CN113651756A

CN113651756A - Genetically encoded photo-crosslinking non-natural amino acid salt and preparation method and application thereof

Info

Publication number: CN113651756A
Application number: CN202110737321.XA
Authority: CN
Inventors: 彭涛; 张雨晴; 王墅塾
Original assignee: Peking University Shenzhen Graduate School
Current assignee: Peking University Shenzhen Graduate School
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2021-11-16
Anticipated expiration: 2041-06-30
Also published as: CN113651756B

Abstract

The invention discloses a genetically encoded photo-crosslinking non-natural amino acid salt, a preparation method and application thereof. After the bisaziridine group is activated by illumination, a high-activity carbene free radical is generated and is crosslinked with surrounding molecules to form a covalent bond; the alkynyl group can perform a click chemistry reaction with a fluorophore containing azide to perform fluorescence detection on the photo-crosslinking complex, or perform a click chemistry reaction with biotin containing azide, and then perform affinity enrichment on the photo-crosslinking complex by using streptavidin agarose (agarose) or magnetic beads. The invention has the advantages that: can be used for complete living cell research, site-specific incorporation of target protein, reduction of detection and enrichment cost price and improvement of efficiency.

Description

Genetically encoded photo-crosslinking non-natural amino acid salt and preparation method and application thereof

Technical Field

The invention relates to the technical field of research on protein-protein interaction in biochemistry, in particular to a living cell photo-crosslinking method for analyzing and enriching protein-protein interaction.

Background

Protein-protein interactions play a key role in the life process, not only constitute a major component of cellular biochemical reaction networks, but also have important significance in regulating cells and their signals. In particular, the formation of transient and low affinity protein interaction complexes is essential in many signaling and regulatory pathways. However, since transient protein-protein interactions are dynamic in nature to enable rapid response to different stimuli and environmental conditions, analysis and characterization of transient protein interaction networks remains very challenging. Although conventional biological methods such as yeast two-hybrid system, phage display technology, fluorescence energy transfer analysis (FRET), co-immunoprecipitation, and affinity purification have been widely used to study protein-protein interactions, they all have certain drawbacks such as low resolution, high false positive, etc. More importantly, none of these methods allow the study of low affinity, weak, transient protein-protein interactions in physiological states (e.g., in living cells).

In recent years, chemical cross-linking agents have begun to be a powerful tool for studying protein-protein interactions. The electrophilic groups of the chemical crosslinkers can chemically react with nucleophilic side chains of the natural amino acids to form covalent bonds, thereby stabilizing low affinity protein-protein interactions, facilitating the validation and characterization of low affinity, transient protein interaction complexes and facilitating structural studies thereof. However, such methods are generally inefficient, toxic to living cells, and place certain requirements on the distance and geometry of the protein interaction complex. Another method of covalent cross-linking of protein-interacting complexes relies on highly reactive intermediates generated in situ by photoactivation, such as diradicals or carbene intermediates generated by benzophenone or diazirine functional groups under uv light irradiation. These photo-excited free radical species are more reactive than chemical cross-linking agents and can form covalent bonds with N-H, O-H, and C-H bonds in nearby protein molecules in intact cells efficiently and irreversibly, a process also known as "photocrosslinking". In addition, the photo-induced generation of free radical intermediates is more reactive and shorter lived than chemical cross-linkers, which potentially increases the efficiency of protein cross-linking and reduces off-target cross-linking. The photocrosslinking strategy combines biochemical analysis and mass spectrum identification, and provides an effective way for researching low-affinity transient protein-protein interaction in a natural environment.

However, the photocrosslinking strategy requires the incorporation of appropriate photocrosslinking groups into the target protein molecule, which limits the broad application of this strategy to some extent. At present, introduction of a photocrosslinking group into a target protein (or polypeptide) is mainly achieved by chemical synthesis (polypeptide), chemical modification, protein posttranslational modification, or residue-specific (residual-specific) unnatural amino acid incorporation. However, there are some problems and challenges with these existing technologies: 1. difficult to use for whole living cell studies. Although the method based on chemical synthesis and modification can introduce the photo-crosslinking group to the protein at a fixed point, the experimental operation is complicated, and the protein obtained by the method is difficult to deliver into the whole living cell. 2. And (3) non-specific doping. Although the unnatural amino acid based on protein posttranslational modification and residue specificity can introduce a photocrosslinking group to a target protein in an intact living cell, the site-specific photocrosslinking labeling cannot be realized, and the photocrosslinking group is introduced to other proteins in the cell, so that potential photocrosslinking background and false positive are caused. 3. Antibody dependent detection and enrichment. Due to the low abundance of the photo-crosslinking complex, the photo-crosslinking complex needs to be enriched in order to realize the biochemical detection and mass spectrum identification of the photo-crosslinking complex. Most of the methods rely on antibodies to detect and enrich target proteins and light cross-linked complexes, but the antibodies are expensive and often have low enrichment efficiency.

