A molecular peptide mutant with high ester bond formation efficiency

Abstract

The present invention relates to a molecular peptide mutant with high ester bond formation efficiency, the amino acid sequence of which is shown in SEQ ID NO: 1. The present invention introduces mutations at three sites without substantially affecting the spatial structure of the Catcher. The mutant EBCatcher is obtained through the combined use of these three mutations. EBCatcher significantly improves the stability of the Catcher in aqueous solution and can enhance its binding efficiency with EBTag.

Description

Technical Field

[0001] The invention belongs to the field of molecular peptide design, and particularly relates to a molecular peptide mutant with high ester bond formation efficiency. Background Art

[0002] In 2014, Edward N. Baker et al. discovered an Ig-like protein that forms an isopeptide bond between Thr and Gln and performed structural analysis. The PDB ID of its crystal structure is 4ni6. In 2017, the team separated it into Catcher and Tag based on 4ni6. In an acidic environment, Thr11 of Catcher and Gln14 of Tag can form a covalent bond (ester bond), but glycerol and CaCl2 need to be added to the system. Unlike ordinary molecular peptide pairs, the pH of the environment is adjusted to 8.0, and glycerol and CaCl2 are added. 2+ After dialysis, the ester bond can be hydrolyzed. This has applications in areas such as protein separation. However, its low efficiency in forming covalent bonds has limited its potential for expanded applications. Summary of the Invention

[0003] The present invention utilizes rational design to transform Catcher into a mutant EBCatcher, which can quickly form a covalent bond with EBTag.

[0004] The present invention introduces three mutations into the Catcher, which can significantly improve the efficiency of covalent bond formation between the Tag and the Catcher. The present invention refers to the modified Catcher as EBCatcher and the Tag as EBTag.

[0005] A molecular peptide mutant with high ester bond formation efficiency, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0006] Another object of the present invention is to provide a gene sequence encoding the above-mentioned molecular peptide mutant.

[0007] Another object of the present invention is to provide the use of the molecular peptide mutant in protein separation.

[0008] Another object of the present invention is to provide a method for purifying the molecular peptide mutant, comprising:

[0009] (1) The gene sequence of the molecular peptide is introduced into the vector to construct a recombinant plasmid, and the recombinant plasmid is introduced into the host bacteria;

[0010] (2) Cultivate the host bacteria containing the recombinant plasmid to OD 600 =0.6-0.8, then IPTG was added for induction;

[0011] (3) After induction, the bacterial culture was centrifuged and the cells were collected, resuspended in phosphate buffer, and disrupted by ultrasonication.

[0012] (4) After ultracentrifugation of the broken liquid, the supernatant was taken and purified by dialysis to obtain the purified protein.

[0013] As a preferred embodiment, in (1), the vector is pET-22b.

[0014] As a preferred embodiment, the restriction enzyme site for the vector connection is Nde I and Xho I.

[0015] As a preferred embodiment, in (1), the host bacteria is Escherichia coli E. coli BL21(DE3).

[0016] As a preferred embodiment, in (2), the Escherichia coli containing the recombinant plasmid is cultured in LB medium.

[0017] As a preferred embodiment, in (4), the supernatant is subjected to protein purification in Ni-NTA resin.

[0018] As a preferred embodiment, the purified protein is dialyzed in a 3000 Da dialysis bag for 24-26 h.

[0019] The modified EBCatcher can still form an isopeptide bond with EBTag, but the efficiency of covalent bond formation is significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the Catcher and EBCatcher sequence alignment.

[0021] Figure 2 is the ligation efficiency of EBCatcher-GFP and EBTag-GFP.

[0022] Figure 3 is the Catcher-GFP and EBTag-GFP ligation efficiency. DETAILED DESCRIPTION

[0023] The original protein crystal structure in the examples was obtained from the PDB database with PDB ID 4ni6. Example 1

[0024] This example specifically illustrates the method for designing mutants.

[0025] By observing the original protein crystal structure, mutations were designed into the Catcher amino acid sequence. At position F31, the calcium binding site, a hydroxyl group was introduced into the Phe residue, converting it to a Tyr residue (F31Y). This increased local hydrophilicity without altering the steric structure, lowering the free energy in aqueous solution and improving stability. Furthermore, within the Catcher, where F94 is located, the Phe residue was mutated into an Ile residue with a smaller side chain, effectively reducing internal steric hindrance and improving folding stability. Finally, a polar amino acid, Glu, was introduced at position Q97 to reduce the folding energy in aqueous solution without significantly altering the structure. By combining these three mutations, the stability of EBCatcher in aqueous solution was significantly improved, enhancing its binding efficiency with EBTag. Example 2

[0026] This example specifically illustrates the method for purifying the mutant.

[0027] The mutated peptide was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and a GFP was added to both EBTag and EBCatcher to increase the molecular weight. The recombinant plasmid pET-22b-EBCatcher-GFP was obtained by cloning into the vector pET-22b. The restriction enzyme cutting site was Nde I and Xho I, the host is Escherichia coli E. coli BL21(DE3). The recombinant vector of EBTag-GFP is pET-22b-EBTag-GFP, and the restriction enzyme cutting sites and host are the same as those of EBCatcher-GFP.

