CN113481173A

CN113481173A - Proximal biotin ligase and application thereof

Info

Publication number: CN113481173A
Application number: CN202110545864.1A
Authority: CN
Inventors: 李栋; 王新禹
Original assignee: Institute of Biophysics of CAS
Current assignee: Institute of Biophysics of CAS
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2021-10-08
Anticipated expiration: 2041-05-19
Also published as: CN113481173B

Abstract

The present invention relates to genetic engineering, in particular to a genetically engineered adjacent biotin ligase, in particular to a protein adjacent labeling using the adjacent biotin ligase and its application. The adjacent biotin ligase is the site-directed insertion of an unnatural amino acid near the catalytic center site of the ATP or biotin binding pocket in the biotin ligase. The obtained adjacent biotin ligase can inhibit the enzymatic activity of its adjacent biotin ligase when it is not irradiated with a specific wavelength of ultraviolet light or incubated with a specific compound. Then, the obtained adjacent biotin ligase is irradiated with a specific ultraviolet wavelength or added with a specific compound to convert the unnatural amino acid into a natural amino acid and restore its enzymatic activity. The adjacent biotin ligase of the present invention can eliminate the non-specific background of the previous biotin ligase, and greatly improve the specificity of identifying the proteome in a specific organelle.

Description

Proximal biotin ligase and application thereof

Technical Field

The invention relates to gene engineering, in particular to a proximity biotin ligase which is transformed by the gene engineering, and specifically relates to a protein proximity marker by using the proximity biotin ligase and application thereof.

Background

Proximity labeling has become an alternative to immunoprecipitation and traditional biochemical separations and, through use in conjunction with proteomics, has been widely used for macromolecular complex component identification, intracellular protein species analysis, and the construction of protein interaction networks. The strategy of proximity labeling technology is generally to express the protein of interest in fusion with a proximity-labeled enzyme, thereby targeting the protein of interest to a specific location within the cell. The proximity-labeled enzyme can catalyze biotinylation covalent link modification of a protein adjacent to the spatial position of the target protein by adding a small molecule substrate such as biotin, and the biotinylation modified protein can be enriched by using streptavidin-coupled beads and further identified by using mass spectrometry.

Currently, researchers have successfully developed the following two broad classes of enzymes for proximity tagging: (1) one type is the proximity-labeled mutant of Escherichia coli biotin ligase BirA, which mainly includes BirA-R118G (hereinafter this protein is referred to as BioID) [1,2] and TurboID [3 ]; (2) another class is soybean ascorbate peroxidase mutants, which mainly include APEX2[4 ].

The APEX2 has the advantage of high labeling speed of adjacent proteins and can be completed within about 1 minute, however, the biotin labeling of the adjacent proteins by the APEX2 needs to add hydrogen peroxide (H) into the culture medium₂O₂) This is toxic to live cell markers and is also difficult to apply to living tissue.

BioID has the advantages of using biotin as a substrate and having no cytotoxicity, and more than 100 reports about application of BioID exist at present. However, the major drawback of BioID is that it takes at least 18 hours for the biotin-labeled adjacent proteins to accumulate enough protein for mass spectrometric identification. TurboID is similar to BioID and is derived from mutation of biotin ligase of Escherichia coli, and the TurboID takes biotin as a substrate, and can shorten the labeling time to 10 min. TurboID has therefore found widespread use in mammalian cells and model organisms [5,6 ].

However, biotin is an essential substance for the growth of most mammalian cells, the gradual accumulation process of expression of TurboID and BioID in cells generally requires about 12 hours, and the gradual accumulation of TurboID and BioID can utilize biotin in cell culture media to generate higher background markers for adjacent proteins, thereby influencing the specificity of mass spectrum identification.

Unlike the classical codon, the amber codon UAG (TAG) can be used as a codon encoding pyrrolysine (lysine derivative), [ 2] in Methanococcus pasteurianus and Methanococcus jannaschii, respectively7]And a sense codon of tyrosine [ alpha ], [ alpha ] and [ alpha ], [ alpha ] and [ alpha ] a8]And not a stop codon. Orthogonal to mammals can be obtained by genetic engineering

And pyrrolidinyl-tRNA synthetase, whereby a derivative of lysine or a derivative of tyrosine is site-specifically inserted into the protein of interest in a mammalian cell.

If the adjacent biotin ligase TurboID can be made inactive in the process of accumulating expression and the activity of the biotin ligase can be recovered under the condition of receiving physical or chemical stimulation, the labeling background can be effectively reduced, the accuracy and specificity of the adjacent labeling can be improved, and the time and space resolution of the TurboID can be retained.

Disclosure of Invention

The invention aims to provide a protein proximity marker by using a light-controlled proximity biotin ligase and application thereof.

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

a proximal biotin ligase which is a site-specific insertion of an unnatural amino acid near the catalytic central site of the ATP or biotin-binding pocket of biotin ligase.

The obtained adjacent biotin ligase can inhibit the enzyme activity of the adjacent biotin ligase when the adjacent biotin ligase is not irradiated by specific wavelength ultraviolet or incubated with a specific type of compound. Then 365nm irradiation is carried out on the adjacent biotin ligase or the adjacent biotin ligase is incubated with a specific compound, the unnatural amino acid is converted into the natural amino acid, and the enzymatic activity of the natural amino acid is recovered.

The adjacent biotin ligase is obtained by mutating one or more sites of 183 th lysine, 132 th tyrosine and 172 th lysine in an amino acid sequence of a mutant of biotin ligase BirA derived from escherichia coli into unnatural amino acid, so that the enzyme activity of the adjacent biotin ligase is inhibited.

The adjacent biotin ligase is one or more of 183 th lysine, 132 th tyrosine and 172 th lysine in an amino acid sequence of biotin ligase TurboID, and is mutated into an unnatural amino acid; wherein the unnatural amino acid is an unnatural amino acid MNPY-lysine or an unnatural amino acid ONB-lysine.

The adjacent biotin ligase is characterized in that the 183 th lysine in the amino acid sequence shown in SEQ ID NO. 1 is mutated into an unnatural amino acid MNPY-lysine or an unnatural amino acid ONB-lysine, namely the amino acid sequence shown in SEQ ID NO. 4 and the amino acid sequence shown in SEQ ID NO. 5;

or the 132 th tyrosine in the amino acid sequence shown in SEQ ID NO. 1 is mutated into the unnatural amino acid ONB-tyrosine, namely the amino acid sequence shown in SEQ ID NO. 7.

Or the 172 th lysine in the amino acid sequence shown in SEQ ID NO. 1 is mutated into the unnatural amino acid MNPY-lysine or the unnatural amino acid ONB-lysine, namely the amino acid sequence shown in SEQ ID NO. 9 and the amino acid sequence shown in SEQ ID NO. 10.

