POP protein modified by non-natural amino acid, preparation method and application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a method for site-specific introduction of non-natural amino acid modification in specific amino acid residues of prolyl oligopeptidase (POP) based on genetic codon expansion technology and bio-orthogonal translation system. The method comprises using genetic codon expansion technology to introduce an early stop codon in the coding region of a POP protein gene, using a bio-orthogonal translation system to read through an amber stop codon UAG, and introducing the non-natural amino acid into the POP protein in the presence of the non-natural amino acid, which can not only conditionally control the expression of the POP protein, but also can use the non-natural amino acid containing a special group to specifically label the POP protein, and form a stable covalent bond between proteins interacting with the cell through light cross-linking, so as to provide a powerful means for studying the interaction between POP proteins in the cell; and can also be used for studying the function and post-translational modification of the POP protein.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a POP protein modified by site-specific modification with orthogonal unnatural amino acids (UAAs) based on the genetic code expansion (GCE) technology and the bio-orthogonal translation system to read through premature termination codons (PTC), as well as a preparation method and application thereof. Background Art

[0002] Prolyl oligopeptidase (POP), also known as prolyl endopeptidase (PREP) or proline-specific endoprotease, is a highly conserved member of the serine protease S9 family, which is widely distributed in different species. The common catalytic preference of this family is to cleave prolyl peptides less than 3 kDa at the C-terminus of the proline residue, and can specifically hydrolyze the peptide bonds of proline residues or a few hydrophobic amino acid residues in polypeptides. Its enzymatic function is mainly involved in the maturation and degradation of various peptide hormones and neuropeptides, such as thyrotropin-releasing hormone (TRH), angiotensin (Ang), α-melanocyte stimulating hormone (α-MSH), arginine vasopressin (AVP), luteinizing hormone-releasing hormone (LHRH), substance P (SP) and neurotensin (NT). The hydrolysis activity of POP is related to the phosphorylation state of the substrate. If the P10 residue of the serine of the POP substrate is phosphorylated, the reaction rate increases. Otherwise, POP cannot even cleave the peptide bond between Ala-Ser. POP not only exerts biological effects through enzymatic activity, but also has important non-enzymatic activity. Studies have shown that POP is closely related to the occurrence of neurological diseases such as Parkinson's disease, is highly expressed in many cancer tissues, and is more expressed in malignant tumors than in benign tumors.

[0003] The genetic codon expansion (GCE) technology developed in recent years is a technology that artificially expands the codon table of protein genetic coding and introduces new coding rules to modify or change proteins. This technology is achieved through engineered aminoacyl-tRNA synthetase (aa-tRNA synthetase, aaRS) / tRNA pairs. The modified aaRS can specifically aminoacylate the homologous tRNA with a specific UAA, and is dedicated to decoding the reallocated codon (usually the stop codon, and the matching aaRS / tRNA pair is called nonsense inhibitory aaRS / tRNA). The tRNA is then recognized by the cellular ribosomes. UAA, allowing UAA to correspond to designated codons during translation and to be site-specifically incorporated into synthetic peptides. All metazoan proteins are synthesized from 20 classical amino acids. Different combinations of these 20 amino acids give proteins a variety of structures and functions, but at the same time limit the development of protein properties. Although post-translational modification mechanisms have relieved this limitation to a certain extent, this relies on extremely robust and complex biological systems. The direct incorporation of UAAs into proteins using genetic codon expansion technology has greatly expanded the properties of proteins, allowing UAAs to be specifically inserted into any site of interest on the protein peptide chain through an orthogonal translation system in living cells. An amber stop codon (UAG) is introduced into the genome of the target protein, and an exogenous UAAs bioorthogonal translation system is used to encode a variety of unnatural amino acids and insert them into living organisms. This technology has successfully site-specifically expressed hundreds of UAAs (including affinity-tagged and photoisomerizable amino acids, carbonyl amino acids, and glycosylated amino acids) in proteins in living cells, giving these proteins novel physical, chemical, and physiological properties. This technology can selectively introduce special chemical groups such as carbonyl, alkynyl and azide groups into proteins to achieve site-specific modification of proteins and improve the properties of proteins. At the same time, this technology can also be applied to site-specific labeling, site-specific modification, and replication control of living organisms (such as viruses and bacteria), and has been applied to many fields, such as the treatment of genetic diseases, protein interactions and biomedicine.

