CN116515799A - Recombinant Sortase A enzyme and immobilization method and application thereof - Google Patents
Recombinant Sortase A enzyme and immobilization method and application thereof Download PDFInfo
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- 108090000250 sortase A Proteins 0.000 title claims abstract description 105
- 238000000034 method Methods 0.000 title claims abstract description 24
- 230000008878 coupling Effects 0.000 claims abstract description 12
- 238000010168 coupling process Methods 0.000 claims abstract description 12
- 238000005859 coupling reaction Methods 0.000 claims abstract description 12
- 239000011347 resin Substances 0.000 claims abstract description 12
- 229920005989 resin Polymers 0.000 claims abstract description 12
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims abstract description 11
- 235000018417 cysteine Nutrition 0.000 claims abstract description 11
- 238000005119 centrifugation Methods 0.000 claims abstract description 8
- 150000003573 thiols Chemical class 0.000 claims abstract 2
- 108090000790 Enzymes Proteins 0.000 claims description 35
- 102000004190 Enzymes Human genes 0.000 claims description 35
- 108090000623 proteins and genes Proteins 0.000 claims description 34
- 102000004169 proteins and genes Human genes 0.000 claims description 30
- 235000018102 proteins Nutrition 0.000 claims description 21
- 230000009145 protein modification Effects 0.000 claims description 15
- 238000012986 modification Methods 0.000 claims description 12
- 230000004048 modification Effects 0.000 claims description 11
- 230000015572 biosynthetic process Effects 0.000 claims description 8
- 238000003786 synthesis reaction Methods 0.000 claims description 8
- 241000672609 Escherichia coli BL21 Species 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 230000003100 immobilizing effect Effects 0.000 claims description 2
- 210000004899 c-terminal region Anatomy 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 20
- 230000000694 effects Effects 0.000 description 19
- 238000001514 detection method Methods 0.000 description 15
- 239000000758 substrate Substances 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 108090000279 Peptidyltransferases Proteins 0.000 description 6
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 5
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 5
- 125000003396 thiol group Chemical group [H]S* 0.000 description 5
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 239000003607 modifier Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 3
- 239000004473 Threonine Substances 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 230000002427 irreversible effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000001742 protein purification Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000010791 quenching Methods 0.000 description 3
- 230000000171 quenching effect Effects 0.000 description 3
- FEWFXBUNENSNBQ-UHFFFAOYSA-N 2-hydroxyacrylic acid Chemical compound OC(=C)C(O)=O FEWFXBUNENSNBQ-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000012742 biochemical analysis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000001215 fluorescent labelling Methods 0.000 description 2
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 229910052759 nickel Inorganic materials 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000001089 thermophoresis Methods 0.000 description 2
- 125000001917 2,4-dinitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C(=C1*)[N+]([O-])=O)[N+]([O-])=O 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
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- 239000000872 buffer Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- JDNTWHVOXJZDSN-UHFFFAOYSA-N iodoacetic acid Chemical group OC(=O)CI JDNTWHVOXJZDSN-UHFFFAOYSA-N 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/2207—Sortase A (3.4.22.70)
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention belongs to the field of bioengineering, and particularly discloses a recombinant Sortase A enzyme, an immobilization method and application thereof, wherein cysteine is introduced at the tail end of the Sortase A enzyme, the Sortase A enzyme is immobilized on commercial coupling resin by utilizing a thiol covalent coupling principle, and after the reaction is finished, the Sortase A enzyme can be removed only by a simple centrifugation method.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to a recombinant Sortase A enzyme, an immobilization method and application thereof.
