CN114685679A - Spyware mutant, preparation method thereof and application thereof in fluorescent protein system - Google Patents

Spyware mutant, preparation method thereof and application thereof in fluorescent protein system Download PDF

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CN114685679A
CN114685679A CN202011619571.5A CN202011619571A CN114685679A CN 114685679 A CN114685679 A CN 114685679A CN 202011619571 A CN202011619571 A CN 202011619571A CN 114685679 A CN114685679 A CN 114685679A
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spyware
mutant
spycatcher
fluorescent protein
tev
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张文彬
王智辉
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Jiangsu Bio Tech Co ltd
Peking University
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Peking University
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

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Abstract

The invention discloses a spyware mutant, a preparation method thereof and application thereof in a fluorescent protein system. Belongs to the technical field of biological coupling. The spyware mutant SpyCatcher-N is obtained by connecting a flexible amino acid sequence with the original N end and C end of the spyware SpyCatcher, and a TEV protease recognizable sequence is inserted into the flexible amino acid sequence in the spyware mutant SpyCatcher-N to obtain the spyware mutant SpyCatcher-NTEV. The spyware mutant prepared by the invention has the advantages that on one hand, the spyware high reactivity is kept, and on the other hand, after the ligation reaction, the molecular weight of the Catcher protein can be reduced by protease enzyme digestion. Additional molecular weight was removed by means of proteolytic cleavage.

