A catcher / tag peptide linker with high ester bond formation efficiency

Abstract

The application relates to a Catcher / Tag peptide linker with high ester bond formation efficiency, comprising a binding partner (Reverse Catcher) and a peptide tag (Reverse Tag), wherein the amino acid sequence of the binding partner is shown as SEQ ID NO:1, and the amino acid sequence of the peptide tag is shown as SEQ ID NO:2. The Reverse Tag and the Reverse Catcher have very high covalent binding efficiency, and the binding reaches 100% at a very fast speed, and can be completed within 170 s.

Description

Technical Field

[0001] This invention belongs to the field of molecular peptide design, specifically relating to a Catcher / Tag covalent linker with high ester bond formation efficiency. Background Technology

[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, with its crystal structure having a PDB ID of 4ni6. In 2017, based on 4ni6, the team separated it into Catcher and Tag. Under slightly acidic conditions, Thr11 of Catcher and Gln14 of Tag can form a covalent bond (ester bond), but this requires the addition of glycerol and CaCl2 to the system. Unlike ordinary peptide pairs, the pH of the environment is adjusted to 8.0, and glycerol and CaCl2 are added... 2+ After dialysis, the ester bonds can be hydrolyzed. This will be applied in areas such as protein separation. However, its low efficiency in forming covalent bonds limits its wider application.

[0003] The applicant's previous research, Chinese patent CN 116284278 A, discloses a molecular peptide mutant EBCatcher with high ester bond formation efficiency, which can provide binding efficiency with EBtag. Although its binding efficiency is high, the speed is slow and cannot meet the needs of certain applications. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention utilizes rational design to modify EBCatcher / EBTag to obtain a mutant, forming a ReverseCatcher / ReverseTag linker, which can rapidly and efficiently form ester bonds.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A peptide linker with high ester bond formation efficiency includes a reverse catcher and a reverse tag, wherein the amino acid sequence of the reverse catcher is shown in SEQ ID NO: 1 and the amino acid sequence of the reverse tag is shown in SEQ ID NO: 2.

[0006] The amino acid sequence of the reverse catcher of SEQ ID NO: 1 is TIPEVKEGTLKTTVAADGVNGSSEKEALVSYENSKDGVDVKDTIDYKDLVPNEKYNLTGKLMHVKDDGSLEEVATKTTEVTAVENGSGQWELDFGNQKLQVGEKYVVFERAESVEDLIDTDNNYE.

[0007] The amino acid sequence of the peptide tag (ReverseTag) of SEQ ID NO:2 is DTKQVVKHEDKNDKAQTLIVEKPNR.

[0008] Another object of the present invention is to provide a method for purifying the peptide linker, comprising: (1) ReverseTag-GFP and ReverseCatcher-GFP were cloned into a vector to obtain recombinant plasmids, and the recombinant plasmids were introduced into the host bacteria; (2) Culture the host bacteria carrying the recombinant plasmid to OD600=0.6~0.8, and then add IPTG to induce; (3) After induction, the bacterial culture was centrifuged and the cells were collected, PBS was added, and the cells were sonicated and disrupted. (4) Centrifuge the lysate to collect the supernatant, and then purify and dialyze to obtain the purified protein.

[0009] Preferably, the carrier mentioned in step (1) is pET-22b.

[0010] Preferably, the restriction enzyme sites of the vector are Nde I and Xho I.

[0011] Preferably, the host bacterium in step (1) is Escherichia coli. E. coli BL21(DE3).

[0012] Preferably, in step (2), the host bacteria carrying the recombinant plasmid are cultured in LB medium.

[0013] Preferably, the induction conditions in step (2) are 20℃ for 12~14h and the final IPTG concentration is 0.5~1mM.

[0014] Preferably, the centrifugation conditions in step (3) are 12000 rpm for 3~5 min.

[0015] Preferably, the ultrasonic disruption conditions in step (3) are 300~400W and disruption time is 10~15min.

