Highly active hinpi i enzyme mutants, methods of construction and use thereof

Abstract

The application discloses a high-activity HinP1I enzyme mutant and a construction method and application thereof, and belongs to the technical field of genetic engineering and biological medicine. The mutant is obtained by mutating a wild-type HinP1I amino acid sequence shown in SEQ ID NO. 1 according to any one of the following: (a) mutating proline (Asn) at the 87th position into alanine (Ala); (b) mutating proline (Asn) at the 87th position into alanine (Ala) and mutating glycine (Gly) at the 228th position into lysine (Lvs). Experimental results show that, compared with the wild-type enzyme, the mutant enzyme has a significantly improved specific activity, can complete complete enzyme cutting on a substrate DNA in a shorter time, and maintains sensitivity to CpG methylation. The mutant can be widely applied to fields such as DNA methylation detection, gene cloning and high-throughput sequencing library construction.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and biotechnology, and in particular to a site-modified, highly active HinP1I restriction endonuclease mutant, its encoding gene, preparation method, and its application in DNA methylation detection. Background Technology

[0002] DNA methylation, one of the most important forms of epigenetic modification, regulates gene expression by adding a methyl group to the 5th carbon atom of cytosine (C) without altering the DNA sequence. In mammalian genomes, this modification primarily occurs at CpG dinucleotide sites. Numerous studies have shown that abnormal hypermethylation in tumor suppressor gene promoter regions is closely related to the early development of various malignant tumors. Therefore, developing highly sensitive and accurate DNA methylation detection technologies has become a research hotspot and core direction in the fields of early tumor screening, auxiliary diagnosis, and dynamic prognostic monitoring.

[0003] Currently, the mainstream methods for DNA methylation detection mainly include bisulfite conversion and methylation-sensitive restriction endonuclease (MSRE) methods. While the bisulfite method is considered the "gold standard," it suffers from drawbacks such as a harsh chemical process, significant DNA template degradation, cumbersome procedures, and a tendency for incomplete conversion. This is particularly problematic when processing trace amounts of fragmented circulating tumor DNA (cfDNA) samples, often resulting in low detection rates. In contrast, the MSRE method utilizes CpG-sensitive restriction endonucleases, such as HinP1I (recognizing the 5'-G↓CGC-3' sequence), to specifically cleave the unmethylated target sequence while preserving the methylated target sequence for subsequent amplification. This method offers significant advantages, including ease of operation, minimal DNA damage, low detection cost, and ease of automation, demonstrating immense potential for application in clinical molecular diagnostics.

[0004] However, in practical clinical applications, the performance of wild-type HinP1I enzymes still faces several bottlenecks. First, the catalytic efficiency and substrate affinity of wild-type enzymes are insufficient when dealing with complex human genomic backgrounds. Especially in scenarios such as early cancer screening where extremely low proportions (e.g., below 0.1%) of methylation signals need to be detected, if the massive amount of unmethylated background DNA in the reaction system cannot be completely digested, the remaining uncut template will generate severe background noise in subsequent quantitative PCR (qPCR) or digital PCR (dPCR), leading to false positive results. Second, existing HinP1I enzymes have limited tolerance to reaction conditions. In reaction systems containing residual inhibitors from blood or with varying salt ion strengths, enzyme activity fluctuates significantly, and the incubation time required for complete digestion is typically long, making it difficult to meet the needs of rapid clinical diagnosis. Therefore, screening and constructing HinP1I mutants with higher specific activity, stronger environmental tolerance, and more thorough digestion efficiency through protein-directed evolution or rational design is a key technical challenge that urgently needs to be addressed in the field of molecular diagnostics. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the deficiency of low wild-type HinP1I enzyme activity in the prior art by providing a high-activity HinP1I enzyme mutant, its construction method, and its application.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: In its first aspect, the present invention provides a highly active HinP1I restriction endonuclease mutant, said mutant being a mutant protein derived from the restriction endonuclease HinP1I of Haemophilus influenzae P1. More specifically, it is obtained by mutating the wild-type HinP1I amino acid sequence as shown in SEQ ID NO.1 according to any one of the following mutations. The obtained transaminase mutant can further improve the enzymatic digestion efficiency of substrate DNA while maintaining sensitivity to CpG methylation: (a) The proline (Asn) at position 87 is mutated to alanine (Ala), and the corresponding mutant is denoted as HinP1I-N87A; (b) The proline (Asn) at position 87 is mutated to alanine (Ala), and the glycine (Gly) at position 228 is mutated to lysine (Lvs). The corresponding mutant is denoted as HinP1I-N87A / G228K.

