Human PARP-2 catalytic domain mutant efficiently expressed in Escherichia coli system

Abstract

The invention belongs to the fields of gene technology and pharmacy, and discloses a highly expressed human source (Homosapiens) PARP-2 catalytic domain mutant protein and an application thereof. The method comprises the following steps: carrying out sequence alignment on PARP-2 catalytic structural domains of a human source and a mouse source, and designing a mutant. According to the present invention, the Tm value of the mutant catPARP-2SE (the codon of the nucleotide sequence is optimized, and the 349TELQSP354 is mutated to 349SERQGL354) is determined by using a differential scanning fluorescence method (Diffraction ScanningFluorometry), and the Tm value of the mutant catPARP-2SE is increased by about 1.11 DEG C, wherein the codon of the nucleotide sequence is optimized, and the 349TELQSP354 is mutated to 349SERQGL354; compared with a wild type, the mutant has the advantages that the thermal stability is enhanced, the expression quantity in an Escherichiacoli system is improved by more than four times, and the enzyme level activity evaluation and protein crystallization experiment of the PARP inhibitor are favorably carried out. The protein crystallization temperature is increased to 25 DEG C from 4 DEG C, so that the difficulty of obtaining a protein-ligand compound structure is greatly reduced, and the PARP-2 inhibitor design based on a protein structure is favorably developed. The catPARP-2SE has two characteristics of high expression quantity and crystallization in an environment close to room temperature, and can be applied to discovery of PARP-2 selective / non-selective inhibitors or PARP family other subtype selective inhibitors.

Description

Technical Field

[0001] The invention relates to a modified mutant protein of a potential anti-tumor target PARP-2 catalytic domain of human origin (Homo sapiens), belonging to the fields of gene technology and pharmacy. Background Art

[0002] PARP catalyzes adenosine diphosphate (ADP)–ribosylation, which is an important regulatory mechanism in cellular life processes, including transcription of chromatin to protein translation and stability. PARP is regulated by nicotinamide adenine dinucleotide (NADP) + ) as a substrate to covalently link ADP ribose to serine, glutamic acid, aspartic acid, lysine and other residues of the target protein. There are 17 subtypes of human PARP proteins reported. At present, the most widely studied subtypes are PARP-1 and PARP-2. Inhibition of the two can not only inhibit the repair of damaged DNA, overcome tumor resistance and increase chemotherapy / radiotherapy sensitivity, but also can be used in the clinic for the treatment of ovarian cancer and breast cancer with BRCA1 / 2 mutations through the "synthetic lethality" regulatory mechanism with BRCA1 / 2. Currently, there are six drugs on the market, Olaparib (Olaparib, 2014), Rucaparib (Rucaparib, 2016), Niraparib (Niraparib, 2017), Talazoparib (Talazoparib, 2018), Fluzoparib (Fluzoparib, 2020) and Pamiparib (Pamiparib, 2021), all of which are PARP-1 / 2 dual-target inhibitors.

[0003] However, more and more studies have shown that PARP-2 plays a different role from PARP-1 in the process of DNA repair. For example, the results of atomic force microscopy (AFM) imaging experiments show that PARP-1 can bind to single-stranded and double-stranded DNA breaks, while PARP-2 has a stronger specificity for binding to single-stranded DNA breaks. In addition, PARP-2 can inhibit the activity of PARP-1 when binding to broken DNA. At the same time, PARP-2 is an important regulatory protein for T cell survival. By participating in the DNA repair pathway, it increases the expression of TCRα (T-Cell receptorα) and inhibits the expression of pro-apoptotic proteins, but PARP-1 has not been found to play a similar role in this process.

[0004] Many studies have shown that PARP-2 can affect the occurrence and development of cancer through multiple pathways, such as genomic instability, cell metabolism disorders, angiogenesis, inflammatory response, tumor invasion and immune evasion, but many of the mechanisms and details are still unclear; recent studies have shown that PARP-2 expression in prostate cancer tissue is higher than that in benign prostate tissue, and selectively blocking the interaction between PARP-2 and FOXA1 can effectively inhibit the development of androgen receptor-mediated prostate cancer.

[0005] In general, PARP-1 and PARP-2 have differences in biological functions, especially PARP-2 plays a unique role in the occurrence and development of tumors. Therefore, the discovery of PARP-2 selective inhibitors can not only further clarify the function of PARP-2 in the occurrence and development of tumors, but also may discover safer and more effective anti-tumor drugs.

