Method for predicting proportion change of monomer and dimer after PSI protein unit point mutation and PSI protein mutant

Abstract

The invention provides a method for predicting the proportion change of a monomer and a dimer after PSI protein unit point mutation and a PSI protein mutant, and belongs to the technical field of computational biology and structural biology. Three mutant proteins, namely G14A-PSI, W18A-PSI and V22R-PSI, of which the stability of monomers and dimers is remarkably changed are screened out by calculating the change values of Gibbs free energy of monomer conformation and dimer conformation of wild type PSI protein after single-point mutation, and a liquid nuclear magnetic resonance experiment is carried out to obtain mutant proteins of which the stability of the monomers and the dimers is remarkably changed under the conditions that the pH value is 3.0, the temperature is 30 DEG C, the temperature is 30 DEG C, and the temperature is 30 DEG C; according to the two-dimensional 1H-15N HSQC spectrogram under the conditions of pH 5.0 and pH 7.4, results show that compared with wild type PSI, the G14A-PSI mutant protein has the advantage that the dimer proportion is obviously increased.

Description

Technical Field

[0001] The present invention belongs to the technical fields of computational biology and structural biology, and in particular relates to a method for predicting the change in the ratio of monomers and dimers after a single-site mutation of a PSI protein and a mutant of the PSI protein. Background Art

[0002] In plants, Plant Specific Insert (PSI) is a unique insertion sequence of aspartic protease (AP) with antibacterial activity. PSI often exists in plant cells in the form of AP zymogen. After infection by exogenous pathogens, the zymogen is transported to the vacuole and processed into mature AP hydrolase in the acidic environment of the vacuole. In this process, PSI separates from APs, interacts with the biological membrane of the vacuole, and induces membrane fusion between the vacuole membrane and the cell membrane. PSI exists in the form of a monomer-dimer equilibrium in solution. The ratio of monomers and dimers will change after a single point mutation of the PSI protein. How to use computational biology to predict the change in the ratio of monomers and dimers after a single point mutation of the PSI protein to guide protein modification is a problem that needs to be solved at present. Summary of the invention

[0003] The purpose of the present invention is to provide a method for predicting the change in the ratio of monomers and dimers after a single-site mutation of a PSI protein and a mutant of a PSI protein. The method of the present invention can accurately predict the change in the ratio of monomers and dimers after a single-site mutation of a PSI protein.

[0004] The present invention provides a method for predicting the change in the ratio of monomers and dimers after a single-site mutation of a PSI protein, comprising the following steps: collecting the Gibbs free energy change value of the monomer of the mutant after a single-site mutation of the PSI protein, calculated as ΔΔG 单 ; Collect the Gibbs free energy change value of the dimer of the mutant after the single-site mutation of PSI protein, calculated as ΔΔG 二 ; Calculate the relative stability according to the formula shown in Formula 1; When the relative stability is greater than 0, it is judged that the dimer ratio decreases and the monomer ratio increases after the PSI protein single point mutation; When the relative stability is less than 0, it indicates that the dimer ratio increases and the monomer ratio decreases after the PSI protein single point mutation; Relative stability = ΔΔG 单 -ΔΔG 二 Formula 1; the amino acid sequence of the PSI protein is shown in SEQ ID NO.1.

[0005] Preferably, the site of the single point mutation of the PSI protein is position 14, position 18 or position 22.

[0006] Preferably, the single-site mutation of the PSI protein includes: mutation of glycine at position 14 to alanine, mutation of tryptophan at position 18 to alanine, or mutation of valine at position 22 to arginine.

[0007] The present invention also provides a PSI protein mutant, including G14A-PSI, W18A-PSI or V22R-PSI; the amino acid sequences of the G14A-PSI, W18A-PSI and V22R-PSI are shown in SEQ ID NO.2 to SEQ ID NO.4, respectively.

[0008] The present invention also provides a gene encoding the PSI protein mutant described in the above scheme, the nucleotide sequence of the gene encoding G14A-PSI is shown as SEQ ID NO.5; the nucleotide sequence of the gene encoding W18A-PSI is shown as SEQ ID NO.6; the nucleotide sequence of the gene encoding V22R-PSI is shown as SEQ ID NO.7.

[0009] The present invention also provides a recombinant plasmid into which the coding gene described in the above scheme is inserted.

[0010] The present invention also provides a recombinant bacterium comprising the recombinant plasmid described in the above scheme.

[0011] The present invention also provides the use of the PSI protein mutant, the encoding gene, the recombinant plasmid or the recombinant bacteria described in the above scheme in the preparation of tumor targeting drugs and / or tumor diagnostic reagent carriers.