A method for constructing adjacent biotin ligase by using two adjacent ATP of biotin ligase TurboID or near catalytic central sites of biotin binding pocket

And carrying out site-directed unnatural amino acid insertion with pyrrolidinyl-tRNA synthetase to obtain the adjacent biotin ligase.

The amino acid sequence of the biotin ligase TurboID is shown as SEQ ID NO. 1; the unnatural amino acid is an unnatural amino acid MNPY-lysine or an unnatural amino acid ONB-lysine or ONB-tyrosine.

Use of a proximal biotin ligase in mass spectrometry identification.

The adjacent biotin ligase is used for performing biotin labeling on adjacent protein under the condition of ultraviolet illumination with specific wavelength in mass spectrum identification.

A method for carrying out mass spectrometry identification by biotinylation labeling by using adjacent biotin ligase comprises the steps of utilizing the adjacent biotin ligase to be fused and expressed with target protein, locating the target protein to a specific organelle, then irradiating at 365nm to enable the adjacent biotin ligase to recover enzyme activity, further carrying out biotin labeling on the adjacent protein of the target protein, enriching the protein labeled by biotin by virtue of Streptavidin coupled magnetic beads, and carrying out mass spectrometry identification.

The invention has the advantages that:

the light-operated adjacent biotin ligase designed by the invention can completely inactivate the adjacent biotin ligase by mutating amino acid near the active center of the existing adjacent biotin ligase into unnatural amino acid, thereby eliminating the background generated in the accumulation expression process and greatly improving the accuracy and specificity of mass spectrum identification.

Drawings

FIG. 1 is a schematic diagram showing a comparison between a conventional biotin proximity ligase (A) and a light-controlled biotin proximity ligase (B) in accordance with the present invention.

FIG. 2 is a graph of biotinylated immunoblot results of total protein samples obtained under different treatment conditions and total protein detected using Streptavidin-HRP.

FIG. 3 shows the identification of proteins in mitochondrial matrix by using optically controlled biotin ligase PC-mito-TurboID-183-MNPYK obtained in the present invention; wherein (A) SILAC identifies the protein in the mitochondrial matrix, (B) compares the light-controlled proximity biotin ligase PC-mito-TurboID-183-MNPYK (upper half) with the published proximity biotin ligase mito-TurboID (lower half) to identify the number and specificity of the protein in the mitochondrial matrix.

Detailed Description

The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention.

The present invention relates to genetically engineered vicinal biotin ligases. The light-operated adjacent biotin ligase replaces lysine or tyrosine at a specific site with corresponding unnatural amino acids such as lysine or tyrosine derivatives, and the fixed-point insertion of the unnatural amino acids can eliminate the enzyme activity of the adjacent biotin ligase; under the ultraviolet irradiation of specific wavelength, the light-operated non-natural amino acid such as lysine or tyrosine derivative at specific site in the adjacent biotin ligase is converted into lysine or tyrosine, and then the activity of the adjacent biotin ligase is recovered, so that the biotinylation reaction of the adjacent protein is specifically carried out. The light-controlled adjacent biotin ligase can eliminate the non-specific background of the previous biotin ligase, and greatly improve the specificity of identifying proteome in a specific organelle.

The sequence and chemical structural formula of the invention are as follows:

the protein adjacent to biotin ligase TurboID is a mutant of biotin ligase BirA derived from Escherichia coli, and its sequence and DNA sequence are derived from the literature [3], specifically,

1: TurboID the protein sequence of SEQ ID NO:

KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK

2 DNA sequence of TurboID SEQ ID NO:

aaagacaatactgtgcctctgaagctgatcgctctcctggctaatggcgagttccatagtggcgaacagctgggagaaaccctgggcatgtccagggccgctatcaacaagcacattcagactctgcgcgactggggcgtggacgtgttcaccgtgcccggaaagggctactctctgcccgagcctatcccgctgctgaacgctaaacagattctgggacagctggacggcgggagcgtggcagtcctgcctgtggtcgactccaccaatcagtacctgctggatcgaatcggcgagctgaagagtggggatgcttgcattgcagaatatcagcaggcagggagaggaagcagagggaggaaatggttctctccttttggagctaacctgtacctgagtatgttttggcgcctgaagcggggaccagcagcaatcggcctgggcccggtcatcggaattgtcatggcagaagcgctgcgaaagctgggagcagacaaggtgcgagtcaaatggcccaatgacctgtatctgcaggatagaaagctggcaggcatcctggtggagctggccggaataacaggcgatgctgcacagatcgtcattggcgccgggattaacgtggctatgaggcgcgtggaggaaagcgtggtcaatcagggctggatcacactgcaggaagcagggattaacctggacaggaatactctggccgctacgctgatccgagagctgcgggcagccctggaactgttcgagcaggaaggcctggctccatatctgccacggtgggagaagctggataacttcatcaatagacccgtgaagctgatcattggggacaaagagattttcgggattagccgggggattgataaacagggagccctgctgctggaacaggacggagttatcaaaccctggatgggcggagaaatcagtctgcggtctgccgaaaag

chemical structure of MNPY-lysine (MNPYK) and method for inserting unnatural amino acid MNPY-lysine (MNPYK)

And MNPYK-tRNA synthetase DNA sequence please refer to [9]。

Chemical structure of ONB-lysine (ONBK) and insertion of unnatural amino acid ONB-lysine (ONBK)

And the DNA sequence of ONBK-tRNA synthetase [10]。

Chemical structure of ONB-tyrosine (ONBY) and used for inserting unnatural amino acid ONB-tyrosine (ONBY)

And the DNA sequence of ONBY-tRNA synthetase [11]。

Example 1

Technical scheme for expressing and activating light-operated biotin ligase PC-TurboID-183-MNPYK

1) Through site-directed mutagenesis, the codon of 183 th lysine in TurboID is mutated into amber codon TAG, and TurboID-183 plasmid shown as SEQ ID NO. 3 is obtained; the method specifically comprises the following steps:

using NEB corporation

Site-Directed Mutagenesis Kit and NEB online design PCR primer NEBBaseChanger, and the following two primers are designed:

a forward primer: 5'-GCAGGATAGAtAGCTGGCAGG-3'

Reverse primer: 5'-AGATACAGGTCATTGGGC-3'

The pair of PCR primers can mutate 183 lysine AAG codon into succinic acid codon TAG, 1.25 microliter of forward primer and 1.25 microliter of reverse primer, 12.5 microliter of Q5 Hot Start High-Fidelity 2X Master Mix, 1 microliter of template containing DNA sequence of TurboID shown in SEQ ID NO. 2 and 9 microliter of water are mixed uniformly, and PCR amplification is carried out according to the following conditions: step 198 degree for 30 seconds; step 298 deg. 10 seconds; step 362 degree 15 seconds; step 472 ℃ for 5 minutes; step 5 repeats step 2 to step 4 25 times; step 672 degree 2 minutes.