[0006] However, the low expression level of PARP-2 and the protein crystallization at 4°C have always been obstacles to the development of PARP-2 inhibitors. Low expression means that it takes longer and / or more materials to obtain sufficient protein, which increases the cost of enzyme-level inhibitor activity evaluation and crystallization experiments; 4°C protein crystallization requires a special low-temperature laboratory / environment to complete. Therefore, increasing the expression level of PARP-2 and allowing PARP-2 to crystallize at room temperature are of great significance for the design of selective and non-selective inhibitors of PARP-2, and even selective inhibitors of other subtypes of the PARP family. Summary of the invention

[0007] In order to increase the expression level of PARP-2 and form crystals in an environment closer to room temperature, the present invention provides a mutant of the catalytic domain protein of PARP-2. Compared with the wild type, the mutant protein has enhanced thermal stability, T m The value was increased by about 1.11℃, and it was efficiently expressed in the Escherichia coli system. The protein expression amount was increased by more than four times compared with before codon optimization, and protein crystals could be obtained at 16-25℃.

[0008] To achieve the purpose of the present invention, the present invention provides the following technical solutions:

[0009] The first aspect of the technical solution of the present invention is to provide a human PARP-2 catalytic domain mutant protein, characterized in that the human PARP-2 catalytic domain is the catalytic domain part in the full-length amino acid sequence of wild-type PARP-2, and the human PARP-2 catalytic domain mutant protein is a mutant protein formed by mutating the amino acid sequence of the human PARP-2 catalytic domain; the amino acid sequence mutation is an amino acid sequence obtained by mutating threonine, glutamic acid, leucine, glutamine, serine, and proline at positions 349 to 354 of the full-length amino acid sequence of wild-type PARP-2 to serine, glutamic acid, arginine, glutamine, glycine, and leucine, respectively; the full-length amino acid sequence of wild-type PARP-2 is shown in SEQ ID NO.1, and the amino acid sequence of the catalytic domain part of the wild-type PARP-2 is shown in SEQ ID NO.2.

[0010] The amino acid sequence of the human PARP-2 catalytic domain mutant protein is as shown in SEQ ID NO.6.

[0011] The second aspect of the technical solution of the present invention is to provide a nucleic acid molecule encoding the human PARP-2 catalytic domain mutant protein described in the first aspect, characterized in that the sequence of the nucleic acid molecule of the human PARP-2 catalytic domain mutant protein is shown in SEQ ID NO.8.

[0012] Also provided is a nucleic acid molecule encoding the human wild-type PARP-2 catalytic domain described in the first aspect, characterized in that the nucleic acid molecule of the human wild-type PARP-2 catalytic domain is codon-optimized for E. coli, and the sequence is shown in SEQ ID NO.4.

[0013] The third aspect of the technical solution of the present invention is to provide a recombinant plasmid carrying the nucleic acid molecule of the PARP-2 catalytic domain mutant protein described in the second aspect.

[0014] Also provided is a recombinant plasmid carrying the wild-type PARP-2 catalytic domain nucleic acid molecule described in the second aspect.

[0015] The fourth aspect of the technical solution of the present invention is to provide a recombinant bacterium expressing the human PARP-2 catalytic domain mutant protein described in the first aspect.

[0016] Also provided is a recombinant bacterium containing the nucleic acid molecule of the human PARP-2 catalytic domain described in the second aspect.

[0017] The recombinant bacteria uses Escherichia coli as a host bacterium.

[0018] The fifth aspect of the technical solution of the present invention provides a method for crystallizing the human PARP-2 catalytic domain mutant protein described in the first aspect, characterized in that the protein can obtain protein crystals at 16-25°C.

[0019] The sixth aspect of the technical solution of the present invention provides an application of the human PARP-2 catalytic domain mutant protein described in the first aspect in screening PARP-2 inhibitors or other PARP subtype selective inhibitors, characterized in that the PARP-2 catalytic domain mutant protein is used to evaluate the activity of inhibitors at the enzymatic level.

[0020] Also provided is an application of the human PARP-2 catalytic domain mutant protein described in the first aspect in culturing crystals of a compound and a PARP-2 catalytic domain mutant protein complex, characterized in that crystals of a PARP-2 catalytic domain mutant protein and an inhibitor molecule complex are cultivated to guide the design of a PARP-2 selective or non-selective inhibitor based on the protein structure.

