysis.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and particularly relates to a surface display system based on isopeptide bonds, a construction method thereof, and applications thereof Background Art

[0002] Industrial biotechnology characterized by biocatalysis has gradually become the main approach for green manufacturing. Among them, whole-cell biocatalysis has gradually attracted people's attention in recent years, especially its applications in the field of industrial bioprocessing have been increasing. Compared with the catalytic reaction of extracted enzymes, the complete multi-enzyme system in cells can achieve enzyme cascade reactions, thus making up for the deficiency that the cascade catalysis process is not easily achieved in enzymatic catalysis, improving the catalytic efficiency, and at the same time eliminating the cumbersome enzyme purification process, making the preparation simpler and the production cost lower (Chinese Journal of Biotechnology, 2010, 30(4): 110-115.). However, whole-cell biocatalysis also has deficiencies. Due to the mass transfer hindrance of the cytoplasmic membrane, the transmembrane transport of substances will be greatly restricted, making the whole-cell catalytic efficiency far lower than that of free enzyme catalysis

[0003] To reduce the adverse effects of intracellular enzyme mass transfer hindrance on the catalytic rate, many researchers have developed enzyme surface display technologies. Displaying enzymes on the surface of living cells as an immobilized catalyst has great advantages over free enzymes. For example, enzymes and substrates can react with each other without passing through the cell membrane barrier, greatly improving the reaction efficiency of enzymes (ACS Catalysis, 2018, 8(3): 2611-2614). Ice nucleation protein (INP) is the most classic carrier protein in current bacterial surface display systems. The foreign proteins fused and expressed by it can be stably expressed without affecting the growth stability of host cells (CN106754610A). INP is the most stable and effective carrier protein for expressing foreign proteins with a maximum of 60 kDa. However, the surface display efficiency of INP and the display range of target proteins are also limited, and further modification or modification is required. At the same time, some INP variants are sensitive to proteases, unstable, and the surface translocation of fusion proteins is likely to occur. Due to the advantages of specificity, stability, and rapid reaction of isopeptide molecular adhesives, it has opened up a new idea for bacterial surface display. By fusing and expressing the anchor protein and the target protein with isopeptide molecules respectively, the two conformational proteins can self-assemble in the cell and immobilize the target protein on the bacterial surface. At the same time, adding a linker peptide between each functional domain can reduce the mutual interference caused by the spatial conformation and avoid the influence on the activity of the target protein by directly fusing with the anchor protein. The spontaneous reaction of isopeptide molecular adhesives is very efficient. Taking SpyTag and SpyCatcher as examples, the binding rate of the two peptide segments reaches more than 80% after reacting for 15 minutes at 25 °C and pH 7.0 (Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(12): E690-E697). Therefore, developing a new surface display system based on isopeptide bonds is expected to meet the display of complex enzymes and macromolecular proteins on the cell surface. Summary of the Invention

[0004] Technical problems to be solved: In view of the above problems, the present invention provides an isopeptide bond-based surface display system, its construction method and application. The aim is to display target proteins on the surface of Escherichia coli based on the isopeptide bonds formed between lysine and asparagine or aspartic acid residues in the molecular adhesive system, providing a new technology for the display of complex enzymes or macromolecular proteins on the cell surface.

[0005] Technical solution: A surface display system based on isopeptide bonds, comprising an isopeptide bond molecular adhesive, an anchoring protein containing an isopeptide bond, and a target protein; the system uses the isopeptide bond molecular adhesive as a medium for self-assembly, and through the specific reaction between the lysine residues in the isopeptide bond molecular adhesive and the asparagine or aspartic acid residues in the anchoring protein and the target protein, a covalent isopeptide bond is formed, thereby realizing the spontaneous coupling of the anchoring protein and the target protein; the coupling process stably immobilizes the target protein on the cell surface of the host cell to form a surface display system with specific functions.

[0006] The above-mentioned isopeptide bond molecular adhesives include IsopepTag / Pilin-C, SnoopTag / SnoopCatcher, and SpyTag / SpyCatcher.