Proteins are generally composed of 20 natural amino acids. In recent years, artificial Expansion of the Genetic Code has been carried out using Genetic Code Expansion technology (Genetic Code Expansion) to make it possible to incorporate unnatural amino acids having different side chains into proteins during protein translation. In order to specifically incorporate an unnatural amino acid into a specific site in a protein, it is necessary to use an orthogonal aminoacyl-tRNA synthetase (aaRS) that recognizes the unnatural amino acid to its cognate tRNA. This allows the specific incorporation of unnatural amino acids during translation of mRNA in response to an amber stop codon (i.e., TAG) placed at a custom site in the gene of interest. Orthogonality refers to the specific recognition of aaRS and tRNA from each other for recognition of unnatural amino acids in the chosen host without interfering with endogenous synthetases, trnas, and natural amino acids. Orthogonal aaRS/orthogonal synthetases can be obtained by directed evolution methods and so far more than 250 unnatural amino acids can be introduced at specific sites of proteins of interest in prokaryotic and eukaryotic cells using engineered MjTyrRS or PylRS mutants.

Disclosure of Invention

The invention aims to provide a genetically encoded unnatural amino acid to solve the problems that the prior art is difficult to use in complete living cell research, nonspecific incorporation, detection and enrichment, and has high cost, low efficiency and the like.

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

a non-natural amino acid salt YnDiPK having the structural formula:

wherein X is Cl or CF₃COO。

The non-natural amino acid salt YnDiPK is a lysine analog comprising a bis-aziridine photocrosslinking group, and an alkynyl-rich group. As shown in fig. 1, after being activated by illumination, the bis-aziridine group generates a highly active carbene radical which is crosslinked with surrounding molecules to form a covalent bond; the alkynyl group can perform a click chemistry reaction with a fluorophore containing azide to perform fluorescence detection on the photo-crosslinking complex, or perform a click chemistry reaction with biotin containing azide, and then perform affinity enrichment on the photo-crosslinking complex by using streptavidin agarose (agarose) or magnetic beads.

An orthogonal pyrrollysyl tRNA synthetase that recognizes and incorporates the non-natural amino acid salt YnDiPK into the protein of interest in a site-specific manner:

the non-natural amino acid YnDiPK can be introduced into a specific site of a target protein in prokaryotic and eukaryotic cells.

In some embodiments, an orthogonal pyrrolysinyl tRNA synthetase that recognizes and incorporates the non-natural amino acid salt YnDiPK into a protein of interest in a site-specific manner has an amino acid sequence such as: SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3.

The invention also provides a compound containing the non-natural amino acid salt YnDiPK.

The invention also provides a protein expression system, a translation system and a cell comprising the non-natural amino acid salt YnDiPK.

The invention also provides protein expression systems, translation systems and cells comprising the non-natural amino acid salt YnDiPK and the orthogonal pyrrolysyl tRNA synthetase that recognizes and incorporates the non-natural amino acid salt YnDiPK into a protein of interest in a site-specific manner.

The invention also provides application of the non-natural amino acid salt YnDiPK in site-specific protein insertion.

The invention also provides application of the non-natural amino acid salt YnDiPK in analysis, characterization and enrichment of protein-protein interaction.

Compared with the prior art, the invention has the following advantages:

designing and synthesizing a bifunctional unnatural amino acid salt YnDiPK with a photo-crosslinking group (diazirine) and an enrichment group (alkynyl), then screening orthogonal pyrrilinyl tRNA synthetase to make the bifunctional unnatural amino acid salt YnDiPK specific to YnDiPK, and utilizing a Genetic Code Expansion technology (Genetic Code Expansion) to insert YnDiPK into a position coded by an amber stop codon (TAG) in a target protein in a fixed point manner, thereby realizing the site-specific doping of the photo-crosslinking group and the enrichment group in a target protein molecule in an expression host. Under the condition of illumination, the photocrosslinking group is activated to form a high-reactivity free radical intermediate, and the intermediate is crosslinked with the interacting protein nearby to form a covalent protein complex. And then, carrying out click chemical reaction on the enriched group (alkynyl) and the azide-containing fluorophore or biotin to realize fluorescence detection or affinity enrichment of the photocrosslinking complex.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention:

FIG. 1: genetically encoded photo-crosslinked unnatural amino acid YnDiPK in living cells for capturing, detecting and enriching protein-protein interaction diagrams.

FIG. 2: scheme for synthesis of unnatural amino acid YnDiPK.