[0012] The present invention also provides a tumor targeting drug and / or tumor diagnostic reagent carrier, comprising the PSI protein mutant, the encoding gene, the recombinant plasmid or the recombinant bacteria described in the above scheme.

[0013] The present invention also provides the application of W18A-PSI and V22R-PSI in the study of monomer conformation in protein structure; the amino acid sequences of W18A-PSI and V22R-PSI are shown in SEQ ID NO.3 to SEQ ID NO.4 respectively.

[0014] The present invention provides a method for predicting the change in the ratio of monomers and dimers after a single-site mutation of a PSI protein. The method of the present invention collects the Gibbs free energy change value ΔΔG of the monomer after the single-site mutation. 单 and the Gibbs free energy change of the dimer ΔΔG 二 , compare the difference between the two, that is, relative stability (relative stability = ΔΔG 单 -ΔΔG 二 ). When the relative stability is greater than 0, it indicates that the stability of the monomer increases and the stability of the dimer decreases.1 H- 15 The N HSQC spectrum shows that the monomer ratio increases and the dimer ratio decreases; when the relative stability is <0, it indicates that the monomer stability decreases and the dimer stability increases. 1 H- 15 The NHSQC spectrum shows that the monomer ratio is reduced and the dimer ratio is increased. The prediction results of the method of the present invention are consistent with the liquid nuclear magnetic resonance detection results, which can prove the feasibility of the method of the present invention to accurately predict the change in the ratio of monomers and dimers after single-site mutation of PSI protein.

[0015] The present invention also provides mutants of PSI protein including G14A-PSI, W18A-PSI or V22R-PSI, wherein G14A-PSI is obtained by mutating the glycine at the 14th position of the PSI protein to alanine, and its monomer stability is reduced and the dimer stability is improved compared with the wild-type PSI; W18A-PSI is obtained by mutating the tryptophan at the 18th position of the PSI protein to alanine; V22R-PSI is obtained by mutating the valine at the 22nd position of the PSI protein to arginine; compared with the wild-type PSI, the monomer stability of W18A-PSI and V22R-PSI is improved, and the dimer stability is reduced. The present invention combines computational biology with structural biology, predicts and verifies the ability of key amino acid single-point mutations to regulate protein conformational equilibrium, and lays a solid foundation for further transformation of functional PSI proteins in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0017] Figure 1 Schematic diagram of the pET32a(+)-G14A-PSI plasmid map;

[0018] Figure 2 Schematic diagram of the pET32a(+)-W18A-PSI plasmid map;

[0019] Figure 3 is a schematic diagram of the pET32a(+)-V22R-PSI plasmid map;

[0020] Figure 4 This is the gel image of the dried sample of the target protein detected by SDS-PAGE (with 15 N-G14A-PSI as an example);

[0021] Figure 5 for 1 H- 15 N HSQC characterization of the G40 region of wild-type PSI and its mutants;

[0022] Figure 6 for 1 H- 15 N HSQC characterization HSQC 2D spectra of wild-type PSI and its mutants;

[0023] Figure 7 The monomer-dimer ratio of WT-PSI and its mutants under different pH conditions; white represents pH = 3.0, light gray represents pH = 5.0, and dark gray represents pH = 7.4. Twill grids represent monomers, and blank grids represent dimers. DETAILED DESCRIPTION

[0024] The present invention provides a method for predicting the change in the ratio of monomers and dimers after a single-site mutation of a PSI protein, comprising the following steps: collecting the Gibbs free energy change value of the monomer of the mutant after a single-site mutation of the PSI protein, calculated as ΔΔG 单 ; Collect the Gibbs free energy change value of the dimer of the mutant after the single-site mutation of PSI protein, calculated as ΔΔG 二 ; Calculate the relative stability according to the formula shown in Formula 1; When the relative stability is greater than 0, it is judged that the dimer ratio decreases and the monomer ratio increases after the PSI protein single point mutation; When the relative stability is less than 0, it indicates that the dimer ratio increases and the monomer ratio decreases after the PSI protein single point mutation; Relative stability = ΔΔG 单 -ΔΔG 二 Formula 1;

[0025] The amino acid sequence of the PSI protein is shown in SEQ ID NO.1.

[0026] The method of the present invention is a method for predicting the change in the monomer-dimer content ratio of a single amino acid mutation of a PSI protein based on the collection of Gibbs free energy change values ​​(ΔΔG) reflecting the thermodynamic stability of a protein. The method of the present invention can predict a mutant of a membrane fusion-inducing functional protein PSI that is mainly a highly active dimer.

[0027] In the specific implementation of the present invention, the preferred method for collecting the Gibbs free energy is: through the protein Gibbs free energy calculation website https: / / biosig.lab.uq.edu.au / ddmut / .