Background
The transpeptidase Sortase A separated from staphylococcus aureus can specifically identify and bind to the LPXTG sequence at the C end of the protein, and after the peptide bond between threonine and glycine is cut off, the transpeptidase Sortase A is connected with another peptide chain containing glycine residue at the N end through a peptide bond. Based on this function, the SortaseA enzyme can be used for the synthesis and modification of proteins;
in recent years, sortaseA has been widely used for modification of protein termini. By utilizing the transpeptidation reaction catalyzed by Sortase A, researches find that any interesting water-soluble modification group can be added to the tail end of the protein, including functional groups such as biotin, fluorescein, a crosslinking agent and the like;
although the SortaseA enzyme is widely used in the synthesis and modification reaction of proteins because of its transpeptidase activity, the SortaseA enzyme has hydrolase activity, and the modified product is hydrolyzed again by SortaseA as a substrate, so that the modified molecule is dropped, i.e., sortaseA is not easily separated from the reaction system after the reaction is completed. The current solution is to reduce the occurrence of hydrolysis reaction by timely removing redundant Sortase A in the reaction system. A common removal method is to introduce an affinity tag, such as 6His, at the end of Sortase A, and separate the enzyme from the product using the principle of affinity adsorption. The method needs a complete affinity purification process, and has long time consumption and high cost, so the invention provides a method for immobilizing Sortase A enzyme by using cysteine and application thereof.
Disclosure of Invention
In order to solve the above problems, a primary object of the present invention is to provide a recombinant SortaseA enzyme, and an immobilization method and application thereof.
The specific technical scheme of the invention comprises the following steps:
the invention provides a recombinant Sortase A enzyme, which is obtained by introducing a cysteine at the C end of the Sortase A enzyme for sequence modification, wherein the protein sequence of the recombinant Sortase A enzyme is shown as SEQ ID NO. 1.
As a further optimization scheme of the invention, the vector for expressing the recombinant Sortase A enzyme is pET-28a, and the strain for expressing the recombinant Sortase A enzyme is escherichia coli BL21.
The invention also provides the coding gene of the recombinant Sortase A enzyme, and the nucleotide sequence of the coding gene of the recombinant Sortase A enzyme is shown as SEQ ID NO. 2.
The invention also provides an application of the recombinant Sortase A enzyme in protein synthesis and modification.
The invention also provides an immobilization method of the recombinant Sortase A enzyme, which is characterized in that cysteine is utilized to immobilize the Sortase A enzyme on commercial resin through sulfhydryl coupling, an iodoacetic acid group in the commercial resin and the sulfhydryl exposed by the recombinant Sortase A enzyme form covalent and irreversible thioether bonds, and a specific and efficient reaction occurs, so that the recombinant Sortase A enzyme can be permanently connected on the resin.
The invention also provides an application of the immobilized Sortase A enzyme in the synthesis and modification of protein.
As a further optimization scheme of the invention, after the immobilized Sortase A enzyme plays a protein modification function, the immobilized Sortase A enzyme can be removed after centrifugation at 3000rpm for 5min by using a low-speed centrifuge.
According to the invention, by utilizing the principle that cysteine can be coupled with sulfhydryl, cysteine is added at the C end of Sortase A enzyme, and then the recombinant Sortase A is fixed on resin, the immobilization process is simple to operate, the reaction is efficient and specific and strong, in addition, the immobilization method is irreversible, and the immobilized recombinant Sortase A enzyme is not easy to fall off. The immobilized recombinant Sortase A enzyme can still maintain high transpeptidase activity, the activity can be applied to the synthesis and modification processes of proteins, protein modification comprises biotin labeling, fluorescent labeling and the like, and the labeled proteins can be used for biochemical analysis and detection, such as experiments of Fluorescence Resonance Energy Transfer (FRET), surface Plasmon Resonance (SPR), micro thermophoresis instrument (MST), living cell detection and the like; in addition, the immobilized Sortase A enzyme can be removed after the reaction directly through centrifugation, namely, the residual Sortase A enzyme in the reaction can be removed efficiently and rapidly only by centrifugation for 5min at 3000rpm, and no residue exists. The hydrolysis reversible reaction brought by the residual Sortase A is avoided, and the high efficiency of the Sortase A enzyme in the protein modification function is greatly improved.
In summary, the beneficial effects of the invention are as follows:
according to the method, cysteine is introduced at the tail end of the Sortase A enzyme, the recombinant Sortase A enzyme is fixed on commercial coupling resin by utilizing the principle of sulfydryl covalent coupling, and after the reaction is finished, the Sortase A enzyme can be removed only by a simple centrifugal method.
