CN113372418B - Molecular peptide with pH stimulation response behavior - Google Patents

Abstract

The invention relates to a molecular peptide with pH stimulus response behavior, wherein the amino acid sequence of the molecular peptide is shown as SEQ ID NO: 1 is shown. According to the invention, the original molecular peptide is subjected to mutation transformation, the transformed molecular peptide can still form isopeptide bonds with SpyTag, the surface of the modified molecular peptide is charged with a large amount of charges, and the modified molecular peptide has a stimulation response behavior to pH, and can control the pH of the environment before reaction to obtain different connection efficiencies.

Description

Molecular peptide with pH stimulation response behavior

Technical Field

The invention belongs to the field of molecular peptide SpyCatcher design, and particularly relates to a molecular peptide with a pH stimulus response behavior.

Background

In 2010, the Mark Howarth group, the biochemical center of the university of oxford, england, isolated polypeptide fragments that spontaneously form isopeptide bonds in pilin of the gram-positive bacterium Streptococcus pyogenes (Streptococcus pyogenes), referred to as SpyTag (13 amino acids) and SpyCatcher (116 amino acids), respectively, where Asp117 of SpyTag can spontaneously dehydrate to isopeptide bonds with Lys31 of SpyCatcher. The SpyTag/SpyCatcher has been widely used in various fields, such as in the field of protein purification, protein display systems, and the like. However, the original molecular peptide SpyTag/SpyCatcher has a wide range of reaction conditions and does not have a stimulus response behavior to the external environment. Changes in the charge density at the protein surface can alter many properties of the enzyme including aggregation resistance, cell permeability, stimulus response behavior, and the ability to bind to oppositely charged macromolecules. However, amino acid mutations are risky for proteins, and in most cases, inactivation occurs through modification, so that modification of SpyCatcher may result in failure to normally form isopeptide bonds with SpyTag.

Disclosure of Invention

The invention aims to provide a molecular peptide with pH stimulus response behavior, which can obtain a molecular peptide SpyCatcher-21 with pH stimulus response on the basis of not influencing isopeptide bond formation.

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

a molecular peptide with pH stimulation response behavior has an amino acid sequence shown in SEQ ID NO: 1 is shown.

Another object of the present invention is to protect the gene sequence encoding the molecular peptide of claim 1.

The invention also aims to provide the application of the molecular peptide in a two-enzyme or three-enzyme catalytic system.

Specifically, the coupling of SpyCatcher-21 and SpyTag to varying degrees can be used for dual-enzyme catalysis by changing the pH of the environment; or the enzyme is interacted with positively charged enzyme through electrostatic interaction to obtain a three-enzyme coupled catalytic system.

The molecular peptide of the present invention can be purified by the following means, including:

(1) introducing the gene sequence of the molecular peptide into a vector to construct a recombinant plasmid, and introducing the recombinant plasmid into host bacteria;

(2) culturing the host bacterium containing the recombinant plasmid to OD₆₀₀=0.6-0.8, followed by induction with IPTG;

(3) after induction is finished, taking a bacterium liquid, centrifuging, collecting thalli, adding a phosphate buffer solution for resuspension, and carrying out ultrasonic crushing;

(4) and (4) carrying out ultracentrifugation on the broken liquid, taking supernatant, and carrying out purification and dialysis to obtain purified protein.

Further, in the step (1), the vector is pET-22 b; the restriction enzyme site connected with the vector isNdeI andXho I。

further, in the step (1), the host bacterium is Escherichia coliE. coli BL21(DE3)。

Further, in the (2), Escherichia coli containing the recombinant plasmid is cultured in LB medium.

Further, in the (4), the supernatant is subjected to protein purification in Ni-NTA resin.

Further, the purified protein was dialyzed in a dialysis bag of 3000 Da for 24-26 h.