[0007] Wild-type HinP1I amino acid sequence: MNLVELGSKTAKDGFKNEKDIADRFENWKENSEAQDWLVTMGHNLDEIKSVKAVVLSGYKSDINVQVLVFYKDALDIHNIQVKLVSNKRGFNQIDKHWLAHYQEMWKFDDNLLRILRHFTGELPP YHSNTKDKRRMFMTEFSQEEQNIVLNWLEKNRVLVLTDILRGRGDFAAEWVLVAQKVSNNARWILRNINEVLQHYGSGDISLSPRGSINFGRVTIQRKGGDNGRETANMLQFKIDPTELFDI(SEQ ID NO.1).

[0008] In a second aspect, the present invention provides a gene encoding a highly active HinP1I enzyme mutant as described above.

[0009] Preferably, the nucleotide sequence encoding the wild-type ω-transaminase shown in SEQ ID NO.1 is SEQ ID NO.2, and the genes encoding the highly active HinP1I enzyme mutant are all obtained by site-directed mutagenesis based on the nucleotide sequence shown in SEQ ID NO.2.

[0010] Preferably, the gene encoding the mutant HinP1I-N87A is SEQ ID NO.13, and the gene encoding the mutant HinP1I-N87A / G228K is SEQ ID NO.14.

[0011] A third aspect of the present invention provides a recombinant expression vector comprising the polynucleotides described above.

[0012] In a fourth aspect, the present invention provides a host cell comprising the recombinant expression vector described above.

[0013] Preferably, the host cell is *Escherichia coli* (E. coli). E. coli ).

[0014] In a fifth aspect, the present invention provides a method for constructing a highly active HinP1I enzyme mutant as described above, comprising steps such as gene cloning, site-directed mutagenesis, prokaryotic expression, and affinity chromatography purification.

[0015] Preferably, the construction method includes the following steps: S1. Construct a recombinant expression vector containing the mutant coding gene; S2. Transform the recombinant expression vector into host cells; S3. Induce host cells to express the mutant protein; S4. Separate and purify to obtain the highly active HinP1I enzyme mutant.

[0016] In a sixth aspect, the present invention provides the use of the highly active HinP1I enzyme mutant, gene, recombinant expression vector or host cell as described above in the preparation of DNA methylation detection reagents.

[0017] A seventh aspect of the present invention provides a DNA methylation detection reagent comprising the highly active HinP1I enzyme mutant as described above. In an eighth aspect of the present invention, a method for detecting DNA methylation is provided, wherein a DNA sample to be tested is treated with a highly active HinP1I enzyme mutant as described above, and the methylation status of the target site is determined by detecting whether the target site has been cleaved.

[0018] The beneficial effects of this invention are: This invention provides a highly active HinP1I enzyme mutant, its construction method, and its application in DNA methylation detection. Compared with existing technologies, it has at least the following advantages: (1) High activity: Compared with wild-type HinP1I, the mutants of the present invention (especially the N87A / G228K double mutant) exhibit faster enzymatic kinetics under the same unit definition, and can significantly shorten the reaction time (e.g. from 1 hour to 15 minutes). (2) High specificity: The mutant of the present invention retains the ability to strictly recognize the G^CGC site and does not exhibit non-specific star activity. (3) Methylation sensitivity: The mutant of the present invention fully retains the sensitivity to CpG methylation and is suitable for MSRE-PCR, MSRE-qPCR and other applications. Attached Figure Description

[0019] Figure 1 This is a schematic diagram showing the protein crystal structure of the HinP1I enzyme mutant and the distribution of the mutation sites on the crystal structure. Figure 2 The image shows the SDS-PAGE purification electrophoresis diagrams of wild-type HinP1I and mutant HinP1I in Example 2, where: lanes 1 and 2 are HinP1I-WT, lane 3 is HinP1I-N87A, lane 4 is HinP1I-G228K, and lane 5 is HinP1I-N87A / G228K. Figure 3The results are qPCR results for the performance test of the high-activity HinP1I mutant in Example 3, where: A is the result of wild-type cleavage of unmethylated substrate, B is the result of HinP1I-N87A cleavage of unmethylated substrate, C is the result of HinP1I-N87A / G228K cleavage of unmethylated substrate, D is the result of HinP1I-N87A / G228K cleavage of methylated substrate, and E is the result of control group. Figure 4 The results of rapid methylation detection of genomic DNA in Example 4 are shown, where: A is the result of the control group without enzyme, B is the result of wild-type enzyme digestion, and C is the result of HinP1I-N87A / G228K digestion. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0021] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0023] Example 1: Gene construction and vector acquisition of highly active HinP1I enzyme mutant This embodiment aims to construct wild-type HinP1I expression plasmids and mutant expression plasmids containing point mutations. The specific steps are as follows: 1. Wild-type gene acquisition: Based on Haemophilus influenzae from the GenBank database (… Haemophilus influenzae Based on the HinP1I gene sequence (SEQ ID NO.2) of P1), a HinP1I gene fragment optimized with E. coli codons was designed and synthesized. Introduced at the 5' and 3' ends of this fragment... Nde I and Xho I. Restriction site. The synthesized fragment was cloned into the pET-28a(+) vector to construct the recombinant plasmid pET28a-HinP1I-WT.