FIG. 3: results of the pyrrolysinyl tRNA synthetase mutants that insert YnDiPK into GFP-Y39TAG in mammalian cells were examined. (A) Fluorescence (GFP) detection of different pyrrolysinyl tRNA synthetase mutants YnDiPK was inserted into the GFP-Y39TAG protein. (B) Immunoblotting (anti-GFP) detection of different pyrrolysinyl tRNA synthetase mutants YnDiPK was inserted into the GFP-Y39TAG protein.

FIG. 4: alignment chart of amino acid sequences of wild type pyrrolysinyl tRNA synthetase and mutant synthetase.

FIG. 5: results of detecting YnDiPK site-specific insertion of GFP-Y39TAG in mammalian cells (HEK293T cells) by fluorescence spectroscopy and immunoblotting. (A) Fluorescence (GFP) detection YnDiPK was inserted into the GFP-Y39TAG protein in a concentration-dependent manner. (B) Immunoblotting (anti-GFP) detects YnDiPK insertion of GFP-Y39TAG protein in a concentration-dependent manner.

FIG. 6: site-specific insertion of YnDiPK into GFP in E.coli. Immunoblotting (anti-GFP) and Coomassie blue staining (CB) detect YnDiPK site-specific insertion of GFP-Y39TAG protein in E.coli, with the arrow indicating GFP protein.

FIG. 7: a result chart of a complex of a YnDiPK site-specific inserted H2B-V18TAG protein in a mammalian cell (HEK293T cell) and a cross-linked H2B interacting protein under the illumination condition is detected by utilizing immunoblotting and in-gel fluorescence. (A) Immunoblotting (anti-HA) detects the site-specific insertion of YnDiPK into H2B-V18TAG protein and cross-linking of the H2B interacting protein complex under light conditions. (B) In-gel fluorescence (Az-Rho) detection of the site-specific insertion of YnDiPK H2B-V18TAG protein and cross-linking of the H2B interacting protein complex under light conditions, with coomassie blue staining (CB) as loading control.

FIG. 8: the result chart of the condition of the cross-linking H2B interaction protein complex in the mammalian cell (HEK293T cell) under the illumination condition is optimized by utilizing immunoblotting and in-gel fluorescence detection. (A) Immunoblotting (anti-HA) performed at different concentrations of YnDiPK and cross-linked H2B interacting protein complexes under different light time conditions. (B) In-gel fluorescence (Az-Rho) detection of YnDiPK at various concentrations and cross-linking of the H2B interacting protein complex under different light time conditions, with Coomassie blue staining (CB) as a loading control.

FIG. 9: the result of the light-crosslinked protein complex enriched with H2B by alkynyl was detected by immunoblotting (anti-HA).

FIG. 10: the result of detecting H2B-SET photocrosslinking protein complex by immunoblotting (anti-myc) is shown.

FIG. 11: the result chart of the site-specific insertion of H3 protein into YnDiPK in mammalian cells (HEK293T cells) and the cross-linking of H3 interaction protein complex under the illumination condition is detected by utilizing immunoblotting and in-gel fluorescence. (A) Immunoblotting (anti-HA) detects the site-specific insertion of YnDiPK into H3 protein and cross-linking of the H3 interacting protein complex under light conditions. (B) In-gel fluorescence (Az-Rho) detection of YnDiPK site-specific insertion H3 protein and crosslinking of the H3 interacting protein complex under light conditions, Coomassie blue staining (CB) as loading control.

Detailed Description

The present invention will be described in detail with reference to the drawings and specific embodiments, which are illustrative of the present invention and are not to be construed as limiting the present invention.

Definition of partial terms

The term "genetic code" as used herein means: living cells translate the genetic material information encoded in the DNA or mRNA sequence into proteins.

The "genetically encoded unnatural amino acid" of the invention refers to: an unnatural amino acid that can participate in genetic coding.

The term "pyrrolysinyl tRNA synthetase" as used herein refers to: an enzyme that catalyzes activation of pyrrolysine and covalently binds to the 3' end of the corresponding tRNA molecule. Pyrrolysine (Pyl) is found in the methylamine methyltransferase of methanogens and is the currently known amino acid of position 22 involved in protein biosynthesis. Unlike the standard amino acid, pyrrolysine (Pyl) is formed from the sense code for the stop codon UAG. Correspondingly, the methanogens also contain specific pyrrolysinyl-tRNA synthetase (PylRS) and pyrrolysine tRNA (tRNA pyl), which have a special structure different from that of classical tRNA. The methanogen produces pyrrolysinyl-tRNA Pyl (Pyl-tRNA Pyl) via direct and indirect pathways, which may control the UAG encoding to a stop codon or pyrrolysine via special structures on the mRNA and other mechanisms not yet discovered. The wild-type pyrrilinyl-tRNA synthetase (PylRS) can be obtained, for example, from Methanosarcina mazei (Methanosarcina mazei), Methanosarcina pasteurianus (Methanosarcina barkeri), Methanosarcina acetogenis (Methanosarcina acetovorans) and the like, which are methanogenic archaea, but is not limited thereto.