[0028] The single-site mutation or alanine scanning mutation function in the above website was used to calculate the Gibbs free energy change (ΔΔG) of the monomer conformation of the target protein PSI after mutation.单 , ΔΔG WT→Mut(monomer) ); the multi-site mutation function of the above website was used to calculate the Gibbs free energy change value (ΔΔG 二 , ΔΔG WT→Mut(dimer) ). Then, the Gibbs free energy change value of the monomer conformation after mutation and the Gibbs free energy change value of the dimer conformation after mutation under the same mutation are subtracted to obtain a relative value, which can reflect the relative stability of the monomer / dimer after the same mutation. The calculation formula is shown in Formula 2: Relative stability = ΔΔG WT→Mut(monomer) -ΔΔG WT→Mut(dimer) Formula 2. Among them, alanine scanning mutation belongs to single-site mutation, which means that one amino acid is mutated each time, and each amino acid site is mutated to alanine in turn to see the change in Gibbs free energy after mutation. It is used to screen which sites are important in large quantities. Single-site mutation is used for monomers, mutating one amino acid at a time. Multi-site mutation is used for dimers, mutating the same amino acid sites on both chains in the dimer at the same time.

[0029] In the present invention, the amino acid sequence shown in SEQ ID NO.1 is specifically: IVSMECKTIVSQY 14 G EMI 18 W DLL 22 V SGVRPDQVCSQAGLCFVDGAQHVSSNIKTVVERETEGSSVGEAPLCTACEEMAVVWMQNQLKQEGTKEKVLEYVNQLCEKIP.

[0030] In the present invention, the site of the PSI protein single point mutation is preferably position 14, position 18 or position 22. In the present invention, the PSI protein single point mutation preferably includes: glycine at position 14 mutates to alanine, tryptophan at position 18 mutates to alanine or valine at position 22 mutates to arginine.

[0031] The present invention also provides a PSI protein mutant, including G14A-PSI, W18A-PSI or V22R-PSI; the amino acid sequences of the G14A-PSI, W18A-PSI and V22R-PSI are shown in SEQ ID NO.2 to SEQ ID NO.4, respectively.

[0032] In the present invention, the amino acid sequence of G14A-PSI is shown in SEQ ID NO.2; in the present invention, the amino acid sequence shown in SEQ ID NO.2 is specifically:

[0033] IVSMECKTIVSQYAEMIWDLLVSGVRPDQVCSQAGLCFVDGAQHVSSNIKTVVERETEGSSVGEAPLCTACEEMAVVWMQNQLKQEGTKEKVLEYVNQLCEKIP.

[0034] Compared with wild-type PSI, the dimer ratio of G14A-PSI is significantly increased; specifically, under pH 3.0, pH 5.0 and pH 7.4, the dimer ratio of G14A-PSI is higher than that of wild-type PSI.

[0035] In the present invention, the amino acid sequence of W18A-PSI is shown in SEQ ID NO.3; in the present invention, the amino acid sequence shown in SEQ ID NO.3 is specifically: IVSMECKTIVSQYGEMIADLLVSGVRPDQVCSQAGLCFVDGAQHVSSNIKTVVERETEGSSVGEAPLCTACEMAVVWMQNQLKQEGTKEKVLEYVNQLCEKIP

[0036] In the present invention, the amino acid sequence of V22R-PSI is shown in SEQ ID NO.4; in the present invention, the amino acid sequence shown in SEQ ID NO.4 is specifically: IVSMECKTIVSQYGEMIWDLLRSGVRPDQVCSQAGLCFVDGAQHVSSNIKTVVERETEGSSVGEAPLCTACEMAVVWMQNQLKQEGTKEKVLEYVNQLCEKIP.

[0037] The present invention also provides a gene encoding the PSI protein mutant described in the above scheme, wherein the nucleotide sequence of the G14A-PSI encoding gene G14A-PSI is shown in SEQ ID NO.5; the nucleotide sequence of the W18A-PSI encoding gene W18A-PSI is shown in SEQ ID NO.6; and the nucleotide sequence of the V22R-PSI encoding gene V22R-PSI is shown in SEQ ID NO.7.

