CN117417459A - High ester bond formation efficiency latch/Tag peptide connector - Google Patents

Abstract

The invention relates to a high ester bond formation efficiency Catcher/Tag peptide connector, which comprises a binding partner (reverse Catcher) and a peptide Tag (reverse Tag), wherein the amino acid sequence of the binding partner is shown in SEQ ID NO:1, wherein the amino acid sequence of the peptide tag is shown in SEQ ID NO: 2. The reverse tag and the reverse latch of the invention have very high covalent binding efficiency, and the binding speed reaches 100 percent and can be completed within 170 s.

Description

High ester bond formation efficiency latch/Tag peptide connector

Technical Field

The invention belongs to the field of molecular peptide design, and particularly relates to a latch/Tag covalent connector with high ester bond formation efficiency.

Background

In 2014, edward N.Baker et al found that an isopeptide bond Ig-like protein was formed between Thr-Gln, and structural analysis was performed, and the PDB ID of the crystal structure was 4ni6; the team separated it into catchers and tags on a 4ni6 basis in 2017. Under the acidic environment, thr11 of Catcher and Gln14 of Tag can form covalent bond (ester bond), but glycerol and CaCl are required to be added into the system ₂ Unlike the common molecular peptide pair, the pH of the environment was adjusted to 8.0, and glycerin and Ca were added ²⁺ After dialysis out, the ester bonds can be hydrolyzed. This will find application in the field of protein separation and the like. However, the covalent bond formation efficiency is not high, which affects the expansion application.

Our earlier research results, chinese patent CN 116284278A, disclose a molecular peptide mutant EBCatcher with high ester bond formation efficiency, which can provide binding efficiency with EBtag. Although the binding efficiency is high, the speed is slow, and the requirements of certain applications cannot be met.

Disclosure of Invention

Aiming at the problems in the prior art, the invention utilizes rational design to reform and obtain mutants on the basis of EBCatcher/EBTag, and forms a reverse Catcher/reverse Tag connector, so that ester bonds can be formed rapidly and efficiently.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a peptide linker with high ester bond formation efficiency comprising a binding partner (reverse latch) having an amino acid sequence as set forth in SEQ ID NO:1, wherein the amino acid sequence of the peptide tag is shown in SEQ ID NO: 2.

The SEQ ID NO:1 (reverse catch) is TIPEVKEGTLKTTVAADGVNGSSEKEALVSYENSKDGVDVKDTIDYKDLVPNEKYNLTGKLMHVKDDGSLEEVATKTTEVTAVENGSGQWELDFGNQKLQVGEKYVVFERAESVEDLIDTDNNYE.

The SEQ ID NO:2 (reverse tag) is DTKQVVKHEDKNDKAQTLIVEKPNR.

It is another object of the present invention to provide a method for purifying the peptide linker, comprising:

(1) Cloning the reverse tag-GFP and the reverse latch-GFP on a vector to obtain a recombinant plasmid, and introducing the recombinant plasmid into host bacteria;

(2) Culturing host bacteria with recombinant plasmids to OD600 = 0.6-0.8, and then adding IPTG for induction;

(3) After induction, collecting cells after bacterial liquid centrifugation, adding PBS, and performing ultrasonic disruption;

(4) Centrifuging the crushed liquid to collect supernatant, and purifying and dialyzing to obtain purified protein.

Preferably, the vector in step (1) is pET-22b.

Preferably, the cleavage site of the vector isNdeI andXhoI。

preferably, the host bacterium in the step (1) is Escherichia coliE. coliBL21(DE3)。

Preferably, in step (2), the host bacterium harboring the recombinant plasmid is cultured in LB medium.

Preferably, the induction condition in the step (2) is that induction is carried out for 12-14 hours at 20 ℃, and the final concentration of IPTG is 0.5-1 mM.

Preferably, the centrifugation condition in the step (3) is 12000rpm for 3-5 min.

Preferably, the ultrasonic crushing condition in the step (3) is 300-400W, and crushing is carried out for 10-15 min.