Drawings
FIG. 1 shows the results of expression and purification of recombinant Sortase A;
FIG. 2 shows the expression and purification results of wild-type Sortase A;
FIG. 3 shows the results of protein quality assay for recombinant Sortase A enzyme;
FIG. 4 shows the results of protein plasmid detection of wild-type Sortase A;
FIG. 5 shows the results of activity detection of immobilized Sortase A;
FIG. 6 shows the results of activity detection of wild-type Sortase A;
FIG. 7 shows the results of LC-MS detection of protein modification by immobilized recombinant Sortase A;
FIG. 8 shows the results of LC-MS detection of protein modification by immobilized wild-type Sortase A.
Detailed Description
The following detailed description of the present application is provided in conjunction with the accompanying drawings, and it is to be understood that the following detailed description is merely illustrative of the application and is not to be construed as limiting the scope of the application, since numerous insubstantial modifications and adaptations of the application will be to those skilled in the art in light of the foregoing disclosure.
1. Material
The methods used in this example are conventional methods known to those skilled in the art unless otherwise indicated, and the materials such as reagents used are commercially available products unless otherwise indicated.
2. Method of
1. Protein purification and protein quality detection
(1) Artificially synthesizing a recombinant Sortase A enzyme sequence according to a nucleotide sequence shown in SEQ ID No.2, inserting the recombinant Sortase A enzyme sequence into a pET-28a vector, and introducing the recombinant Sortase A enzyme sequence into competent cells of escherichia coli BL21 to obtain a recombinant Sortase A enzyme expression strain; meanwhile, a wild type Sortase A enzyme sequence with a His tag at the C end is used as a control, the wild type Sortase A enzyme sequence with the His tag is inserted into a pET-28a vector and is introduced into competent cells of escherichia coli BL21 to obtain a wild type Sortase A enzyme expression strain, and a protein sequence of the wild type Sortase A enzyme with the His tag at the C end is shown in No.3.
(2) Induction of expression of fusion proteins: the two types of strains which are obviously expressed are respectively inoculated into 50mLLB liquid culture medium for culture at 37 ℃ for overnight, the overnight cultured bacteria are inoculated into 5LLB liquid culture medium according to the proportion of 1:100, 0.5mM IPTG is added when the bacterial liquid OD600 is 0.6-0.8 after the bacterial liquid OD600 is cultured at 37 ℃, the bacterial liquid is cultured at 15 ℃ for overnight, and the bacterial bodies are collected by centrifugation at 5000 rpm.
(3) Protein purification: the two types of cells collected were weighed, and the respective volumes of lysis buffer (50 mM Tris-HCl (pH 8.0), 500mM NaCl,5% glycerol) were added in a mass ratio of 1:10, and the cells were crushed using a high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation at 16000 rpm. The protein is enriched and purified by using affinity chromatography HisFF, the HisFF column is balanced by using a lysis buffer before purification, after all cell supernatants are hung on the column, the eluted protein is collected for SDS-PAGE detection, the protein concentration is measured by using Nanodrop, the protein yield is calculated, the protein purification results of the recombinant Sortase A enzyme and the wild type Sortase A enzyme are respectively shown in figure 1 and figure 2, and the purity of the recombinant Sortase A enzyme is higher than that of the wild type Sortase A enzyme.
(4) Protein quality detection: and respectively taking a small amount of recombinant Sortase A enzyme eluted from imidazole and wild Sortase A enzyme protein for mass spectrum and molecular sieve detection. The molecular weight of the recombinant Sortase A enzyme detected by mass spectrum is 17016.37Da and is very close to the theoretical molecular weight 17017.99Da, which indicates that the recombinant Sortase A protein purified by HisFF is the target protein; the molecular weight of the wild-type Sortase A enzyme is 17780.97Da, which is very close to its theoretical molecular weight 17781.81 Da. In addition, the results of molecular sieves show that both the recombinant SortaseA enzyme and the wild-type SortaseA enzyme exist in the form of monomers in solution, greatly increasing the effective enzyme amount in practical applications. The experimental results are shown in fig. 3 and 4.