[0038] In the present invention, the nucleotide sequence shown in SEQ ID NO.5 is specifically: ATAGTATCAATGGAATGTAAAACGATTGTCTCGCAATATGCCGAAATGATTTGGGACCTGTTGGTTTCTGGTGTTCGTCCGGATCAGGTATGTAGCCAAGCGGGTCTGTGCTTCGTGGACGGCGCGCAGCACGTGAGCTCCAACATCAAAACCGTTGTCGAGCGCGAGACTGAAGGCAGCAGCGTTGGTGAAGCACCGCTGTGCACCGCGTGTGAGATGGCTGTGGTGTGGATGCAAAACCAGTTGAAGCAGGAGGGCACCAAAGAAAAGGTGCTGGAATACGTTAATCAGTTATGCGAGAAGATCCCG。

[0039] In the present invention, the nucleotide sequence shown in SEQ ID NO.6 is specifically: ATCGTTTCCATGGAATGCAAGACTATTGTTTCTCAGTATGGTGAGATGATCGCAGACCTGCTGGTGTCTGGTGTGCGTCCGGACCAGGTGTGTTCTCAGGCAGGTCTGTGCTTCGTGGACGGCGCGCAGCACGTATCCTCCAACATCAAAACCGTTGTTGAGCGCGAGACTGAAGGTTCCAGCGTTGGCGAGGCCCCGCTGTGCACCGCATGCGAGATGGCCGTGGTTTGGATGCAGAACCAGCTGAAACAGGAGGGTACTAAGGAGAAAGTCCTGGAGTACGTTAACCAGCTGTGCGAAAAAATTCCG。

[0040] In the present invention, SEQ ID The nucleotide sequence shown in NO.7 is specifically: ATAGTATCAATGGAATGTAAAACGATTGTTTCCCAATATGGCGAGATGATTTGGGATTTGCTGCGTAGCGGTGTTCGTCCGGACCAGGTTTGTAGCCAAGCCGGTTTATGCTTCGTGGACGGCGCGCAGCACGTGAGCAGCAACATC AAAACCGTCGTGGAACGCGAAACCGAAGGCAGCTCTGTTGGTGAGGCTCCGCTGTGCACCGCGTGTGAGATGGCAGTTGTGTGGATGCAGAATCAACTGAAACAGGAGGGTACTAAGGAAAAGGTGCTGGAATACGTGAACCAGTTGTGCGAGAAGATCCCG.

[0041] In the present invention, the encoding gene is preferably produced by Nanjing GenScript Biotechnology Co., Ltd.

[0042] The present invention also provides a recombinant plasmid into which the coding gene described in the above scheme is inserted.

[0043] In the present invention, the backbone plasmid of the recombinant plasmid is preferably a pET32a(+) vector; the insertion site of the coding gene on the pET32a(+) vector is preferably after the Thrombin restriction site. In the present invention, the TrxA solubilizing tag and the 6×His tag are preferably inserted in sequence after the T7 promoter of the pET32a(+) vector; the TrxA solubilizing tag is used to improve the solubility and stability of the protein; the 6×His tag is used for subsequent purification; a Thrombin restriction site is added after the 6×His tag to remove the solubilizing tag and the His tag. In the present invention, the recombinant plasmid is preferably completed by Nanjing GenScript Biotechnology Co., Ltd.

[0044] The present invention also provides a recombinant bacterium comprising the recombinant plasmid described in the above scheme.

[0045] In the present invention, the original bacteria of the recombinant bacteria are preferably Escherichia coli; the Escherichia coli are preferably competent cells of Escherichia coli Rosetta-gami B (DE3), pLysS. The present invention has no particular restrictions on the construction method of the recombinant bacteria, and conventional methods in the art can be used. In the present invention, the recombinant plasmid can be stably expressed in the recombinant strain.

[0046] The present invention also provides a method for preparing a PSI protein mutant, comprising the following steps:

[0047] The recombinant bacteria described in the above scheme are induced to obtain an induced culture product; the induced culture product is purified to obtain a PSI protein mutant. In the present invention, the purification includes nickel column purification; the nickel column purification is simple and rapid.

[0048] The present invention also provides the use of the PSI protein mutant, the encoding gene, the recombinant plasmid or the recombinant bacteria described in the above scheme in the preparation of tumor targeting drugs and / or tumor diagnostic reagent carriers.

[0049] In the present invention, the PSI protein mutant has tumor targeting properties.

[0050] The dimer structure of PSI protein is a key component for inducing membrane fusion activity, and the increase in the dimer ratio is conducive to the improvement of membrane fusion activity. PSI protein has two major functions: targeting PS-rich phospholipid membranes under acidic conditions and inducing membrane fusion. The monomer and dimer structures of PSI protein both have the targeting function under acidic conditions, and only the dimer is the main component for inducing membrane fusion function. PSI protein exists mainly in the form of dimers in the solution environment, and can recognize phosphatidylserine (PS) on the surface of tumor cells and target tumor cells to promote membrane fusion function; increasing the ratio of dimers of the protein active component of PSI will help further develop efficient tumor-targeted drugs and / or tumor diagnostic reagent carriers, so G14A-PSI has the application prospect of being further developed as an efficient tumor-targeted drug and / or tumor diagnostic reagent carrier.