Template utilization in a PCR reaction system is digested, and KLD mix is configured: 1 microliter of the PCR product, 5 microliter of 2 XKLD reaction, 1 microliter of 10 XKLD enzyme mixture, and 3 microliter of water were added. Incubate for 5 minutes at room temperature.

The above 1. mu.l of KLD mix was added to chemically competent E.coli and incubated on ice for 30 minutes.

The 42 degree heat shock was incubated on ice for 5 minutes after 30 seconds.

LB medium was added and incubated at 37 ℃ for 1 hour.

Competent E.coli cells were plated on agar plates containing the corresponding antibiotics. The plasmid TurboID-183 containing the base mutation shown in SEQ ID NO. 3 was obtained by Sanger DNA sequencing.

2) TurboID-183 plasmid was ligated with that mentioned in reference 9

And MNPYK-tRNA synthetase plasmid are transfected into Hela cell, and at the same time, the final concentration of 1mM unnatural amino acid MNPYK is added to accumulate and express to obtain the light-operated adjacent biotin ligase PC-TurboID-183-MNPYK shown in SEQ ID NO. 4.

After the ligase was expressed for 12 hours, the sample was irradiated with 365 nm-wavelength ultraviolet light for 1 minute to recover the biotin ligase activity by PC-TurboID-183-MNPYK (see FIG. 1).

The DNA sequence of SEQ ID NO 3, TurboID-183 is as follows:

aaagacaatactgtgcctctgaagctgatcgctctcctggctaatggcgagttccatagtggcgaacagctgggagaaaccctgggcatgtccagggccgctatcaacaagcacattcagactctgcgcgactggggcgtggacgtgttcaccgtgcccggaaagggctactctctgcccgagcctatcccgctgctgaacgctaaacagattctgggacagctggacggcgggagcgtggcagtcctgcctgtggtcgactccaccaatcagtacctgctggatcgaatcggcgagctgaagagtggggatgcttgcattgcagaatatcagcaggcagggagaggaagcagagggaggaaatggttctctccttttggagctaacctgtacctgagtatgttttggcgcctgaagcggggaccagcagcaatcggcctgggcccggtcatcggaattgtcatggcagaagcgctgcgaaagctgggagcagacaaggtgcgagtcaaatggcccaatgacctgtatctgcaggatagaTagctggcaggcatcctggtggagctggccggaataacaggcgatgctgcacagatcgtcattggcgccgggattaacgtggctatgaggcgcgtggaggaaagcgtggtcaatcagggctggatcacactgcaggaagcagggattaacctggacaggaatactctggccgctacgctgatccgagagctgcgggcagccctggaactgttcgagcaggaaggcctggctccatatctgccacggtgggagaagctggataacttcatcaatagacccgtga

agctgatcattggggacaaagagattttcgggattagccgggggattgataaacagggagccctgctgctggaac

aggacggagttatcaaaccctggatgggcggagaaatcagtctgcggtctgccgaaaag

4, the amino acid sequence of the PC-TurboID-183-MNPYK is as follows:

KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADKVRVKWPNDLYLQDR(MNPYK)LAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK

example 2

Technical scheme for expressing and activating light-operated biotin ligase PC-TurboID-183-ONBK

TurboID-183 plasmid shown in SEQ ID NO. 3 and that mentioned in reference 10

And ONBK-tRNA synthetase plasmid is transfected into Hela cells, and simultaneously, the unnatural amino acid ONBK with the final concentration of 1mM is added, and the optically controlled adjacent biotin ligase PC-TurboID-183-ONBK shown in SEQ ID NO. 5 is obtained through accumulation and expression.

After the ligase was accumulated and expressed for 12 hours, the sample was irradiated with 365nm ultraviolet light for 1 minute to allow PC-TurboID-183-ONBK to recover the biotin ligase activity (see FIG. 1).

The amino acids of SEQ ID NO 5, PC-TurboID-183-ONBK are as follows:

KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADKVRVKWPNDLYLQDR(ONBK)LAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK

example 3

Technical scheme for expressing and activating light-operated biotin ligase PC-TurboID-132-ONBY

The point mutation method described in example 1 mutated the codon of tyrosine 132 in TurboID to amber codon TAG to obtain TurboID-132 plasmid shown in SEQ ID NO. 6. The primers used for point mutations were as follows:

a forward primer: 5'-CTAACCTGTAgCTGAGTATGTTTTG-3'

Reverse primer: 5'-CTCCAAAAGGAGAGAACC-3'

TurboID-132 plasmid was ligated with that mentioned in reference 11

And ONBY-tRNA synthetase plasmid are transfected into Hela cell, and simultaneously, unnatural amino acid ONBY with the final concentration of 1mM is added, and the light-operated adjacent biotin ligase PC-TurboID-132-ONBY shown in SEQ ID NO. 7 is obtained through accumulation and expression.

After the ligase was accumulated and expressed for 12 hours, the sample was irradiated with 365nm ultraviolet light for 1 minute to recover the biotin ligase activity by PC-TurboID-ONBY-132 (see FIG. 1).

The DNA sequence of SEQ ID NO 6, TurboID-132 is as follows:

aaagacaatactgtgcctctgaagctgatcgctctcctggctaatggcgagttccatagtggcgaacagctgggagaaaccctgggcatgtccagggccgctatcaacaagcacattcagactctgcgcgactggggcgtggacgtgttcaccgtgcccggaaagggctactctctgcccgagcctatcccgctgctgaacgctaaacagattctgggacagctggacggcgggagcgtggcagtcctgcctgtggtcgactccaccaatcagtacctgctggatcgaatcggcgagctgaagagtggggatgcttgcattgcagaatatcagcaggcagggagaggaagcagagggaggaaatggttctctccttttggagctaacctgtaGctgagtatgttttggcgcctgaagcggggaccagcagcaatcggcctgggcccggtcatcggaattgtcatggcagaagcgctgcgaaagctgggagcagacaaggtgcgagtcaaatggcccaatgacctgtatctgcaggatagaaagctggcaggcatcctggtggagctggccggaataacaggcgatgctgcacagatcgtcattggcgccgggattaacgtggctatgaggcgcgtggaggaaagcgtggtcaatcagggctggatcacactgcaggaagcagggattaacctggacaggaatactctggccgctacgctgatccgagagctgcgggcagccctggaactgttcgagcaggaaggcctggctccatatctgccacggtgggagaagctggataacttcatcaatagacccgtgaagctgatcattggggacaaagagattttcgggattagccgggggattgataaacagggagccctgctgctggaacaggacggagttatcaaaccctggatgggcggagaaatcagtctgcggtctgccgaaaag

the amino acid sequence of SEQ ID NO 7, PC-TurboID-132-ONBY is as follows:

KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANL(ONBY)LSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK

example 4

Technical scheme for expressing and activating light-operated biotin ligase PC-TurboID-172-MNPYK

Mutating the codon of the 172 th lysine in the TurboID into an amber codon TAG as described in example 1 to obtain a TurboID-172 plasmid shown as SEQ ID NO. 8;

the primers for point mutations were as follows:

a forward primer: 5'-GGTGCGAGTCtagTGGCCCAATG-3'

Reverse primer: 5'-TTGTCTGCTCCCAGCTTTC-3'

TurboID-172 plasmid was ligated with the plasmid mentioned in reference 9

And MNPYK-tRNA synthetase plasmid are transfected into Hela cell, and at the same time, the final concentration of 1mM unnatural amino acid MNPYK is added to accumulate and express to obtain the light-operated adjacent biotin ligase PC-TurboID-172-MNPYK shown in SEQ ID NO. 9.