[0024] HinPI gene sequence: (SEQ ID NO.2) 2. Primer Design: Mutations are introduced using site-directed mutagenesis. Specific mutagenic primers were designed for the mutation sites involved in this invention (underlined areas indicate the positions of the mutation codons): For the N87A mutation (Aspartate Asn -> Alanine Ala): Forward primer N87A-F: 5'-CGTTAGT GCT AAACGTGGTTTTAATCAGATAGATAAAC-3' (SEQ IDNO.3) Reverse primer N87A-R: 5'-ACGTTT AGC ACTAACGAGCTTAACTTGAATATTATG-3' (SEQ IDNO.4) For the G228K mutation (glycine Gly -> lysine Lvs): Forward primer G228K-F: 5'-CGATAAT AAG AGAGAAACCGCAAATATGTTGCAAT-3' (SEQ IDNO.5) Reverse primer G228K-R: 5'-TTTTCTCT CTT ATTATCGCCCCTTTTTCTTTGAATAG-3' (SEQ IDNO.6) 3. Construction of mutant plasmids: Construction of a single mutant (Mutant1): Using pET28a-HinP1I-WT plasmid as a template, PCR amplification was performed using primer pair F-N87A / R-N87A and high-fidelity DNA polymerase. The PCR program was as follows: (1) denaturation at 98℃ for 10 seconds; (2) annealing at 58℃ for 5 seconds; (3) extension at 72℃ for 1 minute; (4) repeating steps (1) to (3) for 35 cycles; (5) extension at 72℃ for 5 minutes; (6) storage of the amplified product at 4℃. The product was then subjected to... Dpn After digestion with enzyme I to remove the methylated template, it is converted to E. coli DH5α competent cells. Plasmid extraction and sequencing confirmed a mutation at position 87 from Asn to Ala, named pET28a-HinP1I-M1 (N87A), with the following gene sequence: (SEQ ID NO.13).

[0025] Construction of the double mutant (Mutant2): Using the constructed pET28a-HinP1I-M1 plasmid as a template, the above steps were repeated using primer pair F-G228K / R-G228K. Sequencing confirmed that position 87 is Ala and position 228 is Lvs, and the mutant was named pET28a-HinP1I-M2 (N87A / G228K). Its gene sequence is as follows: (SEQ ID NO.14).

[0026] Example 2: Induction and purification of HinP1I mutant protein This embodiment details the specific steps for obtaining high-purity recombinant enzyme protein: 1. Transformation: The plasmids pET28a-HinP1I-WT, pET28a-HinP1I-M1, and pET28a-HinP1I-M2 obtained in Example 1 were transformed into expression hosts, respectively. E. coli In T7 Express (or BL21 (DE3)).

[0027] 2. Expanded culture: Pick a single colony and inoculate it into 5 mL of LB liquid medium containing 50 μg / mL kanamycin and culture overnight. The next day, transfer it to 500 mL of LB medium (containing kanamycin) at a ratio of 1:100 and culture at 37℃ with shaking at 200 rpm until the OD600 reaches 0.6-0.8.

[0028] 3. Low temperature induction: The culture temperature was lowered to 16℃, and isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.4 mM to induce expression for 18 hours.

[0029] 4. Cell collection and lysis: Collect bacterial cells by centrifugation at 6000 rpm for 10 minutes at 4℃. Resuspend the bacterial cells in lysis buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM Imidazole, 5% Glycerol, 1 mM PMSF). Dissolve the bacterial cells using an ultrasonic homogenizer under ice bath conditions (400W power, 3s operation, 5s interval, 20 minutes in total) until the bacterial solution is clear.

[0030] 5. Affinity chromatography purification: Centrifuge the lysate at 12,000 rpm for 30 minutes, and load the supernatant onto a pre-equilibrated Ni-NTA chromatography column.