[0028] The recombinant plasmid E. coli BL21 (DE3) cells were cultured in LB medium at 37°C until OD 600 =0.6, add 1 M IPTG to a final concentration of 0.5 mM, and induce at 20 °C for 12 h.

[0029] After induction, the bacterial cell suspension was centrifuged at 12000 rpm to collect the cells, and 4 mL of phosphate buffer was added, the cells were resuspended by vortexing, and ultrasonically disrupted at a power of 350 W for 15 min.

[0030] The broken liquid was ultracentrifuged at 12000 rpm and 4°C for 20 min, and the supernatant was taken for protein purification using Ni-NTA resin. The purified protein was dialyzed in a 14000 Da dialysis bag for 26 h before use. Example 3

[0031] The only difference between this embodiment and embodiment 1 is that:

[0032] The recombinant plasmid E. coli BL21 (DE3) cells were cultured in LB medium at 37°C until OD 600 =0.8, add 1 M IPTG to a final concentration of 0.5 mM, and induce at 25 °C for 12 h. Example 4

[0033] This example tests the ligation efficiency of EBCatcher-GFP and Catcher-GFP.

[0034] Experimental group: The EBTag-GFP and EBCatcher-GFP described in Example 1 were mixed at a 10 μM concentration (1:1) in 0.1 M pH 6.0 phosphate buffer (containing 20% ​​glycerol and 100 μM CaCl2) and reacted at 20°C for 60 min. Samples were taken every 10 min during the reaction, and the ligation efficiency was measured by SDS-PAGE.

[0035] Control group: Catcher-GFP gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the rest of the methods were the same as those in Example 2 and the experimental group.

[0036] The protein purification method:

[0037] Drain the 20% ethanol buffer from a 1 mL Ni-NTA prepacked column and add 3-4 column volumes of Buffer A to replace the ethanol in the packing. Pour the ultracentrifuged protein sample into the packing and drain completely. Add 3-4 column volumes of Buffer A to elute the sample to remove any contaminants adsorbed to the packing. Then, add 3-4 column volumes of Buffer B to elute the target protein.

[0038] Buffer A is a 0.1 M phosphate buffer at pH 8.0, containing 500 mM NaCl and 20 mM imidazole;

[0039] Buffer B is a 0.1 M phosphate buffer at pH 8.0, containing 500 mM NaCl and 300 mM imidazole;

[0040] 1 mL Ni-NTA prepacked columns were purchased from Sangon Biotech (Shanghai) Co., Ltd. All other reagents were commercially available.

[0041] SDS-PAGE protein gel electrophoresis method:

[0042] Mix 30 μL of sample with 10 μL of 4× loading buffer and incubate in a 100°C metal bath for 10 minutes. After cooling to 4°C, centrifuge at 1000–12000 rpm. Use an SDS-PAGE protein gel kit to prepare a 12% separating gel and a 5% stacking gel. Load 10 μL of the prepared sample, and run the gel at 120 V for 120 minutes. Stain with Coomassie Brilliant Blue for 60–120 minutes, then destain with a destaining buffer until the background is transparent. Gel images were taken using a gel imager, and band density analysis was performed using ImageJ to determine ligation efficiency. The SDS-PAGE protein gel kit was purchased from Beijing Solebold Technology Co., Ltd.; all other reagents were commercially available.

[0043] The results are as follows Figure 2 and Figure 3 As shown (the ordinate represents the protein complex yield), it can be seen that the molecular peptide mutant EBCatcher calculated by the present invention not only retains the ability to form an isopeptide bond with EBTag, but also significantly increases the rate of covalent bond formation. Furthermore, the protein induced expression results in Example 2 differ slightly from those in Example 1, but this has no significant effect on the final rate of covalent bond formation.

Claims

1. A molecular peptide mutant with high ester bond formation efficiency, characterized in that: The amino acid sequence is shown in SEQ ID NO:

1.

2. A gene encoding the mutant of the molecular peptide according to claim 1.

3. The method for purifying the molecular peptide mutant according to claim 1, characterized in that: include: (1) The gene sequence of the molecular peptide is introduced into the vector to construct a recombinant plasmid, and the recombinant plasmid is introduced into the host bacteria; (2) Cultivate the host bacteria containing the recombinant plasmid to OD 600 =0.6-0.8, then IPTG was added for induction; (3) After induction, the bacterial culture was centrifuged and the cells were collected, resuspended in phosphate buffer, and disrupted by ultrasonication. (4) After ultracentrifugation of the broken liquid, the supernatant was taken and purified by dialysis to obtain the purified protein.

4. The method according to claim 3, characterized in that In the above (1), the vector used is pET-22b.

5. The method according to claim 3, wherein The restriction enzyme cutting site for the vector is Nde I and Xho I.

6. The method according to claim 3, wherein In (1), the host bacteria is Escherichia coli E. coli BL21(DE3).

7. The method according to claim 3, characterized in that In the above (2), Escherichia coli containing the recombinant plasmid is cultured in LB medium.

8. The method according to claim 3, characterized in that In (4), the supernatant was subjected to protein purification on Ni-NTA resin.

9. The method according to claim 3, wherein The purified protein was dialyzed in a 3000 Da dialysis bag for 24-26 h.

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