[0051] The stability of the monomer conformation of the W18A-PSI and V22R-PSI mutants is enhanced compared to the wild-type PSI protein, while the stability of the dimer is reduced. Increasing the proportion of monomer structures and reducing the proportion of dimers can effectively weaken the protein's membrane fusion-inducing function, while retaining the protein's acidic targeting property, which is more conducive to the future development of W18A-PSI and V22R-PSI mutants into tool carrier protein tools that specifically target PS-rich cancer cell phospholipid membranes. The above mutants only provide targeting functions, which can be expanded to connect many functional molecules without interfering with the activity of the protein itself. In addition, in addition to function, in terms of structure, because the structural analysis of mixed conformations is difficult, the existence of a single PSI monomer structure is conducive to further guiding the analysis of the monomer three-dimensional structure of the protein, providing more atomic information, and facilitating more sophisticated molecular modifications in the future.

[0052] In the present invention, the pH value of the PSI protein mutant is preferably ≤5, more preferably 3-5.

[0053] The present invention also provides a tumor targeting drug and / or tumor diagnostic reagent carrier, comprising the PSI protein mutant, the encoding gene, the recombinant plasmid or the recombinant bacteria described in the above scheme.

[0054] In the present invention, the tumor targeting drug preferably also includes an anti-tumor active ingredient.

[0055] In one embodiment of the present invention, the tumor-targeted drug comprises liposomes; the surface of the liposomes is modified with the PSI protein mutant of the above scheme; and the liposomes are encapsulated with anti-tumor active ingredients.

[0056] In the present invention, the anti-tumor active ingredients include small molecule compounds; the small molecule compounds include doxorubicin.

[0057] In the present invention, the dosage form of the tumor targeting drug includes injection.

[0058] The present invention also provides the application of W18A-PSI and V22R-PSI in the study of monomer conformation in protein structure; the amino acid sequences of W18A-PSI and V22R-PSI are shown in SEQ ID NO.3 to SEQ ID NO.4 respectively.

[0059] To further illustrate the present invention, a method for predicting the change in the ratio of monomers and dimers after a single-site mutation of a PSI protein and mutants of the PSI protein provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0060] Example 1

[0061] By collecting the Gibbs free energy change value ΔΔG of the monomer after the PSI protein single point mutation 单 and the Gibbs free energy change of the dimer ΔΔG 二 , compare ΔΔG 单 and ΔΔG 二 Relative stability (relative stability = ΔΔG 单 -ΔΔG 二 ). When the relative stability is greater than 0, the monomer stability is determined to be increased and the dimer stability is decreased. 1 H- 15 The N HSQC spectrum shows that the monomer ratio increases and the dimer ratio decreases; when the relative stability is less than 0, it is judged that the monomer stability decreases and the dimer stability increases. 1 H- 15 The N HSQC spectrum showed that the proportion of monomers decreased and the proportion of dimers increased.

[0062] By simulating and calculating the Gibbs free energy change values ​​of monomers or dimers before and after the single-point mutation of the WT-PSI protein, the effect of the mutation on the thermodynamic stability of the two configurations was determined, and three amino acid sites that significantly affected the conformational stability were analyzed. In the simulated alanine scanning mutation, the glycine at position 14 was mutated to alanine; the tryptophan at position 18 was mutated to alanine; and the valine at position 22 was mutated to arginine. The monomer stability of the simulated mutant G14A-PSI was reduced, and the dimer stability was improved; the monomer stability of the simulated mutants W18A-PSI and V22R-PSI was improved, and the dimer stability was reduced.

[0063] Example 2

[0064] 1. Construction of recombinant plasmid

[0065] The DNA sequences of G14A-PSI, W18A-PSI and V22R-PSI were respectively inserted into the pET32a(+) vector after the Thrombin restriction site. Gene synthesis and plasmid construction were completed by Nanjing GenScript Biotechnology Co., Ltd. The G14A-PSI DNA sequence is shown in SEQ ID NO.5, and the G14A-PSI amino acid sequence is shown in SEQ ID NO.2; the W18A-PSI DNA sequence is shown in SEQ ID NO.6, and the W18A-PSI amino acid sequence is shown in SEQ ID NO.3; the V22R-PSI DNA sequence is shown in SEQ ID NO.7, and the V22R-PSI amino acid sequence is shown in SEQ ID NO.4. The plasmid map is shown in Figure 1 to Figure 3 shown.

[0066] The following steps take G14A-PSI as an example. The subsequent operations for W18A-PSI and V22R-PSI are the same.