[0004] PTC technology is a technology developed by introducing premature termination codons (PTC) into the coding region of the target protein, making it dependent on exogenous non-natural amino acids to control the expression of the protein. To truly apply GCE technology and PTC technology to the study of protein function, an urgent problem to be solved is how to select suitable and efficient amino acid mutation sites. Achieving efficient incorporation of exogenous UAAs and high-level expression of target proteins will not only help to study the biological functions of proteins, but will also greatly promote their application in protein interactions and post-translational modifications. The present invention first selects 8 amino acid residues from the amino acid sequence of POP protein for codon point mutation based on the 3D structural analysis of the target protein and software prediction analysis, and screens amino acid sites with higher UAA incorporation efficiency. This mutation method is a non-natural amino acid introduced at a specific site in the POP protein, and its unique azide group can specifically react with a fluorescent marker specific group to click, thereby marking the location of the POP protein in living cells. The method for preparing POP protein modified by non-natural amino acids has the following advantages: 1) the efficient insertion sites of non-natural amino acids are screened in the coding region of POP protein to realize conditional control of POP protein expression in cells; 2) the method of site-specific modification of proteins by non-natural amino acids realizes intracellular fluorescent labeling of POP protein; 3) the site-specific modification of non-natural amino acids by photocrosslinking can be used to identify protein complexes that interact with POP protein, thereby screening key interacting proteins; 4) based on the above-mentioned efficient amino acid mutation sites, it can be used to prepare POP protein modified after reaction, which is helpful for protein function research and has broad application prospects. Summary of the invention

[0005] The present invention finds that by mutating the codon of the amino acid residue at at least one of the P7, W30, F42, F53, L94, L127 and L164 positions of the POP protein having an amino acid sequence as shown in SEQ ID NO.1 to an amber codon TAG, and utilizing a bioorthogonal translation system, it is possible to site-specifically incorporate non-natural amino acids into the POP protein, thereby achieving conditional control of the expression of the POP protein and intracellular fluorescent labeling, and also to capture unknown proteins interacting with the POP protein through a photocrosslinking reaction in living cells.

[0006] The specific content of the invention is:

[0007] In the first aspect, the present invention provides a POP protein modified with a non-natural amino acid, wherein the amino acid at at least one of positions P7, W30, F42, F53, L94, L127, and L164 of the POP protein shown in SEQ ID NO.1 is mutated into a non-natural amino acid.

[0008] Preferably, the unnatural amino acid is selected from NAEK, DiZPK, DiZSeK, Bpa.

[0009] Preferably, the unnatural amino acid is NAEK or DiZPK.

[0010] Second aspect: The present invention provides a nucleic acid molecule encoding a POP protein modified with a non-natural amino acid as described in the first aspect above.

[0011] Preferably, the nucleic acid molecule differs from the nucleic acid molecule encoding the POP protein shown in SEQ ID NO.1 in that the codon encoding at least one of the amino acids at positions P7, W30, F42, F53, L94, L127 and L164 of SEQ ID NO.1 is mutated to an amber codon TAG.