[0021] The beneficial effects of the present invention are:

[0022] The present invention provides a mutant PARP-2 catalytic domain mutant protein catPARP-2SE ( 349 TELQSP 354 Mutation 349 SERQGL 354 ), compared with the wild type, T m The value increased by about 1.11℃. The mutant protein has better thermal stability and is efficiently expressed in the E. coli system. Compared with the wild type without codon optimization, the expression level increased by more than four times, which significantly reduced the time and material costs for obtaining protein, and is conducive to the evaluation of enzyme inhibitor activity and protein crystallization experiments. When culturing protein crystals of this mutant, it was found that the crystallization temperature increased from 4℃ to 25℃, which significantly reduced the requirements of crystallization experiments on equipment / experimental environment and the energy consumption required for temperature control. The discovery of the catPARP-2SE mutant makes it easier to conduct enzyme activity evaluation experiments and crystallization experiments of PARP inhibitors, which is not only beneficial for the screening and structure-based design of PARP-2 selective and non-selective inhibitors, but can also be applied to the study of discovering inhibitors of other subtypes of the PARP family. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Human PARP-2 (UniProt accession number Q9UGN5) and mouse PARP-2 catalytic domain

[0024] Amino acid sequence alignment of structure 1GS0, with significant differences marked by red lines Figure 2Differential scanning fluorescence images of catPARP-2 and its mutants

[0025] Figure 3 Nickel column purified catPARP-2 (expressed after codon optimization)

[0026] Figure 4 Electropherogram of catPARP-2SE purified by nickel column

[0027] Figure 5 Electrophoresis of catPARP-2SE purified by molecular sieve

[0028] Figure 6 catPARP-2SE crystal A crystallized at 18℃ B crystallized at 23.5℃

[0029] Figure 7 The crystal structure of catPARP-2SE (8HLJ; orange) and the structure of catPARP-2

[0030] (4TVJ; cyan) Overlay Mapping, structural superposition and

[0031] RMSD calculations were done using PyMol-open-source

[0032] Figure 8 Fluorescence polarization assay for the effects of AZD5305 and pamiparib (BGB290) on catPARP-1

[0033] and catPARP-2SE activity DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0035] Example 1: Codon optimization of human PARP-2 catalytic domain protein

[0036] The UniProt accession number of human PARP-2 is Q9UGN5, its full-length amino acid sequence is shown in SEQ ID NO.1, the amino acid sequence of the wild-type catalytic domain is shown in SEQ ID NO.2, the nucleic acid sequence of the catalytic domain of human PARP-2 is shown in SEQ ID NO.3, gene synthesis and codon optimization were completed by Nanjing GenScript Biotechnology Co., Ltd., and the sequence after codon optimization is shown in SEQ ID NO.4.

[0037] Example 2: Analysis of mutation sites and design of mutant proteins

[0038] The amino acid sequence of PARP-2 was compared with the amino acid sequence of the crystal structure 1GS0 of mouse PARP-2 (Mus musculus). Figure 1 The red underline indicates the mutation design site, and the wild-type amino acid sequence (Q9UGN5) is shown in SEQ ID NO.1.

[0039] The wild-type PARP-2 catalytic domain is denoted as catPARP-2. Three mutant proteins were designed at these two sites: catPARP-2SI ( 316 RT 317 Mutation 316 SI 317 ;SEQ ID NO.5), catPARP-2SE( 349 TELQSP 354 Mutation 349 SERQGL 354 ; SEQ ID NO.6) and combined mutation catPARP-2SISE (while 316 RT 317 Mutation 316 SI 317 as well as 349 TELQSP 354 Mutation 349 SERQGL 354 ;SEQ ID NO.7).

[0040] Example 3: Construction of mutant genes and recombinant plasmids and transformation of expression hosts

[0041] The recombinant plasmid of the mutant gene was obtained from Beijing Quanshijin Biotechnology Co., Ltd. FastPfu Fly PCR SuperMix(-dye) and Seamless Cloning and AssemblyKit construction.