Description

Spyware mutant, preparation method thereof and application thereof in fluorescent protein system
Technical Field
The invention belongs to the technical field of biological coupling, and particularly relates to a spyware mutant, a preparation method thereof and application thereof in a fluorescent protein system.
Background
The bioconjugation technology is indispensable in the construction of protein-polymer complexes. Classical approaches to building complex protein-macromolecule topologies include "click" chemistry, native chemical ligation, traceless chemical ligation, and enzyme-mediated ligation of inteins, sortase a, sphenoid myxose 1, and the like. Highly reactive, non-natural reactive groups are often required in chemical attachment means, which may lead to protein yield or inactivate proteins. Enzyme-mediated ligation requires only a relatively short recognition sequence for the substrate to mediate the formation of the native peptide bond. However, enzyme-mediated ligation requires recognition sequences at specific positions on the substrate, and it is difficult to mediate the ligation to obtain branched products, which limits its application in constructing protein topologies and protein-based materials.
The genetically encoded polypeptide-protein reaction provides a powerful bioconjugation approach to e.g.SpyTag/SpyCatcher. The SpyTag/SpyCatcher is derived from a pilin domain that spontaneously forms isopeptide bonds, and they are entirely based on natural amino acids. The high irreversible reactivity and stable connection of the SpyTag/SpyCatcher enable the SpyTag/SpyCatcher to be widely applied to the fields of protein hydrogel, protein topological structure, synthetic drug carrier and synthetic biology of the Spy network. The SpyTag/SpyCatcher reaction pair is continuously developing, and has a super-charge and disorder SpyCatcher (-), and the SpyTag-SpyCatcher reaction pair is orthogonal to the SpyTag-SnoopCatcher reaction pair and the SpyTag002-SpyCatcher002 reaction pair with faster reaction rate. The resolution of proteins similar to the CnaB2 domain can result in similar Tag-Catcher reaction pairs, such as SdyTag-Sdycatcher reaction pairs. However, for most Catcher proteins, the reactive group is often near the N-terminus of the protein, and is less frequently located at the C-terminus or in a protein segment. Furthermore, the Catcher protein often leaves a molecular weight of about 10kDa on the complex after the ligation reaction, which may affect the properties of the final product or elicit an immunogenic response in therapy. Recently, a polypeptide-polypeptide coupling mode mediated by spyware ligase spyLigase is developed by the Howarth task group, and spyware is further split into spyware ligase and a K label. This method significantly reduces the molecular weight of the linking moiety, but the reactivity is greatly affected. Therefore, we have developed an alternative method to search for spyware mutants, which on the one hand maintain the high reactivity of spyware and on the other hand after the ligation reaction the Catcher protein can be reduced in molecular weight by protease cleavage. Additional molecular weight was removed by means of proteolytic cleavage.
Disclosure of Invention
The present application addresses the deficiencies of the prior art and aims to provide a spyware mutant, a preparation method thereof and an application thereof in a fluorescent protein system. The spyware mutant prepared by the invention has the advantages that on one hand, the spyware high reactivity is kept, and on the other hand, after the ligation reaction, the molecular weight of the Catcher protein can be reduced by protease enzyme digestion. Additional molecular weight was removed by means of proteolytic cleavage.
The technical scheme of the invention is as follows:
the selected model proteins, yellow fluorescent protein and cyan fluorescent protein, are both derived from green fluorescent protein. Threonine (T203) at the 203 th position of the green fluorescent protein is mutated into tyrosine to obtain yellow fluorescent protein, and pi-pi interaction between a tyrosine lateral group and chromophore enables the excitation wavelength and the emission wavelength of the fluorescent protein to be subjected to red shift, wherein the excitation wavelength is 517nm, and the emission wavelength is 530 nm. The tyrosine 66 (Y66) of the green fluorescent protein is mutated into tryptophan to obtain the cyan fluorescent protein, the side group of the tryptophan enables the chromophore to finally form an indole ring instead of a phenol compound, the excitation wavelength is 435nm, and the emission wavelength is 477 nm.
In addition to the original protein domain re-splitting can get smaller connecting parts, the protein structure cycle arrangement (circular mutation) and protein engineering editing can get the same effect. If cyclic arrangement and protein engineering editing are carried out on the spyware, the original N end and C end in the spyware structure are connected by flexible segments, and the second annular structure in the spyware structure is split to obtain a new protein structure domain N end and a new protein structure domain C end. In terms of the secondary structure of the protein, the sequence before the second loop structure of spyware is edited to the C-terminal of the protein, namely, the lysine of the reaction site is moved to the C-terminal. In a new secondary structure, a Tobacco Etch Virus (TEV) enzyme recognition sequence ENLYFQG is inserted into a glycine and serine flexible region connected with the C end and the first two beta-strips of the spyware, so that enzyme digestion can be carried out after a spyware label reacts with a spyware mutant, and redundant parts of the spyware are removed, thereby achieving the purpose of reducing the molecular weight of the spyware.
A spy catcher mutant, which is spy catcher-NTEVThe amino acid sequence is shown as SEQ ID NO. 1.