[0067] 2. Preparation of Rosetta-gami B (DE3) pLysS E. coli competent cells:

[0068] 500 μL of Rosetta-gami B (DE3) pLysS bacterial solution was inoculated into 50 mL of LB liquid medium containing 50 μg / mL kanamycin, 34 μg / mL chloramphenicol, and 12.5 μg / mL tetracycline. The culture was shaken at 37°C, 220 rpm for 4 h. 600=0.6~0.8, transfer the bacterial solution to a 50mL centrifuge tube, place on ice for 20min, and centrifuge at 4000rpm for 10min at 4℃ to collect the bacteria. Resuspend the bacteria with 10mL pre-cooled 0.1M CaCl2 solution, and centrifuge at 4000rpm for 10min at 4℃ to collect the bacteria; repeat this operation three times. Add 3mL pre-cooled 0.1M CaCl2 to resuspend the cells, and add 10% glycerol to the final concentration to mix well, and finally dispense into 100μL / 1.5mL centrifuge tubes, freeze in liquid nitrogen, and store at -80℃.

[0069] 3. E. coli transformation:

[0070] Pipette 100ng plasmid pET32a-G14A-PSI into 100μL Rosetta-gami B(DE3)pLysS competent cells, mix gently, and place on ice for 30min; heat shock the mixture at 42℃ for 90s, and place on ice for 2min immediately after heat shock; add 1mL LB liquid medium containing 50μg / mL kanamycin, 34μg / mL chloramphenicol and 12.5μg / mL tetracycline, and culture for 60min at 37℃, 220rpm, and shake to activate the culture. After the activation culture, centrifuge at 4℃, 12000rpm, and remove the supernatant. Resuspend the remaining 50μL supernatant with the bacteria, and evenly spread the resuspended bacteria on LB solid medium containing 100μg / mL ampicillin, 50μg / mL kanamycin, 34μg / mL chloramphenicol and 12.5μg / mL tetracycline, and culture overnight at 37℃ with inversion for screening.

[0071] 4. Identification and preservation of positive monoclones:

[0072] The single clones obtained by resistance screening culture were picked, activated and cultured, and then plasmids were extracted and sequenced for verification.

[0073] The sequencing results were consistent with the pET32a(+)-G14A-PSI plasmid sequence, proving that the positive transformed bacteria were successfully obtained; the positive transformed bacteria were inoculated into LB liquid culture medium containing 100μg / mL ampicillin, 50μg / mL kanamycin, 34μg / mL chloramphenicol and 12.5μg / mL tetracycline, and cultured at 37°C, 220rpm, and shaken for 16h. After the culture was completed, glycerol was added to a final concentration of 50%, mixed and packaged, and stored at -80°C after quick freezing in liquid nitrogen.

[0074] 5. Purification of target protein:

[0075] (1) Activation culture of transformed bacteria:

[0076] Take the -80℃ frozen glycerol bacteria, thaw in an ice bath, take 10μL and inoculate into 100mL LB liquid culture medium containing 100μg / mL ampicillin, 50μg / mL kanamycin, 34μg / mL chloramphenicol and 12.5μg / mL tetracycline, and culture at 37℃, 220rpm, shaking for 14h. 100mL culture liquid was then inoculated into 1L LB liquid culture medium containing 100μg / mL ampicillin, 50μg / mL kanamycin, 34μg / mL chloramphenicol and 12.5μg / mL tetracycline, and cultured at 37℃, 220rpm, shaking for 3h, OD 600 =0.6~0.8, 4℃, 8000rpm, centrifuge for 10min to collect bacteria.

[0077] (2) Induction culture of transformed bacteria:

[0078] The activated cultured bacteria were resuspended in 1L of M9 medium containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, 34 μg / mL chloramphenicol and 12.5 μg / mL tetracycline (the carbon source in the medium was 15 N-NH4Cl, using this medium to culture strains can be used to 15 N label), 37℃, 220rpm, transition culture for 30min. After the transition culture, immediately ice bath for 5-10min. Add IPTG with a final concentration of 500μM, and induce culture for 18h at 30℃, 220rpm. After the induction culture, centrifuge at 4℃, 8000rpm for 10min to collect the bacteria.