After the ligase was expressed for 12 hours, the sample was irradiated with 365 nm-wavelength ultraviolet light for 1 minute to recover the biotin ligase activity by PC-TurboID-172-MNPYK (see FIG. 1).

The DNA sequence of SEQ ID NO 8, TurboID-172 is as follows:

aaagacaatactgtgcctctgaagctgatcgctctcctggctaatggcgagttccatagtggcgaacagctgggagaaaccctgggcatgtccagggccgctatcaacaagcacattcagactctgcgcgactggggcgtggacgtgttcaccgtgcccggaaagggctactctctgcccgagcctatcccgctgctgaacgctaaacagattctgggacagctggacggcgggagcgtggcagtcctgcctgtggtcgactccaccaatcagtacctgctggatcgaatcggcgagctgaagagtggggatgcttgcattgcagaatatcagcaggcagggagaggaagcagagggaggaaatggttctctccttttggagctaacctgtacctgagtatgttttggcgcctgaagcggggaccagcagcaatcggcctgggcccggtcatcggaattgtcatggcagaagcgctgcgaaagctgggagcagacaaggtgcgagtcTAGtggcccaatgacctgtatctgcaggatagaaagctggcaggcatcctggtggagctggccggaataacaggcgatgctgcacagatcgtcattggcgccgggattaacgtggctatgaggcgcgtggaggaaagcgtggtcaatcagggctggatcacactgcaggaagcagggattaacctggacaggaatactctggccgctacgctgatccgagagctgcgggcagccctggaactgttcgagcaggaaggcctggctccatatctgccacggtgggagaagctggataacttcatcaatagacccgtgaagctgatcattggggacaaagagattttcgggattagccgggggattgataaacagggagccctgctgctggaacaggacggagttatcaaaccctggatgggcggagaaatcagtctgcggtctgccgaaaag

the amino acid sequence of SEQ ID NO 9, PC-TurboID-172-MNPYK is as follows:

KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADKVRV(MNPYK)WPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK

example 5

Technical scheme for expressing and activating light-operated biotin ligase PC-TurboID-172-ONBK

TurboID-172 plasmid shown in SEQ ID NO. 8 and that mentioned in reference 10

And ONBK-tRNA synthetase plasmid is transfected into Hela cells, and simultaneously, the unnatural amino acid ONBK with the final concentration of 1mM is added, and the optically controlled adjacent biotin ligase PC-TurboID-172-ONBK shown in SEQ ID NO. 10 is obtained through accumulation and expression.

After the ligase was accumulated and expressed for 12 hours, the sample was irradiated with 365nm ultraviolet light for 1 minute to allow PC-TurboID-172-ONBK to recover the biotin ligase activity (see FIG. 1).

The amino acids of SEQ ID NO 10, PC-TurboID-172-ONBK are as follows:

KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADKVRV(ONBK)WPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK

as shown in FIG. 1, the conventional biotin ligase (described in reference 3) is first overexpressed in cells, and after a large amount of biotin is added from an exogenous source, the biotin modification of the adjacent protein (shown on the right side of A) [3 ]. However, during the gradual expression and accumulation process of the traditional adjacent biotin ligase in cells, biotin is carried out on adjacent proteins by using biotin and ATP in a culture medium, a higher background is formed, and the accurate identification of protein species by downstream mass spectrometry is interfered (shown on the left side of A). The light-controlled adjacent biotin ligase obtained in the embodiment of the invention can prevent the combination of the active center of the adjacent biotin ligase and ATP or biotin by inserting the unnatural amino acid MNPYK or ONBK at the 183 th site or 172 th site or inserting the unnatural amino acid ONBY at the 132 th site of the traditional adjacent biotin ligase, thereby inhibiting enzyme activity, eliminating the influence of background biotinylation on the identification result of an effective mass spectrum in the accumulation expression process, and recovering the activity of the biotin ligase by the light-controlled adjacent biotin ligase under the condition of irradiation of specific ultraviolet wavelength.

Example 6

Technical scheme for expressing biotin ligase TurboID-168-MNPYK

The point mutation method as described in example 1 mutated the codon for lysine 168 in TurboID to amber codon TAG and named the vector TurboID-168 plasmid.

The point mutations were performed using primers as follows:

a forward primer: 5'-GGGAGCAGACtAGGTGCGAGT-3'

Reverse primer: 5'-AGCTTTCGCAGCGCTTCT-3'

TurboID-168 plasmid was ligated with that mentioned in reference 9

And MNPYK-tRNA synthetase plasmid are transfected into Hela cell, and at the same time, the final concentration of 1mM unnatural amino acid MNPYK is added, and the adjacent biotin ligase TurboID-168-MNPYK containing the unnatural amino acid insertion shown in SEQ ID NO. 11 is obtained through accumulation and expression.

After the ligase accumulation was expressed for 12 hours, the sample was irradiated with 365 nm-wavelength ultraviolet light for 1 minute, and the change in the enzyme activity before and after irradiation was compared.

The amino acids of SEQ ID NO 11, TurboID-168-MNPYK are as follows:

KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGAD(MNPYK)VRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK

example 7

Technical scheme for expressing biotin ligase TurboID-168-ONBK

TurboID-168 plasmid was ligated with that mentioned in reference 10

And ONBK-tRNA synthetase plasmid is transfected into Hela cells, and simultaneously, the unnatural amino acid ONBK with the final concentration of 1mM is added, and the adjacent biotin ligase TurboID-168-ONBK containing the unnatural amino acid insertion shown in SEQ ID NO. 12 is obtained through accumulation and expression.

After the ligase was accumulated and expressed for 12 hours, the sample was irradiated with 365 nm-wavelength ultraviolet light for 1 minute, and the change in activity before and after irradiation was compared.

12, TurboID-168-ONBK protein sequence as follows:

KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGAD(ONBK)VRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK

example 8

Technical scheme for expressing biotin ligase TurboID-111-ONBY

The point mutation method as described in example 1 mutated the 111 th tyrosine codon in TurboID to amber codon TAG and named the vector TurboID-111 plasmid.