The term "mutant pyrrilysinyl tRNA synthetase" as used herein refers to: the mutant was prepared by introducing a mutation into wild-type pyrrolysinyl-tRNA synthetase (PylRS) by various methods. The types of lysine derivatives that can be activated by the wild-type PylRS are limited, and for example, the pyrrolysyl tRNA synthetase mutant Mm-AF of the present invention belongs to a "pyrrolysyl-tRNA synthetase mutant", and even an unnatural amino acid salt YnDiPK that is inactive when the wild-type pyrrolysyl tRNA synthetase is used, can be efficiently introduced into a protein by the pyrrolysyl-tRNA synthetase mutant of the present invention.

The term "amber stop codon TAG" as used herein means: transcription of the protein gene DNA results in a tripartite polynucleotide, TAG, corresponding to the termination codon UAG on the mRNA. In the present invention, "TAG" and "UAG" refer to amber stop codons on the corresponding DNA and mRNA, respectively.

The term "unnatural amino acid" as used herein refers to: any amino acid, modified amino acid and/or amino acid analog that is not among the 20 common natural amino acids. For example, the non-natural amino acid salt YnDiPK may be used in the present invention.

The term "translation system" as used herein refers to: amino acids are incorporated into the components of an extended polypeptide chain (protein). Components of a translation system can include, for example, ribosomes, trnas, synthetases, mrnas, and the like. The O-tRNA and/or O-RS of the invention can be added to or part of an in vitro or in vivo translation system, e.g., in a non-eukaryotic cell, such as a bacterium (e.g., E.coli), or in a eukaryotic cell, such as a yeast, mammalian cell, plant cell, algal cell, fungal cell, insect cell, etc.

The term "PBS" as used herein means: phosphate buffered saline, phosphate buffer saline, is well known to those skilled in the art and is widely used.

Example 1: chemical synthesis of unnatural amino acid salt YnDiPK

The chemical synthesis route of the non-natural amino acid salt YnDiPK is shown in figure 2, namely alpha-N-Boc-lysine (N)^α-Boc-L-Lys) is subjected to an activated ester reaction with compound 1 under basic conditions to produce lysine derivative compound 2, followed by Boc protecting group removal under acidic conditions (HCl or TFA) to produce the corresponding unnatural amino acid salt YnDiPK (hydrochloride or trifluoroacetate), which is confirmed by nuclear magnetic resonance. The specific process is as follows:

1.1 Synthesis of Compound 2

To compound 1(1eq) CH at 0 deg.C₂Cl₂Pyridine (1.2eq) and 4-nitrophenyl chloroformate (1.2eq) were added to the solution. The reaction mixture was stirred at 25 ℃ for 10h and quenched with water. The layers were separated and the aqueous layer was treated with CH₂Cl₂And (4) extracting. The combined organic layers were saturated with NH₄Washed with aqueous Cl solution and brine, and dried over anhydrous Na₂SO₄Dried and concentrated under reduced pressure to give the crude product as a colorless oil. The crude product is directly dissolved in DMF and N is added dropwise at 0 DEG C^α-Boc-L-Lys (1eq), DIPEA (1.5eq) and DMAP (0.2eq) in DMF. The reaction mixture was stirred at 25 ℃ for 10h and quenched with water. Separation ofEach layer, aqueous layer is substituted with CH₂Cl₂And (4) extracting. The combined organic layers were washed with 1N HCl and brine, dried over anhydrous Na₂SO₄Dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 2 as a white solid (64% yield).¹H NMR(500MHz,MeOD)δ4.11–4.02(m,1H),3.92(t,J＝6.3Hz,2H),3.10(t,J＝6.8Hz,2H),2.26(t,J＝2.6Hz,1H),2.03(td,J＝7.4,2.7Hz,2H),1.84–1.76(m,1H),1.71(t,J＝6.3Hz,2H),1.67–1.62(m,3H),1.55–1.48(m,2H),1.47–1.41(m,11H).¹³C NMR(126MHz,MeOD)δ174.89,157.31,156.74,82.31,79.16,69.07,59.22,53.46,40.08,32.34,32.14,31.13,29.09,27.45,26.19,22.74,12.50.HRMS:C₁₉H₃₀N₄O₆Na[M+Na]⁺Calculated values: 433.2063, found: 433.2058.