[0031] 6. Washing: Use 10 column volumes of washing buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 50 mM Imidazole) to wash away contaminating proteins.

[0032] 7. Elution: Elute the target protein using elution buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 300 mM Midazole).

[0033] 8. Dialysis concentration: The eluted protein is placed into a dialysis bag (molecular weight cutoff 10 kDa) and dialyzed overnight at 4°C in storage buffer (10 mM Tris-HCl pH 7.4, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50% Glycerol).

[0034] 9. Concentration determination: Protein concentration was determined using the Bradford method, and purity was detected by SDS-PAGE electrophoresis. Results showed that both wild-type and mutant proteins exhibited single bands, with purity >95% and a molecular weight of approximately 28.6 kDa. Figure 2 ).

[0035] Example 3 Performance testing of highly active HinP1I mutant This embodiment uses qPCR technology to compare the catalytic rates of wild-type HinP1I and two mutants (N87A and N87A / G228K) under strict control of the same number of enzyme molecules, and verifies its methylation sensitivity. The specific steps are as follows: 1. Experimental Preparation Substrate preparation: Unmethylated substrate (pUC19-Un): extracted from E. coli pUC19 plasmid of DH5α was diluted to a concentration of 20 ng / μL.

[0036] Methylated substrate (pUC19-Me): pUC19 was completely methylated in vitro using CpG methyltransferase (M.SssI), purified, and diluted to 20 ng / μL.

[0037] Enzyme solution calibration: Protein concentrations of wild-type (WT), single mutant (M1, N87A), and double mutant (M2, N87A / G228K) were determined using the BCA method.

[0038] Dilute all three components to a working concentration of 20 nM using a storage buffer.

[0039] qPCR primer and probe sequences: F-pUC19: 5'-TGCCAGCTGCATTAATGAATC-3' (SEQ ID NO.7) R-pUC19: 5'-AGCGCAGCGAGTCAGTGA-3' (SEQ ID NO.8) P-pUC19: 5'-CGTATTGGGCGCTCTTCCGCTT-3' (SEQ ID NO.9) 2. Enzyme digestion reaction procedure (SOP) (1) Preparation of the reaction system: Prepare 5 reaction groups, with the following system (20 μL): Group A: 5 μL pUC19-Un + 2 μL wild-type enzyme (20 nM) + 2 μL 10×Buffer + 11 μL water Group B: 5 μL pUC19-Un + 2 μL N87A (20 nM) +2 μL 10×Buffer + 11 μL water Group C: 5 μL pUC19-Un + 2 μL N87A / G228K (20 nM) +2 μL 10×Buffer + 11 μL water Group D: 5 μL pUC19-Me + 2 μL N87A / G228K (20 nM) + 2 μL 10×Buffer + 11 μL water Group E: 5 μL pUC19-Un + 2 μL ddH2O + 2 μL 10×Buffer+ 11 μL water (2) Reaction and inactivation: Each group was reacted at 37°C for 30 minutes. Immediately after the reaction was completed, the mixture was placed in an 80°C metal bath and heated for 20 minutes to stop the cutting process.

[0040] 3. qPCR quantitative detection The inactivated product was diluted 20-fold and 5 μL was used as a template.

[0041] Program: 95℃ for 4 min; (95℃ for 20 s, 60℃ for 30 s, 72℃ for 20 s) × 40 cycles. Collect Ct values ​​for each group.

[0042] 4. Results and Analysis Based on the ΔCt value (Ct) of each group relative to the blank control group (Group E), Sample - Ct Blank The cutting efficiency is evaluated using ΔCt. Each increase of 1 in ΔCt represents a 50% reduction in residual template. The final test results are as follows: Figure 3 As shown.

[0043] Experimental Data Statistics Table E Enzyme-free Demethylation 30 min 19.72 0 Baseline (0%) A wild type Demethylation 30 min 23.9 4.18 Approximately 94.5% B N87A Demethylation 30 min 28.47 8.75 Approximately 99.7% C N87A / G228K Demethylation 30 min 31.22 11.5 >99.9% D N87A / G228K methylation 30 min 19.96 0.24 0% (uncut) 5. Conclusion Single point mutation effect: The results showed that after introducing only the N87A mutation (group B), ΔCt increased from 4.18 in the wild type to 8.75, indicating that the mutation of aspartic acid at position 87 to alanine can significantly enhance the catalytic activity of HinP1I.

[0044] Dual-site synergistic effect: When N87A and G228K (group C) were introduced simultaneously, ΔCt further increased to 11.5, showing a significant additive effect compared to the single mutant. Within 10 minutes, the dual mutant almost eliminated all amplifiable template, exhibiting extremely high activity.