The primers used for point mutations were as follows:

a forward primer: 5'-TTGCAGAATAgCAGCAGGCAG-3'

Reverse primer: 5'-TGCAAGCATCCCCACTCT-3'

TurboID-111 plasmid was ligated with that of reference 11

And ONBY-tRNA synthetase plasmid is transfected into Hela cell, and simultaneously the unnatural amino acid ONBY with the final concentration of 1mM is added, and the adjacent biotin ligase TurboID-111-ONBY containing the unnatural amino acid insertion shown in SEQ ID NO. 13 is obtained through accumulation and expression.

After 12 hours of the expression of the ligase accumulation, the sample was irradiated with 365 nm-wavelength ultraviolet light for 1 minute, and the change in activity before and after irradiation was compared.

13, TurboID-111-ONBY amino acid sequence as follows:

KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAE(ONBY)

QQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK

biotinylated immunoblots of total protein were detected with Streptavidin-HRP (see fig. 2) with or without biotinylation reaction of samples of Hela cells containing the expression of proximal biotin ligase obtained in examples 1 to 8 above, with or without 365nm light irradiation (see fig. 2), specifically:

cell samples obtained after different treatments were treated with RIPA lysate containing protease inhibitor (Thermo Scientific)^TM89900) on ice for 15 minutes, the protein concentration was determined using the BCA kit, the final protein concentration was adjusted to 1mg/mL for all samples, and NuPAGE was added at a final concentration of 1 ×^TMLDS sample buffer (Thermo Scientific)^TMNP0007), samples were heated at 70 ℃ for 10 minutes, 10. mu.l of each lane, using NuPAGE ^TM4 to 12% 15-well Bis-Tris mini-protein gel (Thermo Scientific)^TMNP0323BOX) were performed on a 1 XNuPAGE^TMSeparating at 80V for 1 hr in MOPS buffer (NP0001) system by NuPAGE^TMTransfer buffer (Thermo Scientific)^TMNP0006) spin-transfer the PVDF membrane at a current of 200mA for 1 hour, block the transferred PVDF membrane with PBS containing 5% BSA for 1 hourIn PBS buffer, according to 1: Streptavidin-HRP (Abcam, ab7403) was diluted at a ratio of 5000(v/v) and incubated at room temperature for half an hour, and the PVDF membrane was washed 5 times for 5 minutes each with PBS. Joining Pierce^TMECL reaction substrate (Thermo Scientific)^TM32106) reaction for 1 minute, and exposing on a weather chemiluminometer (Tanon 4600FS) to collect light signals.

Wherein, the specific sequence of different treatment conditions (i.e. lanes 1-18 in FIG. 2) is:

TurboID plasmid was transfected, 12 hours later, no biotin was added.

TurboID plasmid was transfected, and 12 hours later, biotin was added to a final concentration of 0.5mM for reaction at 37 ℃ for 10 minutes.

Transfection of TurboID-183 and

and MNPYK-tRNA synthetase plasmid and 1mM MNPYK unnatural amino acid are added simultaneously, after 12 hours, the ultraviolet irradiation with 365nm wavelength is not needed, and biotin with the final concentration of 0.5mM is added for reaction at 37 ℃ for 10 minutes.

Transfection of TurboID-183 and

adding 1mM MNPYK unnatural amino acid into MNPYK-tRNA synthetase plasmid, after 12 hours, carrying out ultraviolet irradiation for 1 minute by using 365nm wavelength, adding biotin with the final concentration of 0.5mM at 37 ℃, and reacting for 10 minutes

Transfection of TurboID-183 and

1mM ONBK unnatural amino acid was added simultaneously with the ONBK-tRNA synthetase plasmid, and after 12 hours, biotin was added to the final concentration of 0.5mM for 10 minutes at 37 ℃ without UV irradiation at a wavelength of 365 nm.

Transfection of TurboID-183 and

adding 1mM ONBK unnatural amino acid together with the ONBK-tRNA synthetase plasmid, after 12 hours, ultraviolet irradiating for 1 minute at 365nm wavelength, adding final concentration of 0.5mMBiotin was reacted at 37 ℃ for 10 minutes.

Transfection of TurboID-132 and

and ONBY-tRNA synthetase plasmid, 1mM ONBY unnatural amino acid was added, and after 12 hours, biotin was added to the resulting mixture at 37 ℃ to react for 10 minutes without UV irradiation at 365 nm.

Transfection of TurboID-132 and

and ONBY-tRNA synthetase plasmid, adding 1mM ONBY unnatural amino acid, after 12 hr, irradiating with 365nm ultraviolet light for 1 min, adding biotin at 37 deg.C to final concentration of 0.5mM, and reacting for 10 min.

Transfection of TurboID-168 and

Transfection of TurboID-168 and

adding 1mM MNPYK unnatural amino acid into MNPYK-tRNA synthetase plasmid, after 12 hours, irradiating the MNPYK unnatural amino acid by using 365nm wavelength ultraviolet rays for 1 minute, and adding biotin with the final concentration of 0.5mM to react for 10 minutes at 37 ℃.

Transfection of TurboID-168 and

Transfection of TurboID-168 and

and ONBK-tRNA synthetase plasmidSimultaneously adding 1mM ONBK unnatural amino acid, after 12 hours, carrying out ultraviolet irradiation for 1 minute by using a wavelength of 365 nanometers, and adding biotin with a final concentration of 0.5mM for reacting for 10 minutes at 37 ℃.

Transfection of TurboID-111 and

Transfection of TurboID-111 and

Transfection of TurboID-172 and

Transfection of TurboID-172 and

Transfection of TurboID-172 and

Transfection of TurboID-172 and

and ONBK-tRNA synthetase plasmid simultaneously with 1mM ONBK unnatural amino acid, 12 hours later, using 365nm wavelength ultraviolet radiation for 1 minutes, adding 0.5mM biotin final concentration 37 degrees reaction for 10 minutes.