[0007] The nucleotide sequence of the above-mentioned SpyCatcher is shown in SEQ ID NO:1.

[0008] The nucleotide sequence of the above-mentioned SpyTag is shown in SEQ ID NO:2.

[0009] The above-mentioned anchoring protein includes Lpp-OmpA, and the nucleotide sequence encoding this protein is shown in SEQ ID NO:3.

[0010] The above-mentioned target protein includes RhaB1, and the nucleotide sequence encoding this protein is shown in SEQ ID NO:4.

[0011] The above-mentioned host cell includes Escherichia coli.

[0012] The construction method of the above-mentioned surface display system includes the following steps: (1) Clone the encoding genes of SpyCatcher and Lpp-OmpA, and use the Gibson assembly strategy to ligate them to the pETDueT-1 vector to obtain the recombinant plasmid pETDueT-Lpp-OmpA-SC; (2) Clone the encoding genes of SpyTag and the target protein, and use the Gibson assembly strategy to ligate them to the recombinant plasmid pETDueT-Lpp-OmpA-SC vector to obtain the recombinant plasmid pETDueT-Lpp-OmpA-SC / RhaB1-ST; (3) Transform the surface display recombinant plasmid described in step (2) into Escherichia coli cells, and screen positive transformants according to the screening marker on the expression vector to obtain the Escherichia coli surface display recombinant strain.

[0013] The application of the above-mentioned system in surface-displaying a target protein.

[0014] The application of the above-mentioned system in surface-displaying the protein rhamnosidase RhaB1.

[0015] Beneficial effects: Aiming at the problems of low display efficiency and limited target protein size range in traditional bacterial surface display systems, the present invention has developed a surface display system based on isopeptide bonds, its construction method and application, achieving the efficient display of complex enzymes and macromolecular proteins on the cell surface. At the same time, the surface-displayed recombinant strains are used for biocatalysis, which not only eliminates the cumbersome enzyme purification process, but also improves the stability of the enzyme, with lower production costs, and shows good application prospects in biocatalysis. Description of the Drawings

[0016] Figure 1 Plasmid map of the surface display constructed in Example 1 of the present invention.

[0017] Figure 2 Fluorescence images of different Escherichia coli cells under a fluorescence microscope.

[0018] Figure 3 Results of protease K sensitivity assay of the surface-displayed recombinant strain.

[0019] Figure 4 Results of thermal stability assay of the surface-displayed recombinant strain.

[0020] Figure 5 Results of the determination of the number of reuse times of the surface-displayed recombinant strain. Detailed Embodiments

[0021] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0022] Example 1 Construction of the Surface-Displayed Recombinant Plasmid

[0023] Using isopeptide bond molecular adhesives such as IsopepTag / Pilin-C, SnoopTag / SnoopCatcherh, or SpyTag / SpyCatcher as the medium for self-assembly, the protein IsopepTag, SnoopTag, or SpyTag is fused with the target protein Passenger, and the protein Pilin-C, SnoopCatcherh, or SpyCatcher is fused with the anchor protein Anchor. The two fusion proteins are co-expressed via the vector pETduet, and a covalent isopeptide bond is formed through the specific reaction between the lysine residue in the isopeptide bond molecular adhesive and the asparagine or aspartic acid residue in the anchor protein and the target protein, thereby achieving the spontaneous coupling of the anchor protein Anchor and the target protein Passenger, stably immobilizing the target protein on the cell surface of the host cell, and forming a surface display system with specific functions. (The plasmid map is as Figure 1 shown).