[0012] In a third aspect, the present invention provides a method for preparing a POP protein modified with a non-unnatural amino acid as described in the first aspect, the method comprising:

[0013] (1) mutating the codon encoding at least one amino acid at position P7, W30, F42, F53, L94, L127 and L164 of the sequence shown in SEQ ID NO.1 to an amber codon TAG to obtain a gene sequence encoding a POP protein with site-directed modification of a non-natural amino acid;

[0014] (2) operably linking the gene sequence described in step (1) to a vector to obtain a mutant sequence expression plasmid;

[0015] (3) The mutant sequence expression plasmid and the orthogonal translation system expression plasmid are co-transfected into host cells, and the non-natural amino acid-induced culture is used to screen and obtain the POP protein modified with the non-natural amino acid site.

[0016] Preferably, the unnatural amino acid is selected from NAEK, DiZPK, DiZSeK, Bpa.

[0017] Preferably, the unnatural amino acid is NAEK or DiZPK.

[0018] Preferably, the orthogonal translation system expression plasmid comprises:

[0019] Orthogonal tRNA / aminoacyl-tRNA synthetases;

[0020] The tRNA is derived from a prokaryotic organism;

[0021] The anticodon loop of the tRNA corresponds to the premature stop codon UAG;

[0022] The aminoacyl-tRNA synthetase is capable of binding an unnatural amino acid to the tRNA;

[0023] The tRNA and aminoacyl-tRNA synthetase are located on the same carrier.

[0024] In a fourth aspect, the present invention provides the use of the POP protein site-specifically modified with a non-natural amino acid as described in the first aspect for conditionally controlling the expression of the POP protein in vitro.

[0025] In a fifth aspect, the present invention provides the use of the POP protein modified with a non-natural amino acid site-specifically as described in the first aspect in intracellular fluorescence labeling analysis of POP protein labeled with a non-natural amino acid.

[0026] In a sixth aspect, the present invention provides the application of the POP protein modified with the non-natural amino acid site-specifically described in the first aspect in the study of the function and post-translational modification of the POP protein.

[0027] In a seventh aspect, the present invention provides the use of the POP protein modified with the non-natural amino acid site-specifically as described in the first aspect above in screening interacting proteins that interact with the POP protein.

[0028] Preferably, the POP protein modified with non-natural amino acids is as follows: the amino acid at at least one of positions P7, W30 and F53 of the POP protein shown in SEQ ID NO.1 is mutated to a non-natural amino acid.

[0029] The beneficial effects of the present invention are:

[0030] (1) The present invention found that efficient insertion sites of non-natural amino acids were screened in the coding region of the POP protein whose amino acid sequence is shown in SEQ ID NO. 1, thereby achieving site-specific modification of non-natural amino acids in the POP protein and conditional control of the in vitro expression of the POP protein;

[0031] (2) The method of site-specific modification of proteins with non-natural amino acids achieves intracellular fluorescent labeling of POP proteins;

[0032] (3) The method for site-specific modification of proteins with non-natural amino acids can be used to screen and identify protein complexes that interact with POP proteins by utilizing photo-crosslinking of non-natural amino acids for site-specific modification;

[0033] (4) The highly efficient amino acid mutation sites described above can be used to prepare post-translationally modified POP proteins, which is helpful for protein function research. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1The 3D structure of the POP protein, where the red marks are amino acid residues at positions P7, W30, F42, F53, L94, L127, W150, and L164;

[0035] Figure 2 Results of conservation analysis of the amino acid sequence of POP protein;

[0036] Figure 3 Results of screening for efficient insertion sites of unnatural amino acids into POP proteins;

[0037] Figure 4 Intracellular fluorescent labeling of POP proteins modified with unnatural amino acids;

[0038] Figure 5 Photo-crosslinking of POP proteins modified with unnatural amino acids to identify interacting protein complexes;

[0039] Figure 6 Schematic diagram of protein interaction studies based on POP proteins modified with non-natural amino acids. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the present invention clearer, the embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. However, it will be appreciated by those skilled in the art that in the embodiments of the present invention, many technical details are provided to enable the reader to better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical scheme claimed in the present application can be implemented.