[0079] (3) Nickel column purification:

[0080] Use 50mL buffer (20mM Tris, 100mM NaCl, pH8.0) to resuspend the induced bacteria. Under low temperature conditions, use a high-pressure homogenizer to break the cells until the resuspension becomes clear, and centrifuge at 4°C, 12000rpm for 30min to collect the supernatant. The supernatant is combined with Ni-NTA filler under low temperature oscillation for more than 2h. After the combination is completed, use a gravity column to collect the combined flow-through liquid and the 20mMTris, 100mMNaCl, pH8.0 buffer containing 0mM imidazole, 10mM imidazole, 30mM imidazole, 50mM imidazole, 150mM imidazole, 300mM imidazole. The eluate after washing is respectively washed with 20mMTris, 100mMNaCl, pH8.0 buffer. Sample the flow-through liquid and the eluate of each gradient concentration of imidazole, and SDS-PAGE is used to determine the eluate where the target protein is located. The eluate containing the target protein (i.e., the eluate containing 150mM imidazole) was placed in a dialysis bag with a molecular weight cutoff of 7kDa and dialyzed in 20mM Tris, 150mM NaCl, pH8.0 buffer. After the dialyzed protein, 0.5U thrombin was added per 1mg of protein, and the protein was digested overnight at 37°C. After the digestion, the digestion system was combined with the Ni-NTA filler under oscillation at low temperature for more than 2h. After the combination was completed, the flow-through after the combination was collected using a gravity column, as well as the eluate after washing with 20mM Tris, 100mM NaCl, pH8.0 buffer containing 300mM imidazole. The flow-through and 300mM imidazole eluate were sampled, and SDS-PAGE was used to determine whether the target protein was present in the flow-through. The flow-through obtained in the above steps was placed in a dialysis bag with a molecular weight cut-off of 1 kDa and dialyzed in deionized water. After the dialysis, the protein solution was transferred to an ultrafiltration tube with a molecular weight cut-off of 3 kDa and concentrated to 30 mL. After the concentration was completed, it was divided into packages and freeze-dried under low-temperature vacuum conditions. 15 The N-stable isotope-labeled G14A-PSI protein samples were dried, sealed and stored at -80°C.

[0081] According to the above scheme, W18A-PSI protein dry sample and V22R-PSI protein dry sample were obtained in turn.

[0082] 6. Liquid NMR:

[0083] The obtained G14A-PSI protein dried samples were dissolved in pH 3.0, pH 5.0, and pH 7.4 buffers, respectively; the obtained W18A-PSI protein dried samples were dissolved in pH 3.0, pH 5.0, and pH 7.4 buffers, respectively; the obtained V22R-PSI protein dried samples were dissolved in pH 3.0, pH 5.0, and pH 7.4 buffers, respectively. The protein samples obtained under the three pH conditions were subjected to 2D 1 H- 15N HSQC detection. 10% D2O was added to the above samples during the detection. All experiments were collected on a Bruker Avance 800MHz spectrometer. The chemical shift was calibrated by the chemical shift of the internal standard DSS. 15 N and 1 The ratio of the gyromagnetic ratio of H is used to indirectly calibrate 15 The chemical shift of N, 15 N and 1 The gyromagnetic ratio of H is -0.10132905. The experimental temperature is 25℃, and the collected spectra are processed by Topspin 3.2 and analyzed by CARA software. Through literature research and two-dimensional NMR spectrum identification, it is found that the 40th amino acid glycine in the monomer and dimer of PSI has different chemical shifts. The chemical shift of G40 in the monomer is 1 H 7.44, 15 At N104, the chemical shift of G40 in the dimer is 1 H 7.53, 15 At N 104, the cross peak area of ​​residue G40 is used as a standard to measure the monomer-dimer ratio and determine the monomer-dimer ratio in the target protein. G40 peak graph integration operation: The 2D-HSQC spectrum obtained by liquid NMR scanning is imported into the CARA software. In the CARA operation interface, first select the center position of the target peak, and then find "File" → "Export" → "Planepeaks to Monoscope" in the function area. After marking the target peak, calibrate it, find "Intergrator" → "Tune Peak Model" in the function area → adjust "Width X" and "WidthY" so that the adjusted green peak overlaps with the blue peak → adjust "PeakTol.X" and "PeakTol.Y" to 65%. After calibration, integrate, find "Intergrator" → "Update All Amplitudes" → "Intergrator all" in the function area → display the integrated area of ​​the target peak at "Amp."