1.2 Synthesis of YnDiPK

To a solution of compound 2 in 1, 4-dioxane was added a solution of HCl (4M) in 1, 4-dioxane and the reaction mixture was stirred at 25 ℃ for 2 h. The solvent was removed under reduced pressure to give a white solid of YnDiPK hydrochloride (94% yield).¹H NMR(300MHz,MeOD)δ4.03–3.88(m,3H),3.14(t,J＝6.5Hz,2H),2.29(t,J＝2.7Hz,1H),2.04(td,J＝7.5,2.7Hz,2H),2.02–1.86(m,2H),1.73(t,J＝6.3Hz,2H),1.66(t,J＝7.4Hz,2H),1.62–1.41(m,4H).¹³C NMR(75MHz,MeOD)δ170.53,157.47,82.37,69.15,59.34,52.57,39.94,32.35,32.20,29.89,29.14,26.28,21.93,12.56.HRMS:C₁₄H₂₃N₄O₄[M+Na]⁺Calculated values: 311.1719, found: 311.1720.

or trifluoroacetic acid was added to a dichloromethane solution of compound 2, and the reaction mixture was stirred at 25 ℃ for 2 h. The solvent was removed under reduced pressure to give a pale yellow solid of YnDiPK trifluoroacetate (98% yield).¹H NMR(300MHz,MeOD)δ4.03–3.88(m,3H),3.14(t,J＝6.5Hz,2H),2.29(t,J＝2.7Hz,1H),2.04(td,J＝7.5,2.7Hz,2H),2.02–1.86(m,2H),1.73(t,J＝6.3Hz,2H),1.66(t,J＝7.4Hz,2H),1.62–1.41(m,4H).¹³C NMR(75MHz,MeOD)δ170.53,157.47,82.37,69.15,59.34,52.57,39.94,32.35,32.20,29.89,29.14,26.28,21.93,12.56.HRMS:C₁₄H₂₃N₄O₄[M+Na]⁺Calculated values: 311.1719, found: 311.1720.

example 2: screening of Pyrrolysinyl tRNA synthetase mutants that recognize YnDiPK

In order to obtain the active site mutants of the pyrrollysyl tRNA synthetase capable of being inserted into YnDiPK, a series of active site mutants based on wild type pyrrollysyl tRNA synthetase are constructed and tested by using a conventional molecular cloning method, and the active site mutants are shown in the following table 1 and figure 4.

TABLE 1

In order to test the ability of the above mutants to recognize the inserted YnDiPK, the activity of the GFP of the present invention was examined in mammalian cells (HEK293T cells) using GFP as a model protein. Specifically, HEK293T cells were seeded onto polylysine coated 12-well plates and cultured overnight in 1mL of growth medium. The next day, a GFP plasmid containing the amber codon TAG, GFP-Y39TAG (0.65. mu.g per well) and the corresponding plasmid expressing the pyrrolysinyl tRNA synthetase mutants shown in Table 1 (0.35. mu.g per well) were co-transfected into HEK293T cells using polyethyleneimine (Polyethylenimine, PEI; 2.5. mu.g per well) with or without YnDiPK (1 mM). After 24 hours of transfection, GFP fluorescence was detected by fluorescence microscopy. At least three fields per well were randomly selected for each fluorescence imaging experiment. The fluorescence intensity of each Image was quantified in Image J and grouped for statistical analysis. Thereafter, cells were lysed with 4% SDS lysis buffer containing protease inhibitors by sonication and vortexing. The resulting cell lysate was centrifuged at 16,000x g for 5 minutes at room temperature to remove cell debris. Protein concentration was determined by BCA assay. Finally, cell lysates were separated on conventional SDS-PAGE gels and analyzed by immunoblotting.

As shown in FIG. 3, in mammalian HEK293T cells, the mutants of pyrrolysinyl tRNA synthetases, such as Mm-AF, Mb-ASF and Mb-MGA, were able to insert the unnatural amino acid YnDiPK into the GFP protein, and either fluorescence of GFP was observed by fluorescence microscopy or expression of GFP was detected by immunoblot analysis. In contrast, in the wild-type sample of the pyrrolysinyl tRNA synthetase, GFP fluorescence was not observed by fluorescence microscopy; in line with this, the expression of GFP was not substantially detected by immunoblotting.

Thus, it was discovered that mutants of pyrrolysinyl tRNA synthetases, such as Mm-AF, Mb-ASF and Mb-MGA, efficiently introduce YnDiPK into the GFP-Y39TAG protein. The sequences of Mm-AF, Mb-ASF and Mb-MGA are shown in SEQ ID NO.1, 2 and 3, respectively.