[0041] Description and explanation of relevant terms in the present invention:

[0042] The term "bioorthogonal translation system" refers to an engineered orthogonal aminoacyl-tRNA synthetase (aa-tRNAsynthetase, aaRS) / tRNA pair, in which the aaRS can specifically aminoacylate the homologous tRNA with a specific UAA, which is dedicated to decoding a specific codon (usually the stop codon UAG), and then the tRNA is recognized by the cellular ribosome. CUA , allowing site-specific incorporation of specific codons corresponding to UAA into synthetic peptides during translation.

[0043] The term "vector" refers to a nucleic acid carrier into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can transform, transduce or transfect the host cell so that the genetic material elements it carries can be expressed in the host cell. For example, vectors include: plasmids; bacteriophages; cosmids, etc.

[0044] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.

[0045] Biological materials: PK-15 cells and HEK-293 cells were purchased from ATCC. The plasmid expressing tRNA synthetase / tRNA orthogonal pair (addgene: #91706) and pCMV-HA-POP expression plasmid were constructed and stored by our laboratory. Escherichia coli competent cells DH5α were purchased from Beijing Quanshijin Biotechnology Co., Ltd. Gene synthesis was completed by Beijing Liuhe BGI Gene Technology Co., Ltd. Primer synthesis and DNA sequencing were completed by Yangling Tianrun Aoke Biotechnology Co., Ltd.

[0046] Main reagents: Puromycin, neomycin (G418), and low melting point agarose were purchased from Ameresco; unnatural amino acid NAEK (CAS: 1167421-25-1) was purchased from Shanghai Zhezhi Biological Company; DiZPK (CAS No.: 1337883-32-5) was purchased from MCE Company; DIBO-Alexa 488, Trizol reagent was purchased from Invitrogen; Viral RNA Kit extraction kit and plasmid mini-extraction kit were purchased from OMEGA; plasmid midi extraction kit was purchased from MN (Macherey-nagel) in Germany; agarose gel recovery kit was purchased from Promega; Q5 ultra-fidelity DNA polymerase, endonuclease, and HiFi DNA Assembly Master Mix was purchased from NEB; Onestep PrimeScript RT-PCR Kit (Perfect Real Time), DNA ladder, and T4 DNA ligase were purchased from TaKaRa; DMEM, MEM cell culture medium, 0.25% trypsin, and fetal bovine serum (FBS) were purchased from Gbico; and JetPRIME cell transfection reagent was purchased from Polyplus-transfection, France.

[0047] The unnatural amino acid (UAA) described in the following examples is selected from NAEK or DiZPK.

[0048] Example 1 Selection of POP protein mutation site and construction of mutant expression plasmid

[0049] 1.1 Selection of POP protein mutation sites:

[0050] According to the 3D structure analysis, amino acid conservation analysis and mutation site selection principle of POP protein, the amino acid residues close to the N-terminus of the amino acid sequence are preferentially selected. A suitable amino acid site is selected in the amino acid sequence encoded by the POP protein, and the amino acid codon of the mutation site is mutated to a UAG codon to construct a POP protein mutant expression plasmid.

[0051] 1.2 Construction of mutant expression plasmid:

[0052] According to standard molecular cloning operation techniques, TAG-POP mutant plasmids were constructed, and the codons of amino acids at positions P7, W30, F42, F53, L94, L127, W150 and L164 in the POP coding gene sequence were point mutated to TAG. The TAG-POP mutants were named: P7TAG, W30TAG, F42TAG, F53TAG, L94TAG, L127TAG, W150TAG and L164TAG, respectively.

[0053] Primers for constructing each mutant were designed separately (as shown in Table 1), and the corresponding amino acid sites were subjected to site-directed mutagenesis using the pCMV-HA-POP expression plasmid as a template, and the codons of the selected amino acid sites were mutated to TAG. The target plasmid was amplified by one-step point mutation PCR, and the TAG codon was introduced into the mutation site by PCR. The operation was performed according to the instructions of the kit. The PCR reaction system and reaction procedure are shown in Table 2-3. The specific operation steps are as follows:

[0054] 1) Prepare the PCR reaction system according to the table below and perform PCR amplification of the target fragment.