Preferably, the purification in step (4) is performed in a Ni-NTA resin.

Preferably, the purified protein in step (4) is dialyzed for more than 12 hours in a 4000 Da dialysis bag.

It is a further object of the present invention to provide the use of said peptide linker in protein isolation.

Preferably, the application comprises the steps of:

(1-1) ligating a cysteine Cys residue at the N-terminus of the binding partner (reverse latch) by Linker to give Cys-reverse latch;

(1-2) connecting the N-terminal of the target protein with the reverse tag to obtain a reverse tag-target protein;

(1-3) incubating the Cys-reverse catch with a thiol protein agarose coupling resin in a coupling buffer, and then blocking with Cys to obtain a Cys-reverse catch bound resin;

(1-4) incubating the reverse tag-target protein and the Cys-reverse latch-bound resin in an ester bond reaction solution to obtain a bound resin, and then removing the residual ester bond reaction solution; the ester bond reaction liquid contains glycerol and CaCl ₂ ；

(1-5) incubating the binding resin in an ester bond hydrolysis solution to hydrolyze an ester bond and receive a target protein.

Preferably, the SEQ ID NO:3 is GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS.

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

Preferably, the temperature of the incubation in the step (1-3) is room temperature, and the time is 20-30 min. During this process, cys spontaneously forms thioether linkages with the iodoacetic acid groups of the coupling resin;

preferably, the sealing treatment in the step (1-3) is as follows: and incubating with Cys for 20-30 min. During this process, cys binds to unreacted iodoacetic acid groups.

Preferably, the step (1-3) further comprises a step of washing the Cys-reverse catcher bound resin by: rinse with 10 mM PBS pH 7.2.

Preferably, the formula of the ester bond reaction solution in the step (1-4) is as follows: 100 mM HEPES,20% glycerol, 100. Mu.M CaCl ₂ 。

Preferably, the temperature of the incubation in the step (1-4) is room temperature, and the time is 10-20 min.

Preferably, the ester bond hydrolysis liquid in the step (1-5) is 10 mM Tris,pH 8.0.

Preferably, the temperature of the incubation in the step (1-5) is room temperature, and the time is 20-30 min.

The invention has the beneficial effects that:

1. the peptide connector provided by the invention has very high covalent bonding efficiency of the reverse tag and the reverse latch, and the bonding can be completed within 170 s at a high speed of 100%;

2. the peptide connector provided by the invention is used for purifying protein, and does not need high-concentration imidazole for elution, so that dialysis for desalting is not needed, and the method is more convenient.

3. According to the protein purification method provided by the invention, the problem that the conformation of the reverseCatcher is bound and can not be combined with the reverseTag after the reverseCatcher is combined with the resin can be avoided by introducing the linker of 30 amino acids between the Cys and the reverseCatcher.

Drawings

FIG. 1 is a reverse latch/EBlatch and EBTag/reverse tag sequence alignment.

FIG. 2 is a graph comparing the ligation efficiencies of ReverseCatcher/ReverseTag to those of EBTag/ReverseTag at different concentrations.

Description of the embodiments

Example 1

This example specifically illustrates a method of designing a peptide linker.

The amino acid sequence of EBCatcher was designed by mutation by observing the original protein crystal structure. F30 is a calcium ion binding site, and a hydroxyl group is introduced into Phe to be changed into Tyr (F30Y), so that the local hydrophilicity is improved, the space structure is not changed, the free energy in an aqueous solution is reduced, and the stability is improved; a50P can reduce the flexibility of local areas, helping to promote interactions with tags; N109R and N115D can form two pairs of salt bridges with Tag to promote interaction with Tag; NR two amino acids are introduced at the C-terminal of the EBTag, so that the stability of the EBTag in an aqueous solution can be improved. These modifications to the Tag and the Catcher can greatly enhance the interaction of the Tag and the Catcher, and the efficiency of the covalent attachment of the final system is improved.

Example 2

This example illustrates the purification of mutants.