The sequence of SEQ ID NO.1(Mm-AF) is as follows:

the sequence of SEQ ID NO.2(Mb-ASF) is as follows:

the sequence of SEQ ID NO.3(Mb-MGA) is as follows:

example 3: YnDiPK is inserted into mammalian cell proteins in a concentration-dependent manner

HEK293T cells were seeded onto polylysine coated 12-well plates and cultured overnight in 1mL of growth medium. On the next day, a GFP plasmid containing the amber codon TAG, i.e., GFP-Y39TAG (0.65. mu.g per well) and a plasmid expressing the pyrrolysinyl tRNA synthetase mutant Mm-AF (0.35. mu.g per well) were co-transfected into cells using PEI (2.5. mu.g per well) without or with different concentrations (6.25. mu. M, 12.5. mu. M, 25. mu. M, 50. mu. M, 100. mu. M, 200. mu. M, 400. mu. M, 800. mu. M) of YnDiPK added. After 24 hours of transfection, GFP fluorescence was detected by fluorescence microscopy. At least three fields per well were randomly selected for each fluorescence imaging experiment. The fluorescence intensity of each Image was quantified in Image J and grouped for statistical analysis. Thereafter, cells were lysed with 4% SDS lysis buffer containing protease inhibitors by sonication and vortexing. The resulting cell lysate was centrifuged at 16,000x g for 5 minutes at room temperature to remove cell debris. Protein concentration was determined by BCA assay. Finally, cell lysates from which cell debris was removed were separated on an in-house SDS-PAGE gel and analyzed by immunoblotting.

As a result, as shown in fig. 5, it was found that in mammalian HEK293T cells, the full-length GFP protein was expressed only when YnDiPK was present in the medium, and the expression amount of GFP protein correlated with the concentration of YnDiPK.

The same or equivalent technical effects can be achieved by carrying out the above experimental operations by replacing the plasmid expressing the pyrrolysinyl tRNA synthetase mutant Mm-AF with the plasmid expressing the pyrrolysinyl tRNA synthetase mutant Mb-ASF and the plasmid expressing the pyrrolysinyl tRNA synthetase mutant Mb-MGA, respectively.

Example 4: YnDiPK is inserted into the protein of escherichia coli at fixed points

The Escherichia coli strain BL21(DE3) was co-transformed with a GFP plasmid containing the amber codon TAG (GFP-Y39TAG) and a plasmid expressing the pyrrolysinyl tRNA synthetase mutant Mm-AF. The transformed bacteria were grown overnight at 37 ℃ in LB medium containing kanamycin (40. mu.g/mL) and chloramphenicol (34. mu.g/mL), and then cultured at a temperature of 1: 100 dilutions were inoculated into fresh TB medium (pH 8.0) supplemented with kanamycin (40. mu.g/mL) and chloramphenicol (34. mu.g/mL) and cultured with shaking at 37 ℃. When the OD600 reached 0.6, YnDiPK was added to the bacterial culture at a final concentration of 1 mM. After 1 hour of incubation, protein expression was induced by addition of 1mM isopropyl-1-thio- β -D-galactopyranoside (IPTG) for 10 hours. The bacteria were then harvested by centrifugation and lysed with 4% SDS lysis buffer (4% SDS, 150mM NaCl, 50mM triethanolamine, pH 7.4) at 95 ℃. The resulting bacterial lysate was centrifuged at 16,000x g for 5 minutes at room temperature to remove bacterial debris. Protein concentration was determined by BCA assay. Finally, bacterial lysates were electrophoretically separated on home-made SDS-PAGE gels and analyzed by immunoblotting and coomassie blue staining.

As a result, as shown in FIG. 6, when the blank control group and the YnDiPK group were compared, the full-length GFP protein was expressed only when YnDiPK was present in the medium. Therefore, YnDiPK was successfully integrated into the Y39 site of GFP in E.coli.

Taken together, it was shown that YnDiPK can be site-specifically encoded into GFP protein by the pyrrolysinyl tRNA synthetase mutants (Mm-AF, Mb-ASF, Mb-MGA) in both E.coli and mammalian cells.

Example 5: YnDiPK photocrosslinking H2B interacting protein complex in mammalian cells

HEK293T cells were seeded onto polylysine coated 12-well plates and cultured overnight in 1mL of growth medium. The next day, the H2B plasmid containing the amber codon TAG, namely H2B-V18TAG (0.65. mu.g per well) and the plasmid expressing the pyrrollysyl tRNA synthetase mutant Mm-AF (0.35. mu.g per well) were co-transfected into cells using PEI (2.5. mu.g per well) without or with YnDiPK (0.5 mM). 24 hours after transfection, the medium was changed to PBS solution and treated with 365nm light for 10 min. Cells were lysed with 4% SDS lysis buffer containing protease inhibitors by sonication and vortexing. The resulting cell lysate was centrifuged at 16,000x g for 5 minutes at room temperature to remove cell debris. Protein concentration was determined by BCA assay. Then, the lysate is coupled with azide-rhodamine using copper-catalyzed alkynyl-azide click reaction (clickreaction). Finally, the coupled cell lysates were separated on home-made SDS-PAGE gels and analyzed by in-gel fluorescence and immunoblotting.