[0055] Table 1 Primer sequence list for constructing TAG-POP mutants

[0056]

[0057] Table 2 PCR reaction system

[0058]

[0059] Table 3 PCR reaction program

[0060]

[0061] 2) After the PCR reaction is completed, add 1 μL of Dpn I enzyme, 2 μL of 10× Buffer, and H 2 O 17μL, total volume 20μL, 37℃, digest for 30min.

[0062] 3) Enzyme digestion of the vector plasmid (select appropriate endonuclease sites for double enzyme digestion based on the mutation site).

[0063] 4) 1% agarose gel electrophoresis, gel excision, recovery and purification of DNA fragments.

[0064] 5) Homologous recombination connection: according to HiFi DNA Assembly Master Mix Kit Instructions. Reaction system: 10ul purified DNA + 10ul HiFi DNA Assembly Master; reaction conditions: 50℃, 15-30min.

[0065] 6) The ligation product was transformed into E. coli competent cells Trans5α and cultured overnight in a 37°C incubator for 14 to 16 hours.

[0066] 7) Pick a single clone from the LB plate, shake the bacteria, and send the plasmid to the company for sequencing.

[0067] 8) After the correctly sequenced plasmid is transformed, the bacteria are inoculated into LB medium and the plasmid is extracted (endotoxin-free) after shaking, and then stored at -20°C.

[0068] In order to screen for efficient insertion sites of non-natural amino acids and design expression plasmids for POP protein mutants, we analyzed the 3D structure and amino acid sequence conservation of POP protein. Combining the selection principle of mutation sites, we selected 8 amino acid sites and constructed mutant expression plasmids respectively. The 3D structure of POP protein is shown in Figure 1 , where the red marks are the amino acid residues at positions P7, W30, F42, F53, L94, L127, W150 and L164; the results of the amino acid sequence conservation analysis of the POP protein are shown in Figure 2 .

[0069] Example 2 Selection of sites for site-specific modification of POP protein with non-natural amino acids

[0070] 2.1 Transfection of cells with POP mutant expression plasmid:

[0071] 1) Prepare HEK293 cells at 5×10 5 The cells were plated at a density of 1.54 cells / mL in 6-well cell culture plates.

[0072] 2) The cells were cultured at 37°C and 5% CO 2 The cells were cultured under the same conditions and the transfection complex was prepared when the cell confluence reached 70-80%.

[0073] 3) Follow the instructions of the JetPRIME transfection kit, which is briefly described as follows: add 200 μL of JetPRIME Buffer to a 1.5 mL EP tube, then add 2 μg of TAG-POP mutant plasmid and plasmid expressing tRNA synthetase / tRNA orthogonal pair, mix well, add 4 μL of transfection reagent, and mix well again.

[0074] 4) Let stand at room temperature for 10 min and then add dropwise to the cell wells.

[0075] 5) 6 h after transfection, the medium was replaced with fresh DMEM complete medium containing 10% FBS and supplemented with 1 mM UAA (+UAA). The control group did not add UAA (–UAA).

[0076] 6) 24 h after transfection, cells were harvested and protein expression was detected by Western blotting.

[0077] 2.2 Western blotting:

[0078] 1) 24 hours after transfection, discard the cell culture medium, wash the cells once with PBS, centrifuge at 12000×g for 10 min, and discard the supernatant.

[0079] 2) Lyse cells: add protein lysis buffer, incubate on ice for 30 minutes, and collect the protein lysis buffer.

[0080] 3) Take the supernatant after centrifugation, add 5× Loading Buffer, mix well, and cook at 100℃ for 10 minutes.