(1) ReverseTag-GFP and ReverseCatcher-GFP were manufactured in Biotechnology (Shanghai) Co., ltdPerforming total gene synthesis, cloning the added GFP to increase molecular weight on a vector pET-22b to obtain recombinant plasmids pET-22b-reverse tag-GFP and pET-22b-reverse latch-GFP, wherein enzyme cutting sites areNdeI andXhoi, the host is Escherichia coliE. coliBL21(DE3)。

(2) Will carry the recombinant plasmidE. coli BL21 (DE 3) is cultivated in LB medium at 37 ℃ until OD600 = 0.6-0.8, IPTG of 1M is added to a final concentration of 0.5-1 mM, and induction is carried out at 20 ℃ for 12-14 h.

(3) After the induction is finished, centrifuging at 12000rpm for 3-5 min, discarding the supernatant, collecting cells, adding 3-5 mL of 10 mM PBS, and crushing for 10-15 min on a 300-400W ultrasonic crusher.

(4) Centrifuging the crushing liquid at the rotating speed of 12000rpm for 10-15 min, and collecting supernatant.

(5) And (3) completely draining 20% ethanol protection liquid in the Ni-NTA pre-packed column of 1-mL, and adding 3-4 times of the column volume of BufferA to replace ethanol in the packing. The collected supernatant was poured into the medium and drained. And adding 3-4 column volumes of BufferA for eluting, and removing the impurity proteins adsorbed on the filler. And adding 3-4 times of BufferB in the column volume, and eluting the target protein.

(6) The resulting protein solution was dialyzed overnight in a 4000 Da dialysis bag.

BufferA is phosphate buffer with pH 8.0.1M, dissolved with 500 mM NaCl and 20 mM imidazole;

BufferB is phosphate buffer with pH 8.0.1M, dissolved with 500 mM NaCl and 300 mM imidazole;

1 mL of Ni-NTA pre-loaded column was purchased from Biotechnology (Shanghai) Inc. The remaining reagents were all commercially available.

Example 3

This example tested the connection efficiency of the reverse latch and reverse tag.

Experimental group: the reverse Tag-GFP and reverse Cattcher-GFP purified in example 2 were buffered at a concentration of 10. Mu.M in a 1:1 concentration in 0.1M pH 6.0 phosphate buffer (containing 20% glycerol, 100. Mu.M CaCl) ₂ And 1.5M TMAO), 300 s, 10, 20, 30,60 Samples 120, 180, 240 and 300 s were taken and ligation efficiencies were determined using SDS-PAGE.

Control group: the EBCatcher-GFP and EBTag-GFP genes disclosed in the complete gene synthesis CN 116284278A were carried out in the same manner as in example 2, except that the complete gene synthesis was carried out in the biological engineering (Shanghai) Co., ltd.

Example 4

The present example tested the connection efficiency of the reverse latch and reverse tag and compared to the connection efficiency of the eblatch/EBTag disclosed in CN 116284278A.

The concentrations of ReverseTag-GFP/ReverseCatcher-GFP and EBTag-GFP/EBCatcher-GFP were set at 10,5 and 1. Mu.M. The rest of the procedure was the same as in example 3.

In a total reaction time of 300 s, the EBTag and EBcatcher failed to achieve 100% ligation efficiency, while the ReverseTag and Reversecatcher were fully ligated at 170 s. In reaction 10 s, the ligation efficiency of the reverse tag and the reverse latch was 80% or more, whereas the ligation efficiency of the EBtag and the EBlatch was about 3%. The connection efficiency of the Tag and the latch before and after transformation is greatly improved, and the connection efficiency is not influenced by concentration.

Example 5

This example uses reverse catchers and reverse tags to purify proteins.