2. Activity determination of immobilized Sortase A enzyme
Sortase A is taken as a transpeptidase, can specifically recognize Leu-Pro-x-Thr-Gly (LPXTG) polypeptide sequences, and cuts between threonine Thr and glycine Gly residues at specific sites, and the carboxyl terminal of the threonine can be covalently connected to a target object. The invention uses Abz-LPETGK (Dnp) -NH 2 For a substrate ((available from Shanghai JieNitai Biotechnology Co., ltd.) in which Abz (Ortho-aminobenzene) is the fluorescent group and Dnp (2, 4-Dinitrophenyl) is the quenching group, when the polypeptide substrate sequence is intact, internal fluorescence quenching is exhibited, but when Sortase A cleaves between Thr and Gly residues of the substrate such that the fluorescent group Abz is separated from the quenching group Dnp, the Abz fluorescent signal is released and can be continuously detected, so that the enzyme activity of Sortase A can be quantitatively analyzed using this principle, the specific steps are as follows:
(1) Immobilization of Sortase A enzyme: recombinant Sortase A enzyme is coupled to resin through sulfhydryl according to the commercial resin coupling operation flow, so as to obtain immobilized Sortase A enzyme, and the total amount of the enzyme before coupling and the residual enzyme amount after coupling are measured, so that 15mg of the recombinant Sortase A enzyme can be coupled to 1mL of resin;
the method for fixing the wild-type Sortase A enzyme comprises the steps of coupling the wild-type Sortase A enzyme to a nickel magnetic bead through a His affinity tag according to the operation flow of a commercial nickel magnetic bead by utilizing the fact that the C end of the wild-type Sortase A enzyme is provided with the His tag, and calculating that 10mg of the wild-type Sortase A enzyme can be coupled per 1mL of resin by measuring the total amount of the enzyme before coupling and the amount of the enzyme remained after coupling.
(2) Enzyme activity determination: the Sortase A enzyme activity assay buffer was 50mMHEPESpH7.5, 150mMNaCl,5mM CaCl2, the substrate was 50. Mu. MAbz-LPETGK (Dnp) -NH2, the reaction temperature was room temperature, and the immobilized recombinant Sortase A enzyme and the immobilized wild-type Sortase A enzyme were diluted 2-fold in a gradient from 4. Mu.M to 6 concentrations with the buffer, respectively. Transferring 10 mu L of the substrate into a 384-well plate, setting a multi-well, transferring 10 mu L of Sortase A to be tested into a corresponding well plate, immediately centrifuging and shaking uniformly, and collecting fluorescent signal values generated after the substrate is cut by using a TECANM1000 enzyme-labeled instrument. And carrying out data analysis by using GraphPadPrism9 software to finally obtain the enzyme activity parameters of the protease to be detected, wherein the results are shown in figures 5 and 6.
Analysis of results: the fluorescence signal intensity generated by Sortase A per second is used for expressing the enzyme activity, the immobilized recombinant Sortase A enzyme activity is 0.001064RFU/s/nM, and the immobilized wild type Sortase A enzyme activity is 0.001162RFU/s/nM. According to the enzyme activity parameters, the immobilized recombinant Sortase A enzyme has higher enzyme activity compared with the conventional immobilized wild type Sortase A enzyme, and can be used in industrial production.
3. Removal effect test of immobilized Sortase A enzyme
(1) Preparation of immobilized recombinant SortaseA enzyme requires modification of target protein 1 and modifications. Wherein the molecular weight of the target protein 1 is 24151Da, the protein sequence is NO.4, the modified product is Biotin-LPET-G (2-hydroxy-acrylic acid) -GH (purchased from Beijing Cork-Mitsui optical Biotech Co., ltd.) and the molecular weight is 663Da;
the method is used for detecting the removal effect of immobilized wild Sortase A enzyme, preparing target protein 2 and a modifier which are required to be modified, wherein the molecular weight of the target protein 1 is 18908Da, the protein sequence is No.5, and the modifier is Biotin-LPET-G (2-hydroxy acrylic acid) -GH, and the molecular weight is 663Da;
(2) Mixing the immobilized recombinant Sortase A enzyme or the immobilized wild-type Sortase A enzyme, the target protein and the modified protein according to a molar ratio of 1:20:200, reacting for 1 hour at room temperature, centrifuging at 3000rpm for 5min to remove the enzyme, detecting the molecular weight of the sample in the reaction system before and after the reaction by using LC-MS, and judging whether the Sortase A enzyme marks the modified substance on the target protein, wherein the result is shown in figures 7 and 8.