[0081] 4) The protein samples were separated by SDS-PAGE electrophoresis, and then the proteins were electro-transferred to the NC membrane.

[0082] 5) Place the transferred NC membrane in blocking solution and block at room temperature for 1 to 2 hours.

[0083] 6) Dilute the primary antibody to an appropriate concentration with TBST, transfer the blocked NC membrane into the primary antibody solution, and incubate overnight at 4°C.

[0084] 7) Wash the membrane three times with TBST at room temperature, 10 min each time.

[0085] 8) Dilute the secondary antibody to an appropriate concentration with TBST, transfer the NC membrane into the secondary antibody solution, and incubate at room temperature for 1 h.

[0086] 9) Wash the membrane three times with TBST at room temperature, 10 min each time.

[0087] 10) Take out the NC membrane, add ECL luminescent solution, mix well, and stand at room temperature away from light for 1 to 2 minutes. Use the BIO-RAD protein imaging system to scan the protein bands and save them.

[0088] The results showed that the constructed 8 POP mutant expression plasmids were transfected into cells, and the unnatural amino acid group (+UAA) and the unnatural amino acid group (-UAA) were set up respectively. The Western blotting results showed that each POP mutant +UAA group could express normally, but could not express normally in –UAA group ( Figure 3 ), indicating that non-natural amino acids can be inserted at each site, and the insertion efficiency of each site is different. There are differences in the protein expression levels at the selected 8 mutation sites, especially at P7, W30, F42, F53, L94, L127 and L164 mutation sites. The expression levels of POP protein are similar, while the protein expression level after mutation at W150 site is the lowest ( Figure 3 ). The results indicate that the protein expression can be conditionally controlled by adding or not adding UAA; on the other hand, the sites P7, W30, F42, F53, L94, L127 and L164 described in the present application are efficient insertion sites for unnatural amino acids and do not affect the expression level of POP protein.

[0089] Example 3 Intracellular Fluorescence Labeling of POP Protein Expression by Site-directed Modification of Unnatural Amino Acids

[0090] The POP mutant expression plasmid (taking L94TAG as an example) with the efficient insertion site selected in the results of Example 2 above was co-transfected with the plasmid expressing the tRNA synthetase / tRNA orthogonal pair. 48 hours after cell transfection, the cells were washed twice with 1% FBSDPBS. A certain concentration of DIBO-Alexa fluor-488 was dissolved in 1% FBSDPBS and stained at room temperature in the dark for 1 hour. Washed 3 times with 1% FBSDPBS, then stained the cell nucleus with DIPA, and then washed 3 times with 1% FBSDPBS, observed with a fluorescence microscope and photographed.

[0091] The results showed that after the POP mutant expression plasmid L94TAG was transfected into cells, adding DIBO-Alexafluor-488 to living cells could specifically label the expression of POP protein in cells ( Figure 4 ). This indicates that this technology can successfully achieve fluorescent labeling of proteins in living cells.

[0092] In this example, L94TAG is used as an example to perform intracellular fluorescent labeling of POP protein expression modified with non-natural amino acids. However, based on Example 2, other mutation sites described in this application, such as P7, W30, F42, F53, L127 and L164, can achieve the same intracellular fluorescent labeling effect.

[0093] Example 4 Identification of protein complexes interacting with POP proteins modified by non-natural amino acids

[0094] As in Example 2 above, the POP mutant expression plasmid and the plasmid expressing the tRNA synthetase / tRNA orthogonal pair were co-transfected into HEK293 cells. 24 hours after cell transfection, the culture medium was removed and the cells were rinsed once with PBS. For the cells in the illumination group, they were placed on ice and irradiated with UV365nm. After 10 minutes of 365nm ultraviolet light irradiation, the photo-crosslinking reaction of DiZPK was fully completed, and the covalently cross-linked POP-protein complex band was obtained by Western blotting experiment identification, and the protein complex was further affinity purified, and LC-MS / MS analysis was performed to identify the unknown interacting proteins.