The spyware mutant SpyCatcher-N is obtained by connecting a flexible amino acid sequence with the original N end and C end of the spyware SpyCatcher, and a TEV protease recognizable sequence is inserted into the flexible amino acid sequence in the spyware mutant SpyCatcher-N to obtain the spyware mutant SpyCatcher-NTEVWherein the spyware mutants Spycatcher-N and Spycatcher-NTEVThe new N end and C end are generated in the second ring part of the spyware, namely the second ring area for splitting the spyware, and the new N end and C end of the mutant can be generated.
The TEV protease recognizable sequence is ENLYFQG.
The flexible amino acid sequence is (GGS)3。
The spyware mutant Spycatcher-N has an amino acid sequence shown in SEQ ID NO. 2.
A preparation method of a spy catcher mutant comprises the following steps: the spyware mutant SpyCatcher-N is obtained by connecting a flexible amino acid sequence with the original N end and C end of the spyware SpyCatcher, and the TeV protease recognizable sequence is inserted into the flexible amino acid sequence in the spyware mutant SpyCatcher-N to obtain the spyware mutant SpyCatcher-NTEV。
An application of spyware mutant in a fluorescent protein system.
The spyware mutant is applied to a fluorescent protein model system, and the fluorescent protein is cyan fluorescent protein or yellow fluorescent protein.
The beneficial technical effects of the invention are as follows:
the invention mainly redesigns spyware, reduces the molecular weight after spyware reaction and develops a micro-trace polypeptide-polypeptide coupling method. Protein engineering editing is carried out on the spyware by utilizing a circular arrangement method to obtain a spyware mutant SpyCatcher-N, SpyCatcher-NTEV. Then spy tag-spy catcher mutantSpyCatcher-NTEVThe system is applied to a fluorescent protein plastid system, performs coupling reaction and enzyme digestion reaction, verifies the correctness of corresponding products, and effectively reduces spyware mutant SpyCatcher-NTEVThe molecular weight of (a) and the feasibility of a spyware redundant part method is reduced by a seal cycle arrangement method.
Drawings
Fig. 1 is a topology diagram of a cyclic arrangement of spyware in an embodiment of the present invention.
FIG. 2 shows spyware and spyware mutant SpyCatcher-N, SpyCatcherN in an embodiment of the present inventionTEVAnd the model protein SpyCatcher-NTEV-The gene sequence of CFP.
FIG. 3 shows spy label-yellow fluorescent protein and spy catcher mutant-NTEVCyan fluorescent protein reaction scheme.
FIG. 4 shows exemplary embodiments of the present invention, namely spyware-N, spyware-NTEVAnd (4) amino acid sequence comparison.
FIG. 5 shows the SDS-PAGE analysis of the coupled product and the cleaved product in the examples of the present invention; (b) MALDI-TOF analysis of the molecular weight of the coupled product and the cleaved product.
Detailed Description
The present invention will be described in detail with reference to the following examples and drawings.
Example 1
The gene design is as follows:
the selected model proteins, yellow fluorescent protein and cyan fluorescent protein, are both derived from green fluorescent protein. Threonine 203 (T203) at the 203 th site of the green fluorescent protein is mutated into tyrosine to obtain yellow fluorescent protein, and the side group of the tyrosine interacts with the pi-pi of chromophore to red shift the excitation wavelength and the emission wavelength of the fluorescent protein, wherein the excitation wavelength is 517nm, and the emission wavelength is 530 nm. The tyrosine 66 (Y66) of the green fluorescent protein is mutated into tryptophan to obtain the cyan fluorescent protein, the side group of the tryptophan enables the chromophore to finally form an indole ring instead of a phenol compound, the excitation wavelength is 435nm, and the emission wavelength is 477 nm.
In addition to the original protein domain re-splitting can get smaller connecting parts, the protein structure cycle arrangement (circular mutation) and protein engineering editing can get the same effect. If cyclic arrangement and protein engineering editing are carried out on the spyware, the original N end and C end in the spyware structure are connected by flexible segments, and the second annular structure in the spyware structure is split to obtain a new protein structure domain N end and a new protein structure domain C end. From the secondary structure of the protein, the sequence before the second loop structure of spyware is edited to the C-terminus of the protein, i.e., the lysine at the reaction site is moved to the C-terminus. In the novel secondary structure, a Tobacco Etch Virus (TEV) enzyme recognition sequence ENLYFQG is inserted into a glycine and serine flexible region connected with the C end and the first two beta-bars of the spyware, so that the spyware can be subjected to enzyme digestion after the reaction of a spyware label and a spyware mutant, and the redundant part of the spyware is removed to achieve the purpose of reducing the molecular weight of the spyware. As shown in fig. 1.
After a circularly arranged protein engineering method, the spyware mutant Spycatcher-N, SpyCatcher-NTEVAnd spyware amino acid sequence pairs are shown in FIG. 4. The flexible amino acid (GGS)3 sequence is connected with the original N end and C end of the spyware to obtain a spyware mutant SpyCatcher-N, and the TEV enzyme recognition sequence ENLYFQG is inserted into the flexible chain (GGS)3 sequence of the spyware mutant SpyCatcher-N to obtain a spyware mutant SpyCatcher-NTEV. Spyware mutants SpyCatcher-N and SpyCatcher-NTEVThe new N end and C end are generated in the second ring part of the spyware, namely the second ring region of the spyware is split, so that the new N end and C end of two mutants can be generated. DAHID in the spyware amino acid sequence is not shown in the crystal structure and can be considered as a disordered sequence, and is therefore discarded during the cyclic alignment.
Example 2
The plasmid was constructed as follows:
for exploring spy tags and spy captors-NTEVWhen the fluorescent protein is applied to a model system, yellow fluorescent protein and cyan fluorescent protein are selected as model proteins, and spy tag-yellow fluorescent protein (SpyTag-YFP) and spy captor-N are constructedTEV-Cyan fluorescent protein (BDtag-CFP). To construct spy marksTaking a plasmid containing a yellow fluorescent protein sequence as a template and a DNA single-stranded fragment containing an enzyme cutting site sequence as a primer, and amplifying a spy tag-yellow fluorescent protein target sequence containing an enzyme cutting site by PCR (polymerase chain reaction). And then the spy tag-yellow fluorescent protein fragment is inserted into a pET-28b vector containing the same enzyme cutting site by a double enzyme cutting method. Spy catcher-NTEVThe cyan fluorescent protein (BDTag-CFP) was constructed in the same manner.
TABLE 1 Experimental plasmids
Figure BDA0002872021370000051
TABLE 2 Experimental strains
Name of Strain Source Use of
Escherichia coli DH-5 alpha Kangshi Century (CWBIO) Cloning of genes
Escherichia coli BL21(DE3) Kangshi Century (CWBIO) Protein expression
TABLE 3 primer Gene sequences
Figure BDA0002872021370000052
Example 3
Protein purification and expression
Contains spy tag-yellow fluorescent protein (SpyTag-YFP) and spy catcher-NTEVAfter the plasmid pET-28b of-cyan fluorescent protein (BDtag-CFP) was confirmed by sequencing, it was transferred into BL21(DE3) competent cells and plated, and cultured overnight at 37 ℃ in an incubator. Single colonies were picked on the plates and cultured overnight at 37 ℃ in a shaking incubator in LB medium containing 0.33mg/mL kanamycin. Overnight culture medium was taken as 1: adding 100 proportion of fresh 1L LB culture medium containing 0.33mg/mL kanamycin sulfate, shaking culturing at 37 deg.C to OD6000.4-0.6, changing the temperature of the shaking incubator to 16 ℃ for overnight culture, and simultaneously adding IPTG to the final concentration of 1mM to induce the escherichia coli to express the target protein. After the expression is finished, the bacteria are collected by centrifugation, the supernatant is discarded, and the target protein is purified and eluted by using non-denaturing conditions. The target protein was collected and purified by a gel size exclusion chromatography column (Superdex 200 inch 10/300GL, GE Healthcare) of a protein rapid purification liquid chromatography system, and the mobile phase was 1 XP buffered saline (2mM potassium dihydrogen phosphate, 10mM disodium hydrogen phosphate, 137mM sodium chloride, 2.7mM potassium chloride, pH 7.4).
Example 4
Spy catcher mutant Spycatcher-NTEVApplication to fluorescent protein model systems
Measuring spy tag-yellow fluorescent protein (SpyTag-YFP) and spy catcher mutant-N with ultramicro spectrophotometer (P330, Implen)TEV-concentration of cyan fluorescent protein. Reaction according to spy label-yellow fluorescent protein: spy catcher mutant-NTEVThe molar ratio of-cyan fluorescent protein ═ 1:1.2 (n: n) was reacted at 4 ℃ for 24 hours. The reaction schemes for the extracellular reaction and TEV cleavage reaction are shown in FIG. 3.
When the reaction product is subjected to TEV enzyme digestion reaction, coupling products are adopted: TEV enzyme 1:3 (n: n, molar ratio) was digested at 37 ℃ for 24 hours. The reaction was quenched by addition of 5 Xloading buffer immediately after the reaction time was reached. SDS-PAGE electrophoresis is used for representing reaction mixed liquor and reaction progress, and a multifunctional fluorescence analyzer is used for quantifying the proportion of reactants and products in protein reaction liquor.
The SDS-PAGE result of FIG. 5(a) shows that the band of the coupled product is substantially disappeared after the coupled product is added with TEV enzyme, and a new protein band appears below the band of the coupled product, which is presumed to be an enzyme-cleaved product. Thus, it is considered that the substrate is cleaved to almost completion by cleavage reaction at 37 ℃ for 24 hours. And (3) performing mass spectrum characterization on samples corresponding to the bands of the coupling product and the enzyme digestion product, wherein MALDI-TOF experimental results and theoretical value errors in the figure 5(b) are within an allowable range, and further proving the molecular weights of the coupling product and the enzyme digestion product, thereby indicating that the coupling reaction and the enzyme digestion reaction obtain expected products.
The SpyCatcher-N amino acid sequence is GKTISTWISDGQVKDFYLYPGKYTEVETAAPGDYEVATAITFTVNEQGQVTVNGKATKGGSGGSGGSEDSATHIKFSKRDEDGKELAGATMELRDS;
SpyCatcher-N in the inventionTEVThe amino acid sequence is GKTISTWISDGQVKDFYLYPGKYTEVETAAPGDYEVATAITFTVNEQGQVTVNGKATKGGSENLYFQGGGSEDSATHIKFSKRDEDGKELAGATMELRDS.
SEQUENCE LISTING
<110> Jiangsu Beiottak Biotechnology Ltd
<120> spy catcher mutant, preparation method thereof and application thereof in fluorescent protein system
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 96
<212> PRT
<213> Artificial sequence
<400> 1
Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly Gln Val Lys Asp Phe
1 5 10 15
Tyr Leu Tyr Pro Gly Lys Tyr Thr Glu Val Glu Thr Ala Ala Pro Gly
20 25 30
Asp Tyr Glu Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln Gly
35 40 45
Gln Val Thr Val Asn Gly Lys Ala Thr Lys Gly Gly Ser Gly Gly Ser
50 55 60
Gly Gly Ser Glu Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg Asp
65 70 75 80
Glu Asp Gly Lys Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp Ser
85 90 95
<210> 2
<211> 100
<212> PRT
<213> Artificial sequence
<400> 2
Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly Gln Val Lys Asp Phe
1 5 10 15
Tyr Leu Tyr Pro Gly Lys Tyr Thr Glu Val Glu Thr Ala Ala Pro Gly
20 25 30
Asp Tyr Glu Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln Gly
35 40 45
Gln Val Thr Val Asn Gly Lys Ala Thr Lys Gly Gly Ser Glu Asn Leu
50 55 60
Tyr Phe Gln Gly Gly Gly Ser Glu Asp Ser Ala Thr His Ile Lys Phe
65 70 75 80
Ser Lys Arg Asp Glu Asp Gly Lys Glu Leu Ala Gly Ala Thr Met Glu
85 90 95
Leu Arg Asp Ser
100