[0016] Preferably, the purification described in step (4) is carried out in Ni-NTA resin.

[0017] Preferably, the purified protein in step (4) is dialyzed in a dialysis bag with a pressure of 4000 Da for more than 12 hours.

[0018] Another object of the present invention is to provide the application of the peptide linker in protein separation.

[0019] Preferably, the application includes the following steps: (1-1) A cysteine ​​residue (Cys) is linked to the N-terminus of the reverse catcher via a linker to obtain the Cys-Reverse Catcher; (1-2) Link the N-terminus of the target protein with a ReverseTag to obtain ReverseTag-target protein; (1-3) The Cys-ReverseCatcher was incubated with thiol protein agarose coupling resin in coupling buffer, and then blocked with Cys to obtain the resin bound to Cys-ReverseCatcher. (1-4) The resin binding the ReverseTag-target protein and the Cys-ReverseCatcher is incubated in an ester bond reaction solution to obtain the binding resin, and then the residual ester bond reaction solution is removed; the ester bond reaction solution contains glycerol and CaCl2; (1-5) The binding resin is incubated in an ester hydrolysate to hydrolyze the ester bonds and receive the target protein.

[0020] Preferably, the amino acid sequence of the linker in SEQ ID NO: 3 is GGGGSGGGGSGGGGSGGGGSGGGGSGGGSGGGS.

[0021] Preferably, the coupling buffer in steps (1-3) is 50 mM Tris, 5 mM EDTA-Na, pH 8.5.

[0022] Preferably, the incubation temperature in steps (1-3) is room temperature, and the incubation time is 20-30 min. During this process, Cys spontaneously forms thioether bonds with the iodoacetic acid groups of the coupling resin; Preferably, the sealing treatment described in steps (1-3) is: incubation with Cys for 20-30 min. During this process, Cys binds to unreacted iodoacetic acid groups.

[0023] Preferably, steps (1-3) further include a step of cleaning the resin bound to Cys-ReverseCatcher, wherein the method for cleaning the resin bound to Cys-ReverseCatcher is to rinse with 10 mM pH 7.2 PBS.

[0024] Preferably, the formulation of the ester bond reaction solution in steps (1-4) is: 100 mM HEPES, 20% glycerol, and 100 µM CaCl2.

[0025] Preferably, the incubation temperature in steps (1-4) is room temperature, and the incubation time is 10-20 min.

[0026] Preferably, the ester bond hydrolysate described in steps (1-5) is 10 mM Tris with pH 8.0.

[0027] Preferably, the incubation temperature in steps (1-5) is room temperature, and the incubation time is 20-30 min.

[0028] The beneficial effects of this invention are as follows: 1. The peptide linker provided by the present invention has a very high covalent binding efficiency between its ReverseTag and ReverseCatcher, and the binding speed reaches 100% very quickly, within 170 s; 2. Protein purification using the peptide linker provided by this invention does not require high concentrations of imidazole for elution, thus eliminating the need for dialysis to remove salts, making it more convenient.

[0029] 3. The protein purification method provided by this invention can avoid the problem that the ReverseCatcher cannot bind to the ReverseTag after its conformation is bound to the resin by introducing a 30-amino acid linker between Cys and ReverseCatcher. Attached Figure Description

[0030] Figure 1 It involves sequence alignment between ReverseCatcher / EBCatcher and EBTag / ReversTag.

[0031] Figure 2 This is a comparison chart of the connection efficiency of ReverseCatcher / ReverseTag and EBTag / ReverseTag at different concentrations. Implementation Example 1

[0032] This embodiment specifically illustrates the design method of peptide linkers.