Connecting a cysteine Cys residue at the N-end of the ReverseCatcher through a Linker to obtain a Cys-ReverseCatcher;

5 mg of Cys-reverse latch was incubated with 1 mL of Sulfolink coupling resin (purchased from Soy Broth) in coupling buffer (50 mM Tris,5 mM EDTA-Na, pH 8.5) at room temperature for 30 min, cys spontaneously forming thioether linkages with the iodoacetic acid groups of the coupling resin;

blocking with 50 mM L-Cys for 30 min after incubation, and binding unreacted iodoacetic acid groups;

cys-reverse catch bound resin was washed on a gravity column with 10 mM PBS pH 7.2;

PBS was drained off, and the resin and the reverse tag-target protein crude enzyme solution were reacted in an ester linkage reaction solution (100 mM HEPES,20% glycerol, 100. Mu.M CaCl ₂ ) Incubating for 10 min at room temperature to fully react ester bonds between the reverse tag and the reverse latch;

draining ester bond reaction liquid, adding ester bond hydrolysis liquid (10 mM Tris,pH 8.0), incubating for 30 min at room temperature to fully hydrolyze ester bonds, and receiving target protein;

purified protein purity was verified using SDS-PAGE protein gel electrophoresis.

Example 6

This example uses reverse catchers and reverse tags to purify proteins.

Connecting a cysteine Cys residue at the N-end of the ReverseCatcher through a Linker to obtain a Cys-ReverseCatcher;

3 mg of Cys-reverse latch was incubated with 1 mL of Sulfolink coupling resin (purchased from Soy Broth) in coupling buffer (50 mM Tris,5 mM EDTA-Na, pH 8.5) at room temperature for 20 min, cys spontaneously forming thioether linkages with the iodoacetic acid groups of the coupling resin;

blocking with 50 mM L-Cys for 20 min after incubation, and binding unreacted iodoacetic acid groups;

cys-reverse catch bound resin was washed on a gravity column with 10 mM PBS pH 7.2;

PBS was drained off, and the resin and the reverse tag-target protein crude enzyme solution were reacted in an ester linkage reaction solution (100 mM HEPES,20% glycerol, 100. Mu.M CaCl ₂ ) Incubating for 10 min at room temperature to fully react ester bonds between the reverse tag and the reverse latch;

draining ester bond reaction liquid, adding ester bond hydrolysis liquid (10 mM Tris,pH 8.0), incubating for 30 min at room temperature to fully hydrolyze ester bonds, and receiving target protein;

purified protein purity was verified using SDS-PAGE protein gel electrophoresis.

Example 7

This example uses reverse catchers and reverse tags to purify proteins.

Connecting a cysteine Cys residue at the N-end of the ReverseCatcher through a Linker to obtain a Cys-ReverseCatcher;

3 mg of Cys-reverse latch was incubated with 1 mL of Sulfolink coupling resin (purchased from Soy Broth) in coupling buffer (50 mM Tris,5 mM EDTA-Na, pH 8.5) at room temperature for 30 min, cys spontaneously forming thioether linkages with the iodoacetic acid groups of the coupling resin;

blocking with 50 mM L-Cys for 20 min after incubation, and binding unreacted iodoacetic acid groups;

cys-reverse catch bound resin was washed on a gravity column with 10 mM PBS pH 7.2;

PBS was drained off, and the resin and the reverse tag-target protein crude enzyme solution were reacted in an ester linkage reaction solution (100 mM HEPES,20% glycerol, 100. Mu.M CaCl ₂ ) Incubating for 10 min at room temperature to fully react ester bonds between the reverse tag and the reverse latch;

draining ester bond reaction liquid, adding ester bond hydrolysis liquid (10 mM Tris,pH 8.0), incubating for 30 min at room temperature to fully hydrolyze ester bonds, and receiving target protein;

purified protein purity was verified using SDS-PAGE protein gel electrophoresis.

Comparative example 1

The 20% ethanol protection solution in the 1 mL Ni-NTA pre-packed column was drained and 3 column volumes of BufferA were added to replace the ethanol in the packing.

Pouring the target protein crude enzyme liquid after ultracentrifugation into the middle filler to drain. And adding 3 column volumes of BufferA for eluting, and removing the impurity proteins adsorbed on the packing.