As can be seen from fig. 2, first, comparison of lanes 1 and 2 shows that the biotin ligase TurboID in the vicinity is capable of biotinylation of a neighboring protein with exogenously added biotin within a reaction time of 10 minutes, which is consistent with the conclusion reported in the previous document 3; second, it can be shown from lanes 3, 5, 7, 15 and 17 that after insertion of the unnatural amino acid MNPYK (shown in lane 3) or ONBK (shown in lane 5) at position 183 or insertion of the unnatural amino acid ONBY (shown in lane 7) at position 132 or insertion of the unnatural amino acid MNPYK (shown in lane 15) or ONBK (shown in lane 17) at position 172, biotinylation reaction cannot occur even when biotin is added exogenously, which demonstrates that insertion of the unnatural amino acid at position 183 or position 132 or position 172 of TurboID can block biotin ligase activity; third, comparison of Lane 3 with Lane 4, Lane 5 with Lane 6, Lane 7 with Lane 8, Lane 15 with Lane 16, and Lane 17 with Lane 18 shows that PC-TurboID-183-MNPYK (Lane 3 with Lane 4), PC-TurboID-183-ONBK (Lane 5 with Lane 6), PC-TurboID-ONBY-132 (Lane 7 with Lane 8) PC-TurboID-172-MNPYK (Lane 15 with Lane 16) and PC-TurboID-172-ONBK (Lane 17 with Lane 18) all restore the biotin ligase activity after 1 minute of 365nm illumination, and that TurboID-MNPYK-183 (Lane 4) has a better activity than wild-TurboID (Lane 2) and a better activity than that of TurboID-183-ONBK (Lane 6), PC-TurboID-ONBY-132 (lane 8), PC-TurboID-172-MNPYK (lane 16), and PC-TurboID-172-ONBK (lane 18), which are probably due to the following two reasons: (1) the sensitivity of different unnatural amino acids to 365nm wavelength radiation is different. Specifically, ONBK and ONBY (ONB group modified lysine and tyrosine) are not completely converted into wild-type lysine and tyrosine under the condition of 1 minute of 365nm illumination, while MNPYK (MNPY modified lysine) can be completely converted into wild-type lysine under the condition of 1 minute of 365nm illumination, and (2) the insertion of ONBK and ONBY into 183 or 132 of TurboID or the insertion of MNPYK into 172 of TurboID leads to the reduction of the stability of TurboID and is more easily degraded by proteasome in cells, while the insertion of MNPYK into 183 of TurboID has no influence on the stability of TurboID; fourth, comparison of the 3 rd, 5 th, 7 th, 15 th and 17 th lanes with the 1 st lane shows that the insertion of MNPYK (3 rd lane) and ONBK (5 th lane) at 183 th or the insertion of ONBY (7 th lane) at 132 th or the insertion of MNPYK (15 th lane) and ONBK (17 th lane) at 172 nd of TurboID significantly eliminates the biotinylated background due to the accumulation expression of TurboID (1 st lane); fifthly, comparing the 9 th lane with the 10 th lane, the 11 th lane with the 12 th lane and the 13 th lane with the 14 th lane, it is shown that the insertion of the unnatural amino acid MNPYK (9 th lane and 10 th lane) or ONBK (11 th lane and 12 th lane) at the 168 position of TurboID and the insertion of the unnatural amino acid ONBY (13 th lane and 14 th lane) at the 111 position of TurboID are not changed, whether the lighting is carried out at 365nm for 1 minute, which proves that the insertion of the unnatural amino acid only at the specific site of TurboID can block the biotin ligase activity, and the nonspecific effect of the unnatural amino acid system on the biotin ligase activity of TurboID is excluded.

Application example 1

The light-controlled biotin ligase obtained by the embodiment is used for mass spectrometric identification, and specifically:

taking the example of obtaining the light-controlled biotin ligase PC-TurboID-183-MNPYK in example 1, after fusion expression of the neighboring biotin ligase and the target protein, irradiation is performed at 365nm to restore the enzyme activity of the neighboring biotin ligase, so as to perform biotin labeling on the neighboring protein of the target protein, and the biotin-labeled protein is enriched by Streptavidin coupled magnetic beads for mass spectrometry (see fig. 3). Among them, Stable Isotope Labeling technology (SILAC) under Cell Culture conditions can be used to distinguish cells under different treatment conditions, i.e., 365nm illumination or no illumination.

The method further comprises the following steps:

according to the mitochondrial localization sequence mentioned in reference [3], the mitochondrial matrix localization sequence was fused with TurboID-183 described in SEQ ID NO:3 to obtain mito-TurboID-183 shown in SEQ ID NO:14 by gene synthesis.

The DNA sequence of SEQ ID NO 14mito-TurboID-183 is as follows:

ATGCTCGCCACGAGGGTGTTCTCTCTGGTGGGAAAAAGAGCGATTTCAACCAGTGTGTGTGTCAGAGCCCACGGGGGCAGCGGAGGAaaagacaatactgtgcctctgaagctgatcgctctcctggctaatggcgagttccatagtggcgaacagctgggagaaaccctgggcatgtccagggccgctatcaacaagcacattcagactctgcgcgactggggcgtggacgtgttcaccgtgcccggaaagggctactctctgcccgagcctatcccgctgctgaacgctaaacagattctgggacagctggacggcgggagcgtggcagtcctgcctgtggtcgactccaccaatcagtacctgctggatcgaatcggcgagctgaagagtggggatgcttgcattgcagaatatcagcaggcagggagaggaagcagagggaggaaatggttctctccttttggagctaacctgtacctgagtatgttttggcgcctgaagcggggaccagcagcaatcggcctgggcccggtcatcggaattgtcatggcagaagcgctgcgaaagctgggagcagacaaggtgcgagtcaaatggcccaatgacctgtatctgcaggatagaTagctggcaggcatcctggtggagctggccggaataacaggcgatgctgcacagatcgtcattggcgccgggattaacgtggctatgaggcgcgtggaggaaagcgtggtcaatcagggctggatcacactgcaggaagcagggattaacctggacaggaatactctggccgctacgctgatccgagagctgcgggcagccctggaactgttcgagcaggaaggcctggctccatatctgccacggtgggagaagctggataacttcatcaatagacccgtgaagctgatcattggggacaaagagattttcgggattagccgggggattgataaacagggagccctgctgctggaacaggacggagttatcaaaccctggatgggcggagaaatcagtctgcggtctgccgaaaag

stable Isotope Labeling technology (Stable Isotope Labeling By Amino Acids In Cell Culture, SILAC) under Cell Culture conditions is a technology for quantitative analysis of protein expression By using Stable Isotope labeled Amino Acids In combination with mass spectrometry technology In the Cell Culture process. In this application, L-Arginine-HCl with a final concentration of 73mg/L, 13C6,15N4 and 146 mg/L-Lysine-2 HCl and 13C6 were added to a heavy standard medium, and 73 mg/L-Arginine-HCl and 146 mg/L-Lysine-2 HCl were added to a light standard medium, and cells were cultured for 5 generations in the heavy standard and light standard media, whereby isotopic labeling of 99% or more of the proteins in the cells was achieved.

By co-transfection of mito-TurboID-183 plasmid as shown in SEQ ID NO 11 and the unnatural amino acid insertion System mentioned in reference 9 in heavy-and light-targeted cells

And MNPYK-tRNA synthetase, and adding a final concentration of 1mM unnatural amino acid MNPYK simultaneously, thereby specifically inserting the MNPYK into the 183 rd lysine position of the TurboID to obtain the mitochondrially targeted light-operated adjacent biotin ligase PC-mito-TurboID-183-MNPYK shown in SEQ ID NO. 15.