[0024] Example 2 Construction of Surface Display Recombinant Plasmid

[0025] For the reported genes SpyCatcher, SpyTag, lpp-ompA, and rhaB1, gene synthesis was performed (the nucleotide sequences are shown in Table 1), and they were respectively ligated to the T vector for storage. Primers were designed to amplify the target gene rhaB1-SpyTag encoding the passenger protein and the target gene lpp-ompA-SpyCatcher encoding the anchor protein. Among them, SpyTag and SpyCatcher are a pair of isopeptide bond combinations that can complete self-assembly within the cell. The plasmid pETDuet-1 was double-digested with the restriction enzymes Kpn I and Xho I, and the digested linear vector was recovered. The pETDuet-1 linear vector was ligated with the lpp-ompA-SpyCatcher gene fragment using a seamless cloning kit and transferred into the cloning strain E. coli DH5α by heat shock. Positive clones were picked for expansion culture, and the recombinant plasmid pETDueT-Lpp-OmpA-SC was extracted. The plasmid pETDueT-Lpp-OmpA-SC was double-digested with the restriction enzymes BamH I and Hind III, and the digested linear vector was recovered. The linear vector was ligated with the rhaB1-SpyTag gene fragment using a seamless cloning kit and transferred into the cloning bacterium E. coli DH5α by heat shock. Monoclonal colonies were picked for colony PCR, and those confirmed as positive were further sequenced. The plasmid was extracted from the positive transformants with correct sequencing, and the recombinant plasmid pETDueT-Lpp-OmpA-SC / RhaB1-ST was obtained.

[0026] Table 1 Gene Base Sequences

[0027]

[0028]

[0029]

[0030] Example 3 Construction of Recombinant Strain with Surface Display

[0031] The recombinant plasmid pETDueT-Lpp-OmpA-SC / RhaB1-ST constructed in Example 1 was transferred into E. coli BL21(DE3) competent cells to obtain the expression strain E. coli BL21(DE3)RhaB1-SC / ST. The obtained expression strain was transferred at an inoculum size of 1% into liquid LB medium containing 50 μg / mL ampicillin and cultured in a constant temperature shaking incubator at 37°C until the OD600 reached 0.6 - 0.8. Subsequently, 0.5 mM IPTG was added as an inducer, and low-temperature induction was carried out at 17°C for 16 h.

[0032] The induced expression strain was centrifuged at 4°C and 10,000 rpm for 10 min, washed with PBS buffer, and then resuspended. The cells were suspended in 200 μL of blocking solution and blocked at room temperature for 30 min. Subsequently, 1 mL of mouse anti-His antibody was added and incubated on ice for 2 h. The supernatant was removed by centrifugation, and the cells were washed with PBS buffer. After adding 1 mL of FITC-labeled goat anti-mouse IgG antibody, the cells were incubated in the dark on ice for 30 min. Finally, the supernatant was removed after centrifugation, and the cells were washed and resuspended with PBS buffer and observed under a fluorescence microscope for green fluorescence. The results are as Figure 2 shown. Clear and bright green fluorescence could be seen in the recombinant Escherichia coli with surface display after induction, while almost no fluorescence signal was observed in the control group, indicating that the macromolecular protein rhamnosidase RhaB1 was successfully displayed on the surface of Escherichia coli.

[0033] Example 4 Verification of Recombinant Strain with Surface Display

[0034] The recombinant strain E. coli BL21(DE3)RhaB1-SC / ST constructed in Example 2 was induced for expression. The induced recombinant strain was centrifuged at 4°C and 10,000 rpm for 10 min, and the supernatant was removed to retain the cell pellet. After the recombinant strain was washed with PBS buffer, it was diluted to an OD600 of 1. Proteinase K at 100 μg / mL was added to the bacterial solution, and samples were taken at 0, 5, 15, 30, 60, 90, and 120 min of treatment. The treated samples were centrifuged at 10,000 rpm for 10 min, the supernatant was removed, and the cells were resuspended in the same volume of PBS buffer to measure the activity of the whole-cell catalyst. Proteinase K is a macromolecular protein that cannot pass through the cell wall, can degrade the target protein displayed on the surface of Escherichia coli, and can only act on the proteins on the cell membrane of Escherichia coli, but cannot enter the periplasmic space or act on the cell membrane. The results are as Figure 3 shown. As the treatment time of proteinase was extended, the enzyme activity gradually decreased, indicating that the anchoring protein Lpp-OmpA could direct the rhamnosidase RhaB1 to be located on the surface of Escherichia coli cells.