As shown in fig. 7, both in-gel fluorescence and immunoblot analysis results showed that in mammalian HEK293T cells, full-length H2B protein was expressed only when YnDiPK was present in the culture medium; under light conditions, the formation of high molecular weight photo-crosslinked protein complexes can be observed.

Example 6: condition optimization of YnDiPK photocrosslinking of H2B interacting protein complexes in mammalian cells

HEK293T cells were seeded onto polylysine coated 12-well plates and cultured overnight in 1mL of growth medium. The next day, the H2B plasmid containing the amber codon TAG, namely H2B-V18TAG (0.65. mu.g per well) and the plasmid expressing the pyrrollysyl tRNA synthetase mutant Mm-AF (0.35. mu.g per well) were co-transfected into cells using PEI (2.5. mu.g per well) without or with different concentrations of YnDiPK (final concentrations of 0. mu.M, 25. mu.M, 50. mu.M, 100. mu.M or 200. mu.M, respectively). 24 hours after transfection, the medium was changed to PBS solution and treated with 365nm light for various periods of time (0min, 5min, 10min, 15min, 20 min). Cells were lysed with 4% SDS lysis buffer containing protease inhibitors by sonication and vortexing. The resulting cell lysate was centrifuged at 16,000x g for 5 minutes at room temperature to remove cell debris. Protein concentration was determined by BCA assay. Then, the lysate is coupled with azide-rhodamine using copper-catalyzed alkynyl-azide click reaction (click reaction). Finally, the coupled cell lysates were separated on home-made SDS-PAGE gels and analyzed by in-gel fluorescence and immunoblotting.

As shown in fig. 8, both in-gel fluorescence and immunoblot analysis results showed that in mammalian HEK293T cells, full-length H2B protein was expressed only when YnDiPK was present in the culture medium; under light conditions, the formation of high molecular weight photo-crosslinked protein complexes can be observed. And, as the concentration of YnDiPK increases and the light irradiation time increases, the formation of photo-crosslinked protein complexes gradually increases.

Example 7: YnDiPK photocrosslinking and enrichment of H2B interacting protein complexes in mammalian cells

HEK293T cells were seeded onto polylysine coated 100mm dishes and cultured overnight in 10mL growth medium. The next day, the H2B plasmid containing the amber codon TAG, namely H2B-V18TAG (6.5 μ g) and the plasmid expressing the pyrrolysinyl tRNA synthetase mutant Mm-AF (3.5 μ g), were co-transfected into cells using PEI (25 μ g) with no or no YnDiPK (0.2mM) added. 24 hours after transfection, the medium was changed to PBS solution and treated with 365nm light for 10 min. Cells were lysed with 4% SDS lysis buffer containing protease inhibitors by sonication and vortexing. The resulting cell lysate was centrifuged at 16,000x g for 5 minutes at room temperature to remove cell debris. Protein concentration was determined by BCA assay. Thereafter, the lysate was coupled with azide-biotin using copper-catalyzed alkynyl-azide click reaction (click reaction), and then the biotin-modified protein was enriched using streptavidin agarose (agarose). Finally, the enriched proteins were eluted with SDS-PAGE loading buffer, separated on home-made SDS-PAGE gels and analyzed by immunoblotting.

As shown in fig. 9, the immunoblot analysis results showed that H2B photocrosslinked protein complex formed by YnDiPK under light conditions can be isolated and enriched by streptavidin agarose (pull-down).

Example 8: YnDiPK photocrosslinking H2B-SET interaction protein complex in mammalian cells

To further verify that YnDiPK is capable of photocrosslinking interacting protein complexes in mammalian cells, the potential chaperone protein SET of histone H2B was selected and the ability of YnDiPK photocrosslinking H2B to interact with SET protein-protein was examined.

HEK293T cells were seeded onto polylysine coated 100mm dishes and cultured overnight in 10mL growth medium. The next day, the H2B plasmid containing the amber codon TAG, i.e., H2B-V18TAG (4 μ g), the plasmid expressing the pyrrolysinyl tRNA synthetase mutant Mm-AF (2 μ g), and the plasmid expressing the SET protein (4 μ g) were co-transfected into cells using PEI (25 μ g) with the addition of YnDiPK (0.2 mM). 24 hours after transfection, the medium was changed to PBS solution and treated with no light or 365nm light for 10 min. Cells were lysed with 4% SDS lysis buffer containing protease inhibitors by sonication and vortexing. The resulting cell lysate was centrifuged at 16,000x g for 5 minutes at room temperature to remove cell debris, and the supernatant was diluted with a solution containing 1% Triton X-100, and the protein concentration was determined by BCA assay. Then, the magnetic beads coupled with the HA-tag antibody are used for enriching H2B protein and H2B-SET photo-crosslinked complexes, the enriched protein is eluted by SDS-PAGE loading buffer solution, separated on a home-made SDS-PAGE gel and analyzed by immunoblotting.