[0033] Mutation design was performed on the amino acid sequence of EBCatcher by observing the original protein crystal structure. The F30 position is a calcium ion binding site; introducing a hydroxyl group onto Phe to become Tyr (F30Y) increases local hydrophilicity without altering the spatial structure, lowers the free energy in aqueous solution, and improves stability. A50P reduces the flexibility of the local region, contributing to enhanced interaction with the Tag. N109R and N115D can form two pairs of salt bridges with the Tag to further enhance interaction. Introducing the two amino acids NR at the C-terminus of EBTag improves the stability of the Tag in aqueous solution. These modifications to the Tag and Catcher significantly enhance their interaction, ultimately improving the covalent linkage efficiency of the system. Example 2

[0034] This embodiment specifically illustrates the purification method for mutants.

[0035] (1) ReverseTag-GFP and ReverseCatcher-GFP were synthesized in their entirety at Sangon Biotech (Shanghai) Co., Ltd. GFP was added to increase the molecular weight. The resulting recombinant plasmids pET-22b-ReverseTag-GFP and pET-22b-ReverseCatcher-GFP were cloned into the pET-22b vector. The restriction enzyme sites were: Nde I and Xho I, host is Escherichia coli E. coli BL21(DE3).

[0036] (2) The recombinant plasmid is carried E. coli BL21(DE3) was cultured in LB medium at 37 °C until OD600 = 0.6~0.8, then 1 M IPTG was added to a final concentration of 0.5~1 mM, and induced at 20 °C for 12~14 h.

[0037] (3) After induction, centrifuge at 12000 rpm for 3-5 min, discard the supernatant, collect the cells, add 3-5 mL of 10 mMPBS, and sonicate on a 300-400 W sonicator for 10-15 min.

[0038] (4) Centrifuge the broken liquid at 12,000 rpm for 10-15 min and collect the supernatant.

[0039] (5) Drain the 20% ethanol protective solution from 1 mL of the Ni-NTA pre-packed column, and add 3-4 column volumes of Buffer A to replace the ethanol in the packing material. Pour the collected supernatant into the packing material and drain it completely. Add 3-4 column volumes of Buffer A to elute and remove the impurities adsorbed on the packing material. Add 3-4 column volumes of Buffer B to elute the target protein.

[0040] (6) Dialyze the obtained protein solution overnight in a dialysis bag with a pressure of 4000 Da.

[0041] Buffer A is a 0.1M phosphate buffer with pH 8.0, containing 500 mM NaCl and 20 mM imidazole. Buffer B is a 0.1M phosphate buffer with pH 8.0, containing 500 mM NaCl and 300 mM imidazole. The 1 mL Ni-NTA pre-packed column was purchased from Sangon Biotech (Shanghai) Co., Ltd. All other reagents were commercially available. Example 3

[0042] This embodiment tests the connection efficiency between ReverseCatcher and ReverseTag.

[0043] Experimental group: The ReverseTag-GFP and ReverseCatcher-GFP purified in Example 2 were mixed at a concentration of 10 μM in a 1:1 ratio in 0.1 M pH 6.0 phosphate buffer (containing 20% ​​glycerol, 100 μM CaCl2 and 1.5 M TMAO), and reacted at room temperature for 300 s. Samples were taken at 10, 20, 30, 60, 120, 180, 240 and 300 s, and the ligation efficiency was measured by SDS-PAGE.

[0044] Control group: The EBCatcher-GFP and EBTag-GFP genes disclosed in CN 116284278 A were synthesized in their entirety at Sangon Biotech (Shanghai) Co., Ltd., and the rest of the methods were the same as in Example 2. Example 4

[0045] This embodiment tests the connection efficiency of ReverseCatcher and ReverseTag and compares it with the connection efficiency of EBCatcher / EBTag disclosed in CN 116284278 A.

[0046] The concentrations of ReverseTag-GFP / ReverseCatcher-GFP and EBTag-GFP / EBCatcher-GFP were set to 10, 5, and 1 μM, respectively. All other methods were the same as in Example 3.