The protein sequence of SEQ ID NO 15PC-mito-TurboID-183-MNPYK is as follows:

MLATRVFSLVGKRAISTSVCVRAHGGSGGKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADKVRVKWPNDLYLQDR(MNPYK)LAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK

after accumulating and expressing the PC-mito-TurboID-183-MNPYK for 12 hours, illuminating the cells cultured in the heavy-duty culture medium for 1 minute at 365nm, and not illuminating the cells in the light-duty culture medium; adding biotin with the final concentration of 0.5mM, reacting for 10 minutes at 37 ℃, cracking cells, mixing heavy-label cell lysate and light-label cell lysate with equal mass, enriching biotinylated protein by Streptavidin coupled magnetic beads, and identifying the enriched biotinylated protein by mass spectrometry. The ratio of the abundance of the peptide fragments in the heavy-standard sample and the light-standard sample can reflect the protein biotinylated by the PC-mito-TurboID-183-MNPYK after being specifically photoactivated.

As shown in FIG. 3, the conventional method for constructing TurboID fusion to express mitochondrial signal peptide, mito-TurboID [3] (described in reference 3) can biotinylate proteins in mitochondrial matrix, and researchers have identified 212 mitochondrial matrix proteins using mito-TurboID with specificity of only 67% (FIG. 3B, bottom). Using the same biological assay scheme [3] as reported in the previous literature, PC-mito-TurboID-183-MNPYK was able to identify 194 mitochondrial matrix proteins with specificity as high as 91%, whereas the classical proximal biotin ligase was only 67% specific. Therefore, the light-controlled adjacent biotin ligase is significantly superior to the conventional adjacent biotin ligase in the specificity of identifying proteins in specific organelles.

1.Choi-Rhee,E.,H.Schulman,and J.E.Cronan,Promiscuous protein biotinylation by Escherichia coli biotin protein ligase.Protein Sci,2004.13(11):p.3043-50.

2.Roux,K.J.,et al.,A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.Journal of Cell Biology,2012.196(6):p.801-810.

3.Branon,T.C.,et al.,Efficient proximity labeling in living cells and organisms with TurboID.Nat Biotechnol,2018.36(9):p.880-887.

4.Rhee,H.W.,et al.,Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging.Science,2013.339(6125):p.1328-1331.

5.Mair,A.,et al.,Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID.Elife,2019.8.

6.Zhang,B.,Y.Zhang,and J.L.Liu,Highly effective proximate labeling in Drosophila.G3(Bethesda),2021.

7.Srinivasan,G.,C.M.James,and J.A.Krzycki,Pyrrolysine encoded by UAG in Archaea:charging of a UAG-decoding specialized tRNA.Science,2002.296(5572):p.1459-62.

8.Wang,L.,et al.,Expanding the genetic code of Escherichia coli.Science,2001.292(5516):p.498-500.

9.Gautier,A.,et al.,Genetically encoded photocontrol of protein localization in mammalian cells.J Am Chem Soc,2010.132(12):p.4086-8.

10.Chen,P.R.,et al.,A facile system for encoding unnatural amino acids in mammalian cells.Angew Chem Int Ed Engl,2009.48(22):p.4052-5.

11.Arbely,E.,et al.,Photocontrol of tyrosine phosphorylation in mammalian cells via genetic encoding of photocaged tyrosine.J Am Chem Soc,2012.134(29):p.11912-5.

Sequence listing

<110> institute of biophysics of Chinese academy of sciences

<120> a biotin ligase and uses thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 957

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aaagacaata ctgtgcctct gaagctgatc gctctcctgg ctaatggcga gttccatagt 60

ggcgaacagc tgggagaaac cctgggcatg tccagggccg ctatcaacaa gcacattcag 120

actctgcgcg actggggcgt ggacgtgttc accgtgcccg gaaagggcta ctctctgccc 180

gagcctatcc cgctgctgaa cgctaaacag attctgggac agctggacgg cgggagcgtg 240

gcagtcctgc ctgtggtcga ctccaccaat cagtacctgc tggatcgaat cggcgagctg 300

aagagtgggg atgcttgcat tgcagaatat cagcaggcag ggagaggaag cagagggagg 360

aaatggttct ctccttttgg agctaacctg tacctgagta tgttttggcg cctgaagcgg 420

ggaccagcag caatcggcct gggcccggtc atcggaattg tcatggcaga agcgctgcga 480

aagctgggag cagacaaggt gcgagtcaaa tggcccaatg acctgtatct gcaggataga 540

aagctggcag gcatcctggt ggagctggcc ggaataacag gcgatgctgc acagatcgtc 600

attggcgccg ggattaacgt ggctatgagg cgcgtggagg aaagcgtggt caatcagggc 660

tggatcacac tgcaggaagc agggattaac ctggacagga atactctggc cgctacgctg 720

atccgagagc tgcgggcagc cctggaactg ttcgagcagg aaggcctggc tccatatctg 780

ccacggtggg agaagctgga taacttcatc aatagacccg tgaagctgat cattggggac 840

aaagagattt tcgggattag ccgggggatt gataaacagg gagccctgct gctggaacag 900

gacggagtta tcaaaccctg gatgggcgga gaaatcagtc tgcggtctgc cgaaaag 957

<210> 2

<211> 957

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aaagacaata ctgtgcctct gaagctgatc gctctcctgg ctaatggcga gttccatagt 60

ggcgaacagc tgggagaaac cctgggcatg tccagggccg ctatcaacaa gcacattcag 120

actctgcgcg actggggcgt ggacgtgttc accgtgcccg gaaagggcta ctctctgccc 180

gagcctatcc cgctgctgaa cgctaaacag attctgggac agctggacgg cgggagcgtg 240

gcagtcctgc ctgtggtcga ctccaccaat cagtacctgc tggatcgaat cggcgagctg 300

aagagtgggg atgcttgcat tgcagaatat cagcaggcag ggagaggaag cagagggagg 360

aaatggttct ctccttttgg agctaacctg tacctgagta tgttttggcg cctgaagcgg 420

ggaccagcag caatcggcct gggcccggtc atcggaattg tcatggcaga agcgctgcga 480

aagctgggag cagacaaggt gcgagtcaaa tggcccaatg acctgtatct gcaggataga 540

tagctggcag gcatcctggt ggagctggcc ggaataacag gcgatgctgc acagatcgtc 600

attggcgccg ggattaacgt ggctatgagg cgcgtggagg aaagcgtggt caatcagggc 660

tggatcacac tgcaggaagc agggattaac ctggacagga atactctggc cgctacgctg 720

atccgagagc tgcgggcagc cctggaactg ttcgagcagg aaggcctggc tccatatctg 780

ccacggtggg agaagctgga taacttcatc aatagacccg tgaagctgat cattggggac 840

aaagagattt tcgggattag ccgggggatt gataaacagg gagccctgct gctggaacag 900

gacggagtta tcaaaccctg gatgggcgga gaaatcagtc tgcggtctgc cgaaaag 957

<210> 3

<211> 957

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aaagacaata ctgtgcctct gaagctgatc gctctcctgg ctaatggcga gttccatagt 60