[0035] Analysis of the stability of the surface-displayed recombinant strain in Example 5

[0036] The recombinant strain E. coli BL21(DE3)RhaB1-SC / ST constructed in Example 2 was induced for expression. At different temperatures (4, 10, 20, 25, 30, 35, 40, 45, and 50°C), the thermal stabilities of the free enzyme RhaB1 and the surface-displayed recombinant strain E. coli BL21(DE3)RhaB1-SC / ST were investigated. As Figure 4 shown, the optimal reaction temperature of the surface-displayed recombinant strain was 37°C, and under the high-temperature reaction condition of 50°C, it could still maintain more than 50% of the highest enzyme activity, while the relative activity of the free enzyme had dropped to 22%, indicating that the surface-display system based on isopeptide bonds could significantly improve the temperature stability of the enzyme.

[0037] Analysis of the reusability of the surface-displayed recombinant strain in Example 6

[0038] The recombinant strain E. coli BL21(DE3)RhaB1-SC / ST constructed in Example 2 was induced for expression. After the recombinant strain was washed with PBS buffer, it was diluted to an OD600 of 1 with the buffer. The enzyme activity of the whole-cell catalyst was measured using 0.1 mM 4-nitrotoluene-α-L-rhamnopyranoside as the substrate. After the reaction ended, the whole-cell catalyst was washed three times with PBS buffer, and then a new substrate solution was added again for the next round of reaction, and the enzyme activity at each round of reaction was calculated. As Figure 5As shown, the engineered Escherichia coli with surface display still maintained more than 50% activity after being reused three times, demonstrating that this engineered strain with surface display can be reused multiple times while maintaining high activity and has a very good reusability rate.

Claims

1. A surface display system based on isopeptide bonds, characterized in that: The system comprises an isopeptide bond molecular adhesive, an anchor protein containing an isopeptide bond, and a target protein; the system uses the isopeptide bond molecular adhesive as a medium for self-assembly, and forms a covalent isopeptide bond through a specific reaction between the lysine residue in the isopeptide bond molecular adhesive and the asparagine or aspartic acid residue in the anchor protein and the target protein, thereby realizing spontaneous coupling between the anchor protein and the target protein; The coupling process stably fixes the target protein on the cell surface of the host cell, forming a surface display system with specific functions.

2. The surface display system according to claim 1, characterized in that: The isopeptide bond molecular adhesives include IsopepTag / Pilin-C, SnoopTag / SnoopCatcher and SpyTag / SpyCatcher.

3. The surface display system according to claim 2, characterized in that: The nucleotide sequence of the SpyCatcher is shown in SEQ ID NO:

1.

4. The surface display system according to claim 2, characterized in that: The nucleotide sequence of the SpyTag is shown in SEQ ID NO:

2.

5. The surface display system according to claim 2, characterized in that: The anchoring protein includes Lpp-OmpA, and the nucleotide sequence encoding the protein is shown in SEQ ID NO:

3.

6. The surface display system according to claim 2, characterized in that: The target protein includes RhaB1, and the nucleotide sequence encoding the protein is shown in SEQ ID NO:

4.

7. The surface display system according to claim 1, characterized in that: The host cell includes Escherichia coli.

8. The method for constructing a surface display system according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) The coding genes of SpyCatcher and Lpp-OmpA were cloned and connected to the pETDueT-1 vector using the Gibson assembly strategy to obtain the recombinant plasmid pETDueT-Lpp-OmpA-SC; (2) The coding genes of SpyTag and the target protein were cloned and connected to the recombinant plasmid pETDueT-Lpp-OmpA-SC vector using the Gibson assembly strategy to obtain the recombinant plasmid pETDueT-Lpp-OmpA-SC / RhaB1-ST; (3) The surface display recombinant plasmid described in step (2) was transformed into Escherichia coli cells, and positive transformants were selected according to the selection marker on the expression vector to obtain the Escherichia coli surface display recombinant strain.

9. Use of the system according to any one of claims 1 to 8 in surface display of a target protein.

10. Use of the system according to any one of claims 1 to 8 in surface display of protein rhamnosidase RhaB1.

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