[0047] Within a total reaction time of 300 seconds, EBTag and EBCatcher could not reach 100% connection efficiency, while ReverseTag and ReverseCatcher could fully connect in 170 seconds. Furthermore, after 10 seconds of reaction, the connection efficiency of ReverseTag and ReverseCatcher reached over 80%, while the connection efficiency of EBTag and EBCatcher was approximately 3%. The connection efficiency of the tags and catchers before and after the modification was significantly improved, and the connection efficiency was unaffected by concentration. Example 5

[0048] This embodiment utilizes ReverseCatcher and ReverseTag to purify proteins.

[0049] By linking a cysteine ​​Cys residue to the N-terminus of the ReverseCatcher, we obtain the Cys-ReverseCatcher. 5 mg Cys-ReverseCatcher was incubated with 1 mL SulfoLink coupling resin (purchased from Solarbio) in coupling buffer (50 mM Tris, 5 mM EDTA-Na, pH 8.5) at room temperature for 30 min. Cys spontaneously formed thioether bonds with the iodoacetic acid groups of the coupling resin. After incubation, the mixture was blocked with 50 mM L-Cys for 30 min to remove unreacted iodoacetic acid groups. The Cys-ReverseCatcher-bound resin was rinsed on a gravity column with 10 mM pH 7.2 PBS; After draining the PBS, incubate the resin and the crude enzyme solution of the target protein ReverseTag in an ester bond reaction solution (100 mM HEPES, 20% glycerol, 100 µM CaCl2) at room temperature for 10 min to allow the ester bond between ReverseTag and ReverseCatcher to react fully. After draining the ester bond reaction solution, add ester bond hydrolysis solution (10 mM Tris, pH 8.0) and incubate at room temperature for 30 min to allow the ester bonds to be fully hydrolyzed and the target protein to be received. The purity of the purified protein was verified using SDS-PAGE protein gel electrophoresis. Example 6

[0050] This embodiment utilizes ReverseCatcher and ReverseTag to purify proteins.

[0051] By linking a cysteine ​​Cys residue to the N-terminus of the ReverseCatcher, we obtain the Cys-ReverseCatcher. 3 mg Cys-ReverseCatcher was incubated with 1 mL SulfoLink coupling resin (purchased from Solarbio) in coupling buffer (50 mM Tris, 5 mM EDTA-Na, pH 8.5) at room temperature for 20 min. Cys spontaneously formed thioether bonds with the iodoacetic acid groups of the coupling resin. After incubation, the mixture was blocked with 50 mM L-Cys for 20 min to remove unreacted iodoacetic acid groups. The Cys-ReverseCatcher-bound resin was rinsed on a gravity column with 10 mM pH 7.2 PBS; After draining the PBS, incubate the resin and the crude enzyme solution of the target protein ReverseTag in an ester bond reaction solution (100 mM HEPES, 20% glycerol, 100 µM CaCl2) at room temperature for 10 min to allow the ester bond between ReverseTag and ReverseCatcher to react fully. After draining the ester bond reaction solution, add ester bond hydrolysis solution (10 mM Tris, pH 8.0) and incubate at room temperature for 30 min to allow the ester bonds to be fully hydrolyzed and the target protein to be received. The purity of the purified protein was verified using SDS-PAGE protein gel electrophoresis. Example 7

[0052] This embodiment utilizes ReverseCatcher and ReverseTag to purify proteins.

[0053] By linking a cysteine ​​Cys residue to the N-terminus of the ReverseCatcher, we obtain the Cys-ReverseCatcher. 3 mg Cys-ReverseCatcher was incubated with 1 mL SulfoLink coupling resin (purchased from Solarbio) in coupling buffer (50 mM Tris, 5 mM EDTA-Na, pH 8.5) at room temperature for 30 min. Cys spontaneously formed thioether bonds with the iodoacetic acid groups of the coupling resin. After incubation, the mixture was blocked with 50 mM L-Cys for 20 min to remove unreacted iodoacetic acid groups. The Cys-ReverseCatcher-bound resin was rinsed on a gravity column with 10 mM pH 7.2 PBS; After draining the PBS, incubate the resin and the crude enzyme solution of the target protein ReverseTag in an ester bond reaction solution (100 mM HEPES, 20% glycerol, 100 µM CaCl2) at room temperature for 10 min to allow the ester bond between ReverseTag and ReverseCatcher to react fully. After draining the ester bond reaction solution, add ester bond hydrolysis solution (10 mM Tris, pH 8.0) and incubate at room temperature for 30 min to allow the ester bonds to be fully hydrolyzed and the target protein to be received. The purity of the purified protein was verified using SDS-PAGE protein gel electrophoresis. Comparative Example 1