ggcgaacagc tgggagaaac cctgggcatg tccagggccg ctatcaacaa gcacattcag 120

actctgcgcg actggggcgt ggacgtgttc accgtgcccg gaaagggcta ctctctgccc 180

gagcctatcc cgctgctgaa cgctaaacag attctgggac agctggacgg cgggagcgtg 240

gcagtcctgc ctgtggtcga ctccaccaat cagtacctgc tggatcgaat cggcgagctg 300

aagagtgggg atgcttgcat tgcagaatat cagcaggcag ggagaggaag cagagggagg 360

aaatggttct ctccttttgg agctaacctg tagctgagta tgttttggcg cctgaagcgg 420

ggaccagcag caatcggcct gggcccggtc atcggaattg tcatggcaga agcgctgcga 480

aagctgggag cagacaaggt gcgagtcaaa tggcccaatg acctgtatct gcaggataga 540

aagctggcag gcatcctggt ggagctggcc ggaataacag gcgatgctgc acagatcgtc 600

attggcgccg ggattaacgt ggctatgagg cgcgtggagg aaagcgtggt caatcagggc 660

tggatcacac tgcaggaagc agggattaac ctggacagga atactctggc cgctacgctg 720

atccgagagc tgcgggcagc cctggaactg ttcgagcagg aaggcctggc tccatatctg 780

ccacggtggg agaagctgga taacttcatc aatagacccg tgaagctgat cattggggac 840

aaagagattt tcgggattag ccgggggatt gataaacagg gagccctgct gctggaacag 900

gacggagtta tcaaaccctg gatgggcgga gaaatcagtc tgcggtctgc cgaaaag 957

<210> 4

<211> 957

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aaagacaata ctgtgcctct gaagctgatc gctctcctgg ctaatggcga gttccatagt 60

ggcgaacagc tgggagaaac cctgggcatg tccagggccg ctatcaacaa gcacattcag 120

actctgcgcg actggggcgt ggacgtgttc accgtgcccg gaaagggcta ctctctgccc 180

gagcctatcc cgctgctgaa cgctaaacag attctgggac agctggacgg cgggagcgtg 240

gcagtcctgc ctgtggtcga ctccaccaat cagtacctgc tggatcgaat cggcgagctg 300

aagagtgggg atgcttgcat tgcagaatat cagcaggcag ggagaggaag cagagggagg 360

aaatggttct ctccttttgg agctaacctg tacctgagta tgttttggcg cctgaagcgg 420

ggaccagcag caatcggcct gggcccggtc atcggaattg tcatggcaga agcgctgcga 480

aagctgggag cagacaaggt gcgagtctag tggcccaatg acctgtatct gcaggataga 540

aagctggcag gcatcctggt ggagctggcc ggaataacag gcgatgctgc acagatcgtc 600

attggcgccg ggattaacgt ggctatgagg cgcgtggagg aaagcgtggt caatcagggc 660

tggatcacac tgcaggaagc agggattaac ctggacagga atactctggc cgctacgctg 720

atccgagagc tgcgggcagc cctggaactg ttcgagcagg aaggcctggc tccatatctg 780

ccacggtggg agaagctgga taacttcatc aatagacccg tgaagctgat cattggggac 840

aaagagattt tcgggattag ccgggggatt gataaacagg gagccctgct gctggaacag 900

gacggagtta tcaaaccctg gatgggcgga gaaatcagtc tgcggtctgc cgaaaag 957

Claims

1. An adjacent biotin ligase, characterized in that: the adjacent biotin ligase is a site-specific insertion of unnatural amino acids near the catalytic center site of ATP or a biotin binding pocket in the biotin ligase.

2. The adjacent biotin ligase according to claim 1, wherein the adjacent biotin ligase is the 183rd lysine in the amino acid sequence of the mutant of the biotin ligase BirA derived from Escherichia coli, One or several sites of tyrosine at position 132 and lysine at position 172 are mutated into unnatural amino acids, thereby inhibiting the enzymatic activity of the adjacent biotin ligase.

3. according to the described adjacent biotin ligase of claim 2, it is characterized in that: described adjacent biotin ligase is the 183rd lysine, the 132nd tyrosine, the 183th lysine, the 132nd tyrosine in the amino acid sequence of the biotin ligase TurboID One or several sites in the 172-position lysine are mutated into an unnatural amino acid; wherein, the unnatural amino acid is the unnatural amino acid MNPY-lysine or ONB-lysine or ONB-tyrosine.

4. The adjacent biotin ligase according to claim 3, wherein the adjacent biotin ligase is that the 183rd lysine in the amino acid sequence shown in SEQID NO:1 is mutated to an unnatural amino acid MNPY-lysine amino acid or unnatural amino acid ONB-lysine, namely the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence shown in SEQ ID NO:5;

Or, in the amino acid sequence shown in SEQ ID NO:1, the tyrosine at position 132 is mutated to the unnatural amino acid ONB-tyrosine, that is, the amino acid sequence shown in SEQ ID NO:7;

Or, the 172nd lysine in the amino acid sequence shown in SEQ ID NO:1 is mutated to unnatural amino acid MNPY-lysine or unnatural amino acid ONB-lysine, that is, the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:9 The amino acid sequence shown in ID NO: 10.

5. the construction method of the described adjacent biotin ligase of claim 1, is characterized in that: to the adjacent ATP of biotin ligase TurboID or two sites near the catalytic center site of biotin binding pocket, utilize positive made

Site-directed insertion of unnatural amino acids with pyrrolidinyl-tRNA synthetase to obtain adjacent biotin ligase.

6. by the construction method of the described adjacent biotin ligase of claim 5, it is characterized in that: described biotin ligase TurboID amino acid sequence is as shown in SEQ ID NO: 1; Unnatural amino acid is unnatural amino acid MNPY-lysine amino acid or the unnatural amino acid ONB-lysine or ONB-tyrosine.

7 . The application of the adjacent biotin ligase according to claim 1 , wherein the application of the adjacent biotin ligase in mass spectrometry identification. 8 .

8. by the application of the described adjacent biotin ligase of claim 7, it is characterized in that: record any adjacent biotin ligase in the described claim 1-4 under specific ultraviolet wavelength illumination conditions, carry out biological treatment to adjacent protein Element labeling and its application in mass spectrometry identification.

9. a kind of method that utilizes adjacent biotin ligase to carry out biotinylation labeling and then carries out mass spectrometry identification, it is characterized in that: utilize any adjacent biotin ligase to record in claim 1-4, after fusion expression with target protein, locate After reaching a specific cell structure, and then irradiating with a specific ultraviolet wavelength, the adjacent biotin ligase restores the enzymatic activity, and then biotin-labels the adjacent protein of the target protein. The biotin-labeled protein is enriched by Streptavidin-coupled magnetic beads for mass spectrometry. identification.