According to the invention, the interaction force among amino acids is fully considered to design and transform the SpyCatcher, the mutant is obtained under the condition that the core structure is not changed as much as possible, and the connection efficiency is not greatly influenced. On the basis of an original molecule SpyCatcher, the SpyCatcher is subjected to negative charge modification, the original potential is increased from-9 to-21, 10 acidic amino acid mutations are introduced on the surface of the SpyCatcher, and a molecular peptide with a response to pH stimulation is obtained on the basis of not influencing isopeptide bond formation. The modified SpyCatcher-21 can still form isopeptide bonds with SpyTag, and in contrast to the SpyCatcher, the surface of the SpyCatcher-21 is provided with a large amount of charges, and the SpyCatcher-21 can also have a stimulus response behavior to pH, so that the pH of the environment can be controlled before reaction to obtain different connection efficiencies. The designed SpyCatcher-21 with pH response can be used for obtaining coupling with different degrees by changing the pH of the environment to obtain double-enzyme catalysis on the premise of objectively needing; or can be interacted with positively charged enzyme through electrostatic interaction to obtain a three-enzyme coupled catalytic system.

Drawings

FIG. 1 is an alignment of the SpyCatcher and SpyCatcher-21 sequences.

FIG. 2 is a SpyCatcher surface charge density plot.

FIG. 3 is a graph of SpyCatcher-21 surface charge density.

FIG. 4 shows the ligation efficiency of SpyCatcher-21 in different pH environments.

FIG. 5 is the ligation efficiency of SpyCatcher in different pH environments.

Detailed Description

Example 1

This example specifically describes the method for designing Spycatcher-21.

The original SpyCatcher protein crystal structure used in the examples was obtained from the PDB database with a PDB ID of 4 mli.

The structure of the Spycatcher is introduced into a calculation software Rosetta, the Spycatcher is calculated, the surface potential is set to be-21, and the set mutant amino acid positions are all on the surface of the protein, so that the Spycatcher-21 is obtained, as shown in FIG. 1. The APBS and the VMD are used for calculating the charge density of the protein surface, as shown in FIG. 2 and FIG. 3, it can be seen that the surface of the molecular peptide Spycatcher-21 obtained by modification is provided with a large amount of charges.

Example 2

This example specifically illustrates the purification method of SpyCatcher-21.

(1) The mutated molecular peptide is subjected to whole-gene synthesis in Biotechnology engineering (Shanghai) GmbH, and cloned on a vector pET-22b to obtain a recombinant plasmid SC-21-pET-22b, wherein the restriction enzyme site isNdeI andXhoi, the host is Escherichia coliE. coli BL21(DE3)。

(2) Will carry recombinant plasmidsE. coliBL21(DE3) was cultured in LB medium at 37 ℃ to OD₆₀₀=0.6, add 1M IPTG to final concentration 0.5 mM, induce 10 h at 20 ℃.

(3) After induction, collecting thalli by centrifuging bacteria liquid at 8000 rpm, adding 4mL of phosphate buffer solution, shaking for resuspension, and carrying out ultrasonication for 10 min under the power of 300W.

(4) And (3) ultracentrifuging the crushed solution at 12000 rpm and 4 ℃ for 10 min, taking supernatant, purifying the protein in Ni-NTA resin, and dialyzing the purified protein in a 3000 Da dialysis bag for 24 h for later use.

Example 3

This example tested the efficiency of the ligation of SpyCatcher-21 and SpyCatcher at different pH.

Experimental groups: after mixing SpyCatcher-21 and SpyTag-GFP at a final concentration of 10. mu.M and adding buffers (0.1M) of pH =4, 5, 6, 7, 8, and 9, respectively, to the mixture and reacting the mixture at 25 ℃ for 180 minutes, the ligation efficiency was determined by SDS-PAGE.

Control group:

the SpyCatcher gene was synthesized in its entirety by Biotechnology engineering (Shanghai) Co., Ltd. in the same manner as in example 2.

Wherein the method for purifying the protein comprises the following steps:

1 mL of Ni-NTA pre-packed column was drained of 20% ethanol guard and 3-4 column volumes of BufferA were added to replace the ethanol in the packing. The ultracentrifuged protein sample was poured into the packing and drained. Then adding 3-4 column volumes of BufferA for elution to remove the foreign protein adsorbed on the filler. Then adding 3-4 times column volume of BufferB to elute the target protein.