Claims (8)

1. The spyware mutant is characterized by being SpyCatcher-NTEVThe amino acid sequence is shown as SEQ ID NO. 1.
2. The spyware mutant according to claim 1, wherein the spyware mutant SpyCatcher-N is obtained by linking a flexible amino acid sequence to the original N-terminal and C-terminal of spyware SpyCatcher, and a TEV protease recognizable sequence is inserted into the flexible amino acid sequence of spyware mutant SpyCatcher-N to obtain spyware mutant SpyCatcher-NTEVWherein the spyware mutants SpyCatcher-N and SpyCatcher-NTEVAnd the new N end and C end are generated in the second ring part of the spyware, namely the second ring region for splitting the spyware, so that the new N end and C end of the mutant can be generated.
3. The spyware mutant according to claim 1, wherein the TEV protease recognizable sequence is ENLYFQG.
4. The method for preparing low-temperature normal-pressure sintered silicon carbide composite ceramic according to claim 1, wherein the flexible amino acid sequence is (GGS)3。
5. The method for preparing the low-temperature normal-pressure sintered silicon carbide composite ceramic according to claim 1, wherein the spyware mutant SpyCatcher-N has an amino acid sequence shown in SEQ ID No. 2.
6. A method for preparing spyware mutants according to any one of claims 1 to 5, wherein the method comprises: the spyware mutant SpyCatcher-N is obtained by connecting a flexible amino acid sequence with the original N end and C end of the spyware SpyCatcher, and the TEV protease recognizable sequence is insertedObtaining spyware mutant SpyCatcher-N from the flexible amino acid sequence in spyware mutant SpyCatcher-NTEV。
7. Use of a spy-catch mutant according to any one of claims 1-5 in a fluorescent protein system.
8. Use of spyware mutant according to claim 7 in a fluorescent protein model system, wherein the fluorescent protein is cyan fluorescent protein or yellow fluorescent protein.
CN202011619571.5A 2020-12-30 2020-12-30 Spyware mutant, preparation method thereof and application thereof in fluorescent protein system Pending CN114685679A (en)

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