[0042] Primers are listed in Table 1. Generally, primers 28a_F and 28a_R are used to amplify the linearized vector, primers catPARP-2_F and catPARP-2SI_R or catPARP-2SE_R are used to amplify the upstream gene fragment of the mutant, and primers catPARP-2SI_F or catPARP-2SE_F and catPARP-2_R are used to amplify the downstream gene fragment of the mutant. The amplification system is as follows: The PCR amplification procedure was as shown in Table 2. The product was -Basic Seamless Cloning and Assembly Kit was used to recombinant the upstream and downstream gene fragments and the linearized plasmid according to the instructions, and transformed into E. coli BL21-CodonPlus (DE3)-RIL to obtain the recombinant plasmids and recombinant bacteria of catPARP-2SI and catPARP-2SE. Similarly, based on the mutant catPARP-2SI, the primers catPARP-2_F and catPARP-2SE_R were used to amplify the upstream gene fragment of the mutant, and the primers catPARP-2SE_F and catPARP-2_R were used to amplify the downstream gene fragment of the mutant. After the recombination treatment, the recombinant plasmid and recombinant bacteria of catPARP-2SISE were obtained.

[0043] Table 1. Primers used to construct mutant gene recombinant plasmids

[0044]

[0045] Table 2. PCR amplification program

[0046]

[0047] The gene fragment was inserted into the pET-28a plasmid to obtain the recombinant plasmid. The PreScission protease cleavage site was inserted between the histidine tag and the protein sequence of the nickel column purification, which can be used for subsequent target protein cleavage verification. Since the target protein is often eluted with impurities when the nickel column is eluted, the PreScission protease cleavage removes the His-tag, which can not only be used for the verification of the target protein, but also can adsorb impurities and penetrate the target protein during the second nickel column purification, further improving the protein purity. The recombinant bacterial host is E. coli BL21-CodonPlus (DE3) -RIL, which can be induced for expression by IPTG.

[0048] Example 4: Differential Scanning Fluorimetry (DSF) experiment of mutants

[0049] Improved protein stability is more conducive to protein preparation and crystallization experiments. Here, differential scanning fluorimetry was used to calculate the T of the mutant. m The mutant stability was evaluated.

[0050] The recombinant bacteria were cultured with seed solution for 16 hours one day in advance, 0.5% of the seed solution was inoculated into 1 L of LB medium, cultured at 37°C for 6 hours, and induced to express at 18°C ​​for 20 hours.

[0051] The protein was obtained by ultrasonic disruption and lysis of the bacteria, with 3 seconds of ultrasonication, 7 seconds interval, and repeated for 10 minutes. The target protein was captured using a pre-packed column HisTrapHP (Cytiva, Uppsala, Sweden), nickel column A solution, 25mM Tris-HCl, 200mM NaCl, pH7.7, nickel column B solution, 25mM Tris-HCl, 200mM NaCl, 500mM imidazole, pH 7.7. SDS-PAGE electrophoresis was used for purity verification and preliminary estimation of expression level. The expression levels (nickel column recovery amount) were catPARP-2SE, catPARP-2SISE, catPARP-2SI, and catPARP-2 from high to low.

[0052] Differential scanning fluorescence detection was performed on samples containing almost no impurities to ensure that the main peak was significant and there was no interference from impurities. The labeled fluorescent probe was Sypro Orange (Sigma-Aldrich, Merck, USA), the working concentration was 500 times the original solution, the protein working concentration was 0.15 mg / mL, and the total volume was 100 uL. The detection instrument was qTOWER (Jena, Germany) fluorescence quantitative PCR, and the temperature was increased from 25°C to 95°C, increasing by 0.5°C per second and recording the fluorescence intensity. T was obtained by fitting with formula 1 m value.

[0053]

[0054] Y = fluorescence intensity (RFU); X = rising temperature (°C); Y max = maximum fluorescence intensity (RFU); Y min = minimum fluorescence intensity (RFU); T m The initial values ​​of the fitting for and c were set to 25 °C and 1, respectively.

[0055] T of codon-optimized catPARP-2, catPARP-2SI, catPARP-2SE, and catPARP-2SISE m The values ​​are 48.33℃, 48.25℃, 49.44℃ and 48.89℃ respectively. The original data are as attached. Figure 2 As shown, it is obvious that the beneficial mutation is catPARP-2SE, catPARP-2SI is a negative mutation, and the combination of the two mutations is in between.

[0056] Example 5: Purification method of catPARP-2SE

[0057] Because catPARP-2SE has the highest expression level, the best thermal stability, and carries fewer mutant amino acids than catPARP-2SISE, and is closer to the wild type, catPARP-2SE was selected for large-scale protein preparation, and its nucleic acid sequence is shown in SEQ ID NO.8. The nucleic acid sequence of the N-terminal connected histidine tag and the PreScission protease cleavage site is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10. The expression and purification methods are similar to the method for preparing wild-type catPARP-2, and the expression method has been described in Example 4. The purification steps are nickel column adsorption of the target protein, PreScission removal of His-tag, nickel column adsorption of impurities, anion column purification and molecular sieve purification. The peak of the molecular sieve reaches electrophoresis purity, and this purity can make catPARP-2SE crystallize.