And adding 3 times of BufferB with the column volume, and eluting the target protein.

BufferA is phosphate buffer with pH 8.0.1M, dissolved with 500 mM NaCl and 20 mM imidazole;

BufferB is phosphate buffer with pH 8.0.1M, dissolved with 500 mM NaCl and 300 mM imidazole;

1 mL of Ni-NTA pre-loaded column was purchased from Biotechnology (Shanghai) Inc. The remaining reagents were all commercially available.

Example 8

Protein purity was determined by SDS-PAGE protein gel electrophoresis:

mixing 30 μl of sample with 10 μl of 4×loading buffer, maintaining in metal bath at 100deg.C for 10 min, cooling to 4deg.C, and centrifuging at 1000-12000 rpm. 12% separation gel and 5% concentration gel were prepared using SDS-PAGE protein gel kit. The prepared sample was loaded at a voltage of 120V and an electrophoresis time of 120 min by 10. Mu.L. After the completion of the dyeing, the color is dyed by coomassie brilliant blue dye solution for 60 to 120 minutes, and then the color is decolorized by a decolorizing solution until the background is transparent. Wherein the SDS-PAGE protein gel kit is purchased from Beijing Soy Bao technology Co., ltd, and the rest reagents are all commercially available.

Group of	Protein purity/%
		Example 5	98.8
Example 6	97.9
		Example 7	98.1
Comparative example 1	67.8

The results indicate that the proteins purified by the purification system designed by the reverse tag and reverse catcher have higher purity, while the proteins purified by the HisTag and Ni-NTA are not sufficiently high.

Claims (10)

1. A latch/Tag peptide linker with high ester bond formation efficiency, comprising a binding partner having an amino acid sequence as set forth in SEQ ID NO:1, wherein the amino acid sequence of the peptide tag is shown in SEQ ID NO: 2.

2. The method for purifying a latch/Tag peptide linker of claim 1, comprising:

(1) Cloning the peptide tag and the binding partner on a vector to obtain a recombinant plasmid, and introducing the recombinant plasmid into host bacteria;

(2) Culturing host bacteria with recombinant plasmids to OD600 = 0.6-0.8, and then adding IPTG for induction;

(3) After induction, collecting cells after bacterial liquid centrifugation, adding PBS, and performing ultrasonic disruption;

(4) Centrifuging the crushed liquid to collect supernatant, and purifying and dialyzing to obtain purified protein.

3. The method of claim 2, wherein the vector of step (1) is pET-22b.

4. The method of claim 2, wherein the cleavage site of the vector isNdeI andXho I。

5. the method according to claim 2, wherein the host bacterium in the step (1) is E.coliE. coliBL21(DE3)。

6. The method of claim 2, wherein the purification in step (4) is performed in a Ni-NTA resin.

7. The method according to claim 2, wherein the purified protein of step (4) is dialyzed for more than 12 hours in a dialysis bag of 4000 Da.

8. Use of the Catcher/Tag peptide linker of claim 1 in protein isolation.

9. The application according to claim 8, characterized in that it comprises the steps of:

(1-1) ligating a cysteine Cys residue at the N-terminus of said binding partner via Linker to yield Cys-reverse catch;

(1-2) connecting the N-terminal of the target protein with the peptide tag to obtain a reverse tag-target protein;

(1-3) incubating the Cys-reverse catch with a thiol protein agarose coupling resin in a coupling buffer, and then blocking with Cys to obtain a Cys-reverse catch bound resin;

(1-4) incubating the reverse tag-target protein and the Cys-reverse latch-bound resin in an ester bond reaction solution to obtain a bound resin, and then removing the residual ester bond reaction solution; the ester bond reaction liquid contains glycerol and CaCl ₂ ；

(1-5) incubating the binding resin in an ester bond hydrolysis solution to hydrolyze an ester bond and receive a target protein.

10. The use according to claim 9, wherein the Linker has the amino acid sequence of SEQ ID NO:3.