As shown in fig. 10, the results of the immunoblot analysis showed that the H2B-SET photocrosslinked complex with high molecular weight was observed only under light conditions and the molecular weight was about the sum of the molecular weights of H2B and SET protein, confirming that YnDiPK can indeed photocrosslink protein-protein interactions in mammalian cells.

Example 9: YnDiPK photocrosslinking H3 interacting protein complex in mammalian cells

HEK293T cells were seeded onto polylysine coated 12-well plates and cultured overnight in 1mL of growth medium. The next day, the H3 plasmid containing the amber codon TAG (0.65. mu.g per well) and the plasmid expressing the pyrrolysinyl tRNA synthetase mutant Mm-AF (0.35. mu.g per well) were co-transfected into cells using PEI (2.5. mu.g per well) with the addition of YnDiPK (0.2 mM). 24 hours after transfection, the medium was changed to PBS solution and treated with no light or 365nm light for 10 min. Cells were lysed with 4% SDS lysis buffer containing protease inhibitors by sonication and vortexing. The resulting cell lysate was centrifuged at 16,000x g for 5 minutes at room temperature to remove cell debris. Protein concentration was determined by BCA assay. Then, the lysate is coupled with azide-rhodamine using copper-catalyzed alkynyl-azide click reaction (click reaction). Finally, the coupled cell lysates were separated on home-made SDS-PAGE gels and analyzed by in-gel fluorescence and immunoblotting.

As shown in fig. 11, both in-gel fluorescence and immunoblot analysis results indicate that in mammalian HEK293T cells, the formation of a photocrosslinked interacting protein complex with high molecular weight H3 protein was observed only under light conditions.

The technical solutions provided by the embodiments of the present invention are described in detail above, and the principles and embodiments of the present invention are explained herein by using specific examples, and the descriptions of the embodiments are only used to help understanding the principles of the embodiments of the present invention; meanwhile, for a person skilled in the art, according to the embodiments of the present invention, there may be variations in the specific implementation manners and application ranges, and in summary, the content of the present description should not be construed as a limitation to the present invention.

Sequence listing

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Claims

1. A non-natural amino acid salt YnDiPK has the following structural formula:

wherein X is Cl or CF₃COO。

2. An orthogonal pyrrollysyl tRNA synthetase that recognizes and incorporates the non-natural amino acid salt YnDiPK of claim 1 into a protein of interest in a site-specific manner, characterized in that:

the non-natural amino acid YnDiPK can be specifically introduced into a specific site of a target protein in prokaryotic and eukaryotic cells.

3. The orthogonal pyrrollysyl tRNA synthetase for identifying and incorporating the non-natural amino acid salt YnDiPK into a protein of interest in a site-specific manner according to claim 2, wherein:

the amino acid sequence is SEQ ID NO. 1:

MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLANYLRKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL。

4. the orthogonal pyrrollysyl tRNA synthetase for identifying and incorporating the non-natural amino acid salt YnDiPK into a protein of interest in a site-specific manner according to claim 2, wherein:

the amino acid sequence is SEQ ID NO. 2:

MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVNNSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISLNMAKPFRELEPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVDRGFLEIKSPILIPAEYVERMGINNDTELSKQIFRVDKNLCLRPMLAPTLYNYARKLDRILPGPIKIFEVGPCYRKESDGKEHLEEFTMVNFSQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVFGDTLDIMHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNL。

5. the orthogonal pyrrollysyl tRNA synthetase for identifying and incorporating the non-natural amino acid salt YnDiPK into a protein of interest in a site-specific manner according to claim 2, wherein:

the amino acid sequence is SEQ ID NO. 3:

MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVNNSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISLNMAKPFRELEPELVTRRKNDFQRLYTNDREDYLGKLERDITKFFVDRGFLEIKSPILIPAEYVERMGINNDTELSKQIFRVDKNLCLRPMLAPTLMNYGRKLDRILPGPIKIFEVGPCYRKESDGKEHLEEFTMVNFAQMGSGCTRENLEALIKEFLDYLEIDFEIVGDSCMVYGDTLDIMHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNL。

6. a compound comprising the non-natural amino acid salt YnDiPK of claim 1.

7. A protein expression system, translation system and cell comprising the non-natural amino acid salt YnDiPK of claim 1.

8. A protein expression system, a translation system and a cell comprising the non-natural amino acid salt YnDiPK according to claim 1 and an orthogonal pyrrollysyl tRNA synthetase according to any of claims 2-5 that recognizes and incorporates the non-natural amino acid salt YnDiPK into a protein of interest in a site-specific manner.

9. The use of the non-natural amino acid salt YnDiPK of claim 1 for site-directed insertion of a protein.

10. Use of the non-natural amino acid salt YnDiPK of claim 1 for analysis, characterization and enrichment of protein-protein interactions.