Buffer pH 8.00.1M phosphate buffer with 500 mM NaCl and 20 mM imidazole;

BufferB is phosphate buffer solution with pH 8.00.1M, and 500 mM NaCl and 300 mM imidazole are dissolved in the buffer solution;

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

SDS-PAGE protein gel electrophoresis method:

mixing 30 mu L of sample with 10 mu L of 4 × loading buffer, preserving the temperature in a metal bath at 100 ℃ for 10 min, cooling to 4 ℃ after the preservation, and centrifuging at 12000 rpm at 1000-. The SDS-PAGE protein gel kit is used for preparing 12% separating gel and 5% concentrated gel. 10 mu L of the prepared sample is loaded, the voltage is 120V, and the electrophoresis time is 120 min. After the dyeing is finished, the dyeing is carried out for 60-120 min by using Coomassie brilliant blue staining solution, and then the background is decolored by using decoloring solution until the background is transparent. And (4) taking a picture in a gel imager, and analyzing the density of the strips by using ImageJ to obtain the connection efficiency. The SDS-PAGE protein gel kit is purchased from Beijing Solaibao science and technology limited company, and other reagents are all sold in the market.

As a result, as shown in fig. 4 and 5, the molecular peptide of the present invention still has the ability to form isopeptide bonds with SpyTag, has a large amount of negative charges on the surface, and can respond to pH stimulation.

Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Sequence listing

<110> Nanjing university of industry

<120> a molecular peptide having pH stimulus response behavior

<130> xb21051201

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ala Met Val Asp Thr Leu Ser Gly Leu Ser Ser Glu Gln Gly Gln Ser

1 5 10 15

Asp Asp Met Thr Ile Glu Glu Asp Ser Ala Thr His Ile Glu Phe Ser

20 25 30

Lys Arg Asp Glu Asp Gly Lys Glu Leu Ala Gly Ala Thr Met Glu Leu

35 40 45

Arg Asp Ser Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly Asp

50 55 60

Val Lys Asp Phe Tyr Leu Tyr Pro Gly Glu Tyr Thr Phe Val Glu Thr

65 70 75 80

Glu Ala Pro Asp Gly Tyr Glu Val Asp Asp Ala Ile Thr Phe Thr Val

85 90 95

Asn Glu Asp Gly Gln Val Thr Glu Glu Gly Lys Ala Thr Lys Gly Asp

100 105 110

Ala His Ile

115

Claims (9)

1. A molecular peptide with pH stimulation response behavior is characterized in that the amino acid sequence is shown as SEQ ID NO: 1 is shown.

2. A gene encoding the molecular peptide of claim 1.

3. A method for purifying a molecular peptide according to claim 1, comprising:

(1) introducing the gene of the molecular peptide into a vector to construct a recombinant plasmid, and introducing the recombinant plasmid into host bacteria;

(2) culturing the host bacterium containing the recombinant plasmid to OD₆₀₀=0.6-0.8, followed by induction with IPTG;

(3) after induction is finished, taking a bacterium liquid, centrifuging, collecting thalli, adding a phosphate buffer solution for resuspension, and carrying out ultrasonic crushing;

(4) and (4) carrying out ultracentrifugation on the broken liquid, taking supernatant, and carrying out purification and dialysis to obtain purified protein.

4. The method according to claim 3, wherein in (1), the vector is pET-22 b.

5. The method of claim 3, wherein the vector is ligated at a cleavage site selected from the group consisting ofNdeI andXho I。

6. the method according to claim 3, wherein in (1), the host bacterium is Escherichia coliE. coliBL21(DE3)。

7. The method according to claim 6, wherein in (2), the recombinant plasmid-containing Escherichia coli is cultured in LB medium.

8. The method according to claim 3, wherein in the step (4), the supernatant is subjected to protein purification in Ni-NTA resin.

9. The method of claim 3, wherein the purified protein is dialyzed in a 3000 Da dialysis bag for 24-26 h.

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