Analysis of results: the molecular weight of the sample after the target protein 1 is reacted is increased by 662.41Da, and the molecular weight corresponds to the molecular weight of the modifier, which indicates that the modification is successful. Furthermore, as can be seen from the range of LC-MS detection, no distinct peak was found at the location of 17017Da, 17017Da corresponding to the protein molecular weight of recombinant SortaseA, indicating that recombinant SortaseA enzyme has been removed. The molecular weight of the sample after the target protein 2 is reacted is increased by 666.6Da, and the molecular weight corresponding to the molecular weight is the molecular weight of the modifier, which indicates that the modification is successful. However, according to the range of LC-MS detection, obvious peaks appear at 17780Da and 1847 Da after the reaction, and the two peaks correspond to the molecular weight of the wild-type Sortase A enzyme and the molecular weight of the modified wild-type Sortase A enzyme marked respectively, which indicates that the immobilized wild-type Sortase A enzyme is not removed completely and the hydrolysis reaction occurs in the reaction system.
3. Conclusion(s)
According to the invention, by utilizing the principle that cysteine can be coupled with sulfhydryl, cysteine is added at the C end of Sortase A enzyme, and then the recombinant Sortase A is fixed on resin, the immobilization process is simple to operate, the reaction is efficient and specific and strong, in addition, the immobilization method is irreversible, and the immobilized recombinant Sortase A enzyme is not easy to fall off. The immobilized recombinant Sortase A enzyme can still maintain high transpeptidase activity, the activity can be applied to the synthesis and modification processes of proteins, protein modification comprises biotin labeling, fluorescent labeling and the like, and the labeled proteins can be used for biochemical analysis and detection, such as experiments of Fluorescence Resonance Energy Transfer (FRET), surface Plasmon Resonance (SPR), micro thermophoresis instrument (MST), living cell detection and the like;
in addition, the immobilized recombinant Sortase A enzyme can be directly removed after the reaction by centrifugation, namely, the residual Sortase A enzyme in the reaction can be efficiently and rapidly removed by centrifugation for 5min at 3000rpm without residue. The hydrolysis reversible reaction brought by the residual Sortase A is avoided, and the high efficiency of the Sortase A enzyme in the protein modification function is greatly improved.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that modifications can be made without departing from the spirit of the invention, which are within the scope of the invention.
Claims (8)
1. The recombinant Sortase A enzyme is characterized in that the recombinant Sortase A enzyme is obtained by introducing a cysteine at the C terminal of the Sortase A enzyme for sequence modification, and the protein sequence of the recombinant Sortase A enzyme is shown as SEQ ID NO. 1.
2. The recombinant sortase a enzyme of claim 1, wherein the vector expressing the recombinant sortase a enzyme is pET-28a and the strain expressing the recombinant sortase a enzyme is escherichia coli BL21.
3. A gene encoding a recombinant SortaseA enzyme according to claim 1, wherein the nucleotide sequence of the gene encoding the recombinant SortaseA enzyme is shown in SEQ ID No. 2.
4. Use of the recombinant sortase a enzyme of claim 1 in the synthesis and modification of proteins.
5. A method of immobilizing a recombinant sortase a enzyme according to claim 1, wherein sortase a enzyme is immobilized to a commercial resin via thiol coupling using cysteine.
6. An immobilized SortaseA enzyme prepared by the immobilization process of claim 5.
7. Use of the immobilized sortase a enzyme of claim 6 in the synthesis and modification of proteins.
8. The use of the immobilized SortaseA enzyme according to claim 7 in the synthesis and modification of proteins, wherein after the immobilized SortaseA enzyme is used to perform a protein modification function, the immobilized SortaseA enzyme is removed by centrifugation at 3000rpm for 5min using a low-speed centrifuge.
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