[0095] The results showed that after expressing DiZPK-tagged POP protein in cells for a period of time, ultraviolet UV365nm irradiation was used to induce the photo-crosslinking reaction of DiZPK, forming a covalent bond between the DiZPK-tagged POP protein and its interacting protein. The formed photo-crosslinked complex was detected by protein immunoblotting ( Figure 5 ). It was also found that the molecular weight of the photo-crosslinked complex was 130kDa-170kDa, among which the POP protein with mutation sites at W30, F53 and P7 captured the most obvious photo-crosslinked complex, while the POP protein with mutation sites at L94, L127 and L146 did not observe photo-crosslinked complexes, indicating that the mutation sites described in the present application at W30, F53 and P7 can be used to capture protein complexes after the introduction of non-natural amino acids, and can be used for the identification of protein complexes that interact with POP protein. Among them, the model diagram of protein interaction research based on POP protein modified by non-natural amino acids is shown in Figure 6 shown.

[0096] In summary, the present application found that by screening out efficient insertion sites of non-natural amino acids in the coding region of the POP protein with an amino acid sequence as shown in SEQ ID NO.1, the non-natural amino acid site-specific modification of the POP protein and the conditional control of the in vitro expression of the POP protein were achieved, which can be used to prepare post-translationally modified POP proteins and is helpful for protein function research; the method for site-specific modification of proteins with non-natural amino acids achieves intracellular fluorescent labeling of POP proteins; and the site-specific modification of non-natural amino acids by photocrosslinking can be used to screen and identify protein complexes that interact with POP proteins.

Claims

1. A POP protein modified by non-natural amino acids, characterized in that: The codon encoding the amino acid at any one of positions P7, W30, F42, F53, L94, L127 and L164 of the POP protein shown in SEQ ID NO.1 is mutated to an amber codon TAG.

2. A nucleic acid molecule encoding the POP protein modified with the non-natural amino acid according to claim 1.

3. The method for preparing a POP protein modified by a non-natural amino acid site-specific modification as claimed in claim 1, characterized in that: The method comprises: (1) Mutating the codon encoding any one of the amino acids at positions P7, W30, F42, F53, L94, L127 and L164 of the sequence shown in SEQ ID NO.1 to an amber codon TAG to obtain a gene sequence encoding a POP protein with site-directed modification of a non-natural amino acid; (2) operably linking the gene sequence described in step (1) to a vector to obtain a mutant sequence expression plasmid; (3) The mutant sequence expression plasmid and the orthogonal translation system expression plasmid are co-transfected into the host cells, and the non-natural amino acid-induced culture is used to screen and obtain the POP protein with site-specific modification of the non-natural amino acid.

4. The method according to claim 3, characterized in that The orthogonal translation system expression plasmid comprises: Orthogonal tRNA / aminoacyl-tRNA synthetases; The tRNA is derived from a prokaryotic organism; The anticodon loop of the tRNA corresponds to the premature stop codon UAG; The aminoacyl-tRNA synthetase is capable of binding an unnatural amino acid to the tRNA; The tRNA and aminoacyl-tRNA synthetase are located on the same carrier.

5. Use of the non-natural amino acid site-specifically modified POP protein as claimed in claim 1 for conditional control of POP protein expression in vitro, or use of non-natural amino acid labeled POP protein in intracellular fluorescent labeling analysis; the non-natural amino acid site-specifically modified POP protein is: the codon encoding the amino acid at position L94 of the POP protein shown in SEQ ID NO.1 is mutated to an amber codon TAG.

6. Use of the POP protein modified with non-natural amino acids as claimed in claim 1 in screening interacting proteins that interact with the POP protein; the POP protein modified with non-natural amino acids is: the codon encoding the amino acid at any one of the positions P7, W30, and F53 of the POP protein shown in SEQ ID NO.1 is mutated to the amber codon TAG.

Images