[0058] The nickel column pre-packed column was HisTrap HP (Cytiva, Uppsala, Sweden), solution A: 25mM Tris-HCl, 200mM NaCl, pH 7.7, solution B: 25mM Tris-HCl, 200mM NaCl, 500mM imidazole, pH 7.7; the anion column pre-packed column was HiTrap Q HP (Cytiva, Uppsala, Sweden), solution A: 20mM Tris-HCl, pH 7.2, solution B: 20mM Tris-HCl, 1M NaCl, pH 7.2; the molecular sieve pre-packed column was Superdex G75 increase (Cytiva, Uppsala, Sweden), the buffer was 20mM HEPES, 300mM NaCl, 10% glycerol, 2mM TCEP, pH 7.5.

[0059] Codon-optimized wild-type catPARP-2 and mutant catPARP-2SE were purified by nickel column as described in Figure 3 and 4 As shown, the peak of catPARP-2SE in the last molecular sieving step is Figure 5 As shown, the final preparation amount of catPARP-2SE is about 1.5 mg / L, and about 9 mg of protein is obtained in 6L of culture medium, while the final preparation amount of wild-type catPARP-2 is about 0.3 mg / L, and about 2 mg of protein is obtained in 6L of culture medium, and the final preparation amount of codon-optimized wild-type catPARP-2 is about 0.6 mg / L, and about 4 mg of protein is obtained in 6L of culture medium. The high expression amount of mutant catPARP-2SE reduces the material consumption required for protein preparation.

[0060] Example 6: Crystallization method of catPARP-2SE

[0061] The crystallization method of catPARP-2SE is similar to that of catPARP-2. 10 mM 3-aminobenzamide is added to the catPARP-2SE protein solution and concentrated to a protein concentration of 35-60 mg / mL. It is crystallized in 25% PEG-3350, 0.1 M Tris-HCl pH 8.5. The difference is that the wild type of catPARP-2 co-crystallizes with tarazopanib at 16°C, and the crystals of the remaining complex structures are obtained by crystallization at 4°C. The improved thermal stability of catPARP-2SE allows it to crystallize at 16-25°C. In this case, the success rate of crystallization is higher at 16, 18, 22 to 25°C, as shown in Figure 2 . Figure 6 catPARP-2SE can be crystallized at temperatures close to room temperature, making crystallization experiments easier to carry out while greatly reducing the electrical energy consumption required to control low temperatures during crystallization.

[0062] After catPARP-2SE crystals were co-incubated with the marketed drug olaparib for 16 hours, diffraction experiments were completed at the Shanghai Synchrotron Radiation Light Source, AlphaFold built the initial structure, and the molecular replacement method was used to analyze it. It has been uploaded to the Protein Data Bank (RCSB Protein DataBank) with the accession number 8HLJ. 8HLJ was superimposed on the reported catPARP-2 wild-type structure 4TVJ, and the RSMD was Attached Figure 7 Therefore, from the three-dimensional structural level, it is believed that the catPARP-2SE mutant protein is consistent with the catPARP-2 wild-type structure and can be used as a substitute for the catPARP-2 wild-type. The complex structure is analyzed by crystallization and applied to the design of PARP-2 selective and non-selective inhibitors based on protein structure.

[0063] Example 7: Fluorescence polarization assay to measure the activity of AZD5305 and pamiparib (BGB290) on catPARP-1 and catPARP-2SE

[0064] The marketed drug pamiparib and the selective inhibitor AZD5305 were used to test whether catPARP-2SE can be used to evaluate the activity of compounds at the enzymatic level. The method was a fast and efficient fluorescence polarization method. The test buffer was 10mM Na2HPO4, 2mMNaH2PO4, 135mM NaCl, 4.7mM KCl, pH7.3, 100nM BSA. The test instrument was BioTek synergy H1 (BioTek Instruments Inc., Winooski, VT, USA). The test data were fitted and plotted in Prism using Formula 2.

[0065]

[0066] Y = fluorescence anisotropy (Anisotropy, A or mA); X = logarithmic value of compound concentration (Log (μM)); A0 = Y min ; A max =Y max -Y min ; Cl = probe concentration (0.0125 μM); K d = probe equilibrium dissociation constant (PARP-1, 0.00059 μM / PARP-2, 0.00065 μM); K i = equilibrium dissociation constant of the compound (μM).