[0054] Drain the 20% ethanol protective solution from the 1 mL Ni-NTA pre-packed column and add 3 column volumes of Buffer A to replace the ethanol in the packing material.

[0055] After ultracentrifugation, the crude enzyme solution of the target protein was poured into the medium packing material and allowed to drain completely. Then, 3 column volumes of Buffer A were added for elution to remove impurities adsorbed on the packing material.

[0056] Add 3 column volumes of Buffer B to elute the target protein.

[0057] Buffer A is a 0.1M phosphate buffer with pH 8.0, containing 500 mM NaCl and 20 mM imidazole. Buffer B is a 0.1M phosphate buffer with pH 8.0, containing 500 mM NaCl and 300 mM imidazole. The 1 mL Ni-NTA pre-packed column was purchased from Sangon Biotech (Shanghai) Co., Ltd. All other reagents were commercially available. Example 8

[0058] Protein purity was determined using SDS-PAGE protein gel electrophoresis. Mix 30 μL of sample with 10 μL of 4× loading buffer and incubate at 100 ℃ in a metal bath for 10 min. After incubation, cool to 4 ℃ and centrifuge at 1000-12000 rpm. Prepare a 12% separating gel and a 5% stacking gel using an SDS-PAGE protein gel electrophoresis kit. Load 10 μL of the prepared sample onto the gel, electrophoresis at 120 V for 120 min. After electrophoresis, stain with Coomassie Brilliant Blue for 60-120 min, then destain until the background is clear. The SDS-PAGE protein gel electrophoresis kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., and all other reagents were commercially available.

[0059] Example 5 98.8 Example 6 97.9 Example 7 98.1 Comparative Example 1 67.8 The results showed that the protein purified by the purification system designed with ReverseTag and ReverseCatcher had higher purity, while the protein purified using HisTag and Ni-NTA had insufficient purity.

Claims

1. A Catcher / Tag peptide linker with high ester bond formation efficiency, characterized in that, It consists of a binding partner and a peptide tag, wherein the amino acid sequence of the binding partner is shown in SEQ ID NO: 1 and the amino acid sequence of the peptide tag is shown in SEQ ID NO:

2.

2. The purification method for the Catcher / Tag peptide linker according to claim 1, characterized in that, include: (1) The peptide tag and the binding coupler clone were cloned into a vector to obtain a recombinant plasmid, and the recombinant plasmid was introduced into the host bacteria; (2) Culture the host bacteria carrying the recombinant plasmid to OD600=0.6~0.8, and then add IPTG to induce; (3) After induction, the bacterial culture was centrifuged and the cells were collected, PBS was added, and the cells were sonicated and disrupted. (4) Centrifuge the lysate to collect the supernatant, and then purify and dialyze to obtain the purified protein.

3. The method according to claim 2, characterized in that, The carrier mentioned in step (1) is pET-22b.

4. The method according to claim 2, characterized in that, The restriction enzyme sites of the vector are Nde I and Xho I.

5. The method according to claim 2, characterized in that, In step (1), the host bacterium is Escherichia coli. E. coli BL21(DE3).

6. The method according to claim 2, characterized in that, The purification described in step (4) is carried out in Ni-NTA resin.

7. The method according to claim 2, characterized in that, The purified protein in step (4) was dialyzed in a dialysis bag with a pressure of 4000 Da for more than 12 hours.

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