[0084] Results Figure 5 , Figure 6 and Figure 7 . Figure 5 The peak graphs of G40 of different mutants under three pH gradients are shown, and the changes in the proportion of single dimers are judged based on the peak intensity, thereby verifying the correctness of the calculation. Figure 6 The HSQC 2D spectra of different mutants under three pH gradients are shown. Figure 7It can be seen that under different pH conditions, by comparing the G40 cross peaks of the mutant G14A-PSI and WT-PSI, it is found that the dimer ratio in G14A-PSI is significantly higher than that of wild-type PSI, and the monomer structure of the mutant even disappears under pH5.0 and pH3.0 conditions, indicating that after the 14th glycine of the wild-type PSI is mutated to alanine, its dimer stability is enhanced and the monomer stability is reduced. The monomer stability of W18A-PSI and V22R-PSI is improved, and the dimer stability is reduced. Taking W18A as an example: under different pH conditions, by comparing the G40 cross peaks of the mutant W18A-PSI and WT-PSI, it is found that the monomer ratio in W18A-PSI is significantly higher than that of wild-type PSI, and the dimer structure of the mutant disappears under pH7.4, pH5.0 and pH3.0 conditions, and only monomers exist. It indicates that after the 18th tryptophan of the wild-type PSI is mutated to alanine, its dimer stability is reduced and the monomer stability is improved. In addition, the G40 cross peaks of mutants W18A-PSI and V22R-PSI under different pH solution conditions show that the proportion of monomer conformation in the protein structure is increased, and the dimer conformation is greatly reduced under pH7.4 and pH5.0 conditions, indicating that the stability of the monomer conformation of W18A-PSI and V22R-PSI mutants is enhanced compared with the wild-type PSI protein, and the dimer stability is reduced. These results are consistent with the results of protein thermodynamic stability calculation based on Gibbs free energy, proving the feasibility of the protein single dimer regulation method based on thermodynamic stability simulation calculation in the present invention. The present invention combines computational biology with structural biology to predict and verify the ability of key amino acid single point mutations to regulate protein conformational equilibrium, laying a solid foundation for further transformation of functional PSI proteins in the future.

[0085] In summary, the present invention simulates and calculates the change values ​​of the Gibbs free energy of the monomer conformation and dimer conformation of the wild-type PSI protein after the single-point mutation, screens out three mutant proteins with significant changes in monomer and dimer stability: G14A-PSI, W18A-PSI and V22R-PSI, and obtains the two-dimensional stability of each mutant at pH 3.0, pH 5.0 and pH 7.4 through liquid nuclear magnetic resonance experiments. 1 H- 15 NHSQC spectrum, the results showed that the dimer ratio of G14A-PSI mutant protein was significantly increased compared with wild-type PSI.

[0086] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for predicting the change in the ratio of monomers and dimers after a single-site mutation of a PSI protein, characterized in that: The following steps are involved: The Gibbs free energy change value of the monomer of the mutant after the single-site mutation of the PSI protein is collected and calculated as ΔΔG 单 ; The Gibbs free energy change of the dimer of the mutant after the single-site mutation of PSI protein is collected and calculated as ΔΔG 二 ; The relative stability is calculated according to the formula shown in Formula 1; When the relative stability is greater than 0, it indicates that the dimer ratio decreases and the monomer ratio increases after the PSI protein single point mutation; when the relative stability is less than 0, it indicates that the dimer ratio increases and the monomer ratio decreases after the PSI protein single point mutation; Relative stability = ΔΔG 单 -ΔΔG 二 Formula 1; The amino acid sequence of the PSI protein is shown in SEQ ID NO.

1.

2. The method according to claim 1, characterized in that The site of the PSI protein single point mutation is the 14th, 18th or 22nd position.

3. The method according to claim 2, characterized in that The PSI protein single-site mutation includes: the 14th glycine mutates to alanine, the 18th tryptophan mutates to alanine, or the 22nd valine mutates to arginine.

4. A PSI protein mutant, characterized in that: It includes G14A-PSI, W18A-PSI or V22R-PSI; the amino acid sequences of G14A-PSI, W18A-PSI and V22R-PSI are shown in SEQ ID NO.2 to SEQ ID NO.4 respectively.

5. The gene encoding the PSI protein mutant according to claim 4, characterized in that: The nucleotide sequence of the gene encoding the G14A-PSI is shown in SEQ ID NO.5; the nucleotide sequence of the gene encoding the W18A-PSI is shown in SEQ ID NO.6; and the nucleotide sequence of the gene encoding the V22R-PSI is shown in SEQ ID NO.

7.

6. A recombinant plasmid, characterized in that The coding gene according to claim 5 is inserted.

7. A recombinant bacterium, characterized in that: Comprising the recombinant plasmid according to claim 6.

8. Use of the PSI protein mutant according to claim 4, the coding gene according to claim 5, the recombinant plasmid according to claim 6 or the recombinant bacteria according to claim 7 in the preparation of tumor targeting drugs and / or tumor diagnostic reagent carriers.

9. A tumor targeting drug and / or tumor diagnostic agent carrier, characterized in that: It includes the PSI protein mutant according to claim 4, the encoding gene according to claim 5, the recombinant plasmid according to claim 6 or the recombinant bacteria according to claim 7.

10. Application of W18A-PSI or V22R-PSI in the study of monomer conformation in protein structure; the amino acid sequences of W18A-PSI and V22R-PSI are shown in SEQ ID NO.3 to SEQ ID NO.4 respectively.

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