[0067] Test results: catPARP-1 has a positive effect on the K of pamiparib and AZD5305 d The values ​​are: 3.7 and 3.4 nM respectively. The data in the literature (IC 50 ) were 1.3 nM (J. Med. Chem. 2020, 63, 15541-15563) and 3 nM (J. Med. Chem. 2021, 64, 14498-14512), respectively. The K of catPARP-2SE for pamiparib and AZD5305 d The values ​​are: 0.92nM and 765.8nM respectively. The data in the literature (IC 50 ) are 0.9nM (J.Med.Chem.2020,63,15541-15563) and 1400nM (J.Med.Chem.2021,64,14498-14512), respectively. The original data are as follows Figure 8 .

[0068] The above test results can illustrate two points. First, in the enzymatic activity evaluation experiment, the test results of catPARP-2SE are equivalent to the wild type reported in the literature. Second, the activity tests of catPARP-1 and catPARP-2SE compounds were carried out simultaneously. For the non-selective inhibitor pamiparib, the test activities of catPARP-1 and catPARP-2SE were equivalent, while for the selective inhibitor AZD5305, the selectivity ratio of catPARP-1 to catPARP-2SE was about 225 times. It can be concluded that the performance of catPARP-2SE in the tests of non-selective and PARP-1 selective inhibitors is consistent with that of the wild type, and the mutant protein has a high application value for the activity evaluation of PARP-2 selective and non-selective inhibitors and selective inhibitors of other subtypes of the PARP family.

Claims

1. A human PARP-2 catalytic domain mutant protein, characterized in that: The human PARP-2 catalytic domain is the catalytic domain part in the full-length amino acid sequence of wild-type PARP-2, and the human PARP-2 catalytic domain mutant protein is a mutant protein formed by mutating the amino acid sequence of the human PARP-2 catalytic domain; the amino acid sequence mutation is an amino acid sequence obtained by mutating threonine, glutamic acid, leucine, glutamine, serine, and proline at positions 349 to 354 of the full-length amino acid sequence of wild-type PARP-2 to serine, glutamic acid, arginine, glutamine, glycine, and leucine, respectively; the full-length amino acid sequence of wild-type PARP-2 is shown in SEQ ID NO.1, and the amino acid sequence of the catalytic domain part of the wild-type PARP-2 is shown in SEQ ID NO.

2.

2. The human PARP-2 catalytic domain mutant protein according to claim 1, characterized in that: The amino acid sequence of the human PARP-2 catalytic domain mutant protein is as shown in SEQ ID NO.

6.

3. A nucleic acid molecule encoding the human PARP-2 catalytic domain mutant protein according to any one of claims 1 to 2, characterized in that: The sequence of the nucleic acid molecule is shown in SEQ ID NO.

8.

4. A nucleic acid molecule encoding the human PARP-2 catalytic domain of claim 1, characterized in that: The sequence of the nucleic acid molecule is shown in SEQ ID NO.

4.

5. A recombinant plasmid carrying the nucleic acid molecule of claim 3. A recombinant plasmid carrying the nucleic acid molecule of claim 4.

7. A recombinant bacterium expressing the human PARP-2 catalytic domain mutant protein according to any one of claims 1 to 2.

8. A recombinant bacterium containing the human PARP-2 catalytic domain nucleic acid molecule according to claim 4.

9. The recombinant bacterium according to any one of claims 7 to 8, characterized in that The recombinant bacteria uses Escherichia coli as a host bacterium.

10. A method for crystallizing the human PARP-2 catalytic domain mutant protein according to any one of claims 1 to 2, characterized in that: The protein can be obtained into protein crystals at 16-25°C.

11. A use of the human PARP-2 catalytic domain mutant protein according to any one of claims 1 to 2 in screening PARP-2 inhibitors or other PARP subtype selective inhibitors, characterized in that: The PARP-2 catalytic domain mutant protein is used to evaluate the inhibitor activity at the enzymatic level.

12. A use of the human PARP-2 catalytic domain mutant protein according to claim 1 in culturing a crystal of a compound and a PARP-2 catalytic domain mutant protein complex, characterized in that: Cultivate crystals of PARP-2 catalytic domain mutant protein and inhibitor molecule complexes to guide the design of PARP-2 selective or non-selective inhibitors based on protein structure.

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