[0045] Methylation sensitivity: Although the double mutant has extremely strong catalytic ability, its Ct value is completely consistent with the enzyme-free control when treating methylated substrates (Group D), proving that the mutant's recognition specificity for CpG methylation is not affected by the enhanced activity.

[0046] The above experimental results demonstrate that the HinP1I mutant provided by this invention significantly improves the cleavage rate while maintaining MSRE characteristics, representing a significant technological advancement.

[0047] Example 4: Application in rapid methylation detection of genomic DNA (MSRE-qPCR) This embodiment simulates a real-world application scenario to test the performance of the mutant in MSRE-qPCR.

[0048] Sample source: Human genomic DNA (extracted from HEK293T cell line).

[0049] Target gene: The promoter region of the VIM gene was selected.

[0050] qPCR primers: F-VIM: 5'-TGCCAGCTGCATTAATGAATC-3' (SEQ ID NO.10) R-VIM: 5'-AGCGCAGCGAGTCAGTGA-3' (SEQ ID NO.11) P-VIM: 5'-CGTATTGGGCGCTCTTCCGCTT-3' (SEQ ID NO.12) Experimental procedure: 100 ng of genomic DNA was treated with wild-type HinP1I and mutant N87A / G228K, respectively.

[0051] Rapid procedure: Digest at 37℃ for 30 minutes, then inactivate at 80℃ for 20 minutes.

[0052] Use 2 μL of the digestion product as a template for qPCR amplification, with primers crossing a key GCGC site.

[0053] qPCR results as follows Figure 4 As shown.

[0054] Unenzyme control (Input): The average Ct value was 25.55.

[0055] Wild-type enzyme treatment (15 min): The average Ct value was 29.44. The Ct value only increased by 3.89, indicating that a portion of the template was not cleaved (because this region is unmethylated in normal cells and should theoretically be cleaved, thus preventing amplification).

[0056] HinP1I-N87A / G228K treatment (15 min): The average Ct value was 35.46. The Ct value increased by nearly 10 cycles, approaching that of the template-free control (NTC). This indicates that within just 15 minutes, HinP1I-N87A / G228K completely cleaved the unmethylated target sites in the genome, thereby blocking PCR amplification.

[0057] The above experimental results demonstrate that, compared to the wild type, the mutant enzyme of this invention has significant advantages in rapid screening for methylation at the genomic level, which can greatly shorten the detection process and improve the accuracy of the results (reducing false positive background).

[0058] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A highly active HinP1I restriction endonuclease mutant, characterized in that, It is obtained by mutating the wild-type HinP1I amino acid sequence as shown in SEQ ID NO.1 according to any of the following mutations: (a) The proline (Asn) at position 87 is mutated to alanine (Ala); (b) The proline (Asn) at position 87 is mutated to alanine (Ala), and the glycine (Gly) at position 228 is mutated to lysine (Lvs).

2. A gene encoding a highly active HinP1I enzyme mutant as described in claim 1.

3. The gene according to claim 1, characterized in that, The nucleotide sequence encoding the wild-type ω-transaminase shown in SEQ ID NO.1 is SEQ ID NO.

2. The genes encoding the highly active HinP1I enzyme mutant were all obtained by site-directed mutagenesis based on the nucleotide sequence shown in SEQ ID NO.

2.

4. A recombinant expression vector comprising the polynucleotide as described in claim 2 or 3.

5. A host cell comprising the recombinant expression vector of claim 4.

6. The host cell according to claim 5, characterized in that, The host cell is preferably Escherichia coli.

7. A method for constructing a highly active HinP1I enzyme mutant as described in claim 1, characterized in that, Includes the following steps: S1. Construct a recombinant expression vector containing the mutant coding gene; S2. Transform the recombinant expression vector into host cells; S3. Induce host cells to express the mutant protein; S4. Separate and purify to obtain the highly active HinP1I enzyme mutant.

8. The use of a highly active HinP1I enzyme mutant as described in claim 1, a gene as described in any one of claims 2-3, a recombinant expression vector as described in claim 4, or a host cell as described in any one of claims 5-6 in the preparation of a DNA methylation detection reagent.

9. A DNA methylation detection reagent, characterized in that, It includes the highly active HinP1I enzyme mutant as described in claim 1.

10. A method for detecting DNA methylation, characterized in that, The DNA sample to be tested is treated with the highly active HinP1I enzyme mutant as described in claim 1, and the methylation status of the target site is determined by detecting whether the target site is cut.

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