Method for improving the recombination efficiency of Bxb1 enzyme by optimization

Abstract

The present invention discloses a method for improving the recombination efficiency of the Bxb1 enzyme by optimizing the enzyme. The method comprises at least the following steps: (1) selecting mutation sites that affect the activity of the Bxb1 enzyme based on the structure of the Bxb1 enzyme; (2) performing directed mutagenesis on the mutation sites selected in step (1) to obtain different Bxb1 enzyme mutants; and (3) establishing a fluorescence assay in host cells to evaluate the recombination efficiency of the different Bxb1 enzyme mutants in step (2). The present invention successfully obtained three Bxb1 enzyme mutants, namely Bxb1(L82W), Bxb1(D13R), and Bxb1(T15R), by using directed mutagenesis and fluorescence assay evaluation. Compared with the wild-type Bxb1 enzyme, the three Bxb1 enzyme mutants all showed improved cell line recombination efficiency.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a method for improving the recombination efficiency of Bxb1 by optimizing and improving its enzyme activity. Background Art

[0002] In the field of molecular biology, the mobile genetic elements of many prokaryotes and the cellular defense systems against them have formed a wealth of tools for DNA manipulation. Examples include commonly used restriction endonucleases, DNA polymerases, ligases, transposases, and the CRISPR-Cas system. Enzyme-mediated DNA site-specific recombination systems are a major category. Depending on the type of enzyme mediating DNA recombination, they can be further divided into tyrosine recombinase systems and serine recombinase systems. Serine recombinase systems have two unique properties: 1) directional recombination; and 2) simple recombination sites, requiring only 40-50 base pairs of DNA. These systems have become important tools for mediating gene recombination in cell biology.

[0003] The serine protease family can be further divided into large serine proteases and small serine proteases based on their structure and function. To date, numerous large serine proteases have been discovered, including Bxb1. Bxb1 has been widely used in cell line development. For example, Chinese patent CN114107380A discloses that the applicant has constructed a Bxb1-based CHO-S.ATTP-GFP.A12 host cell system based on its PDEC-CELL platform. However, the wild-type Bxb1 enzyme has a recombination efficiency of only approximately 3%. This leads to low recombination efficiency and prolonged antibiotic pooling times when using wild-type Bxb1 in cell line development. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for improving the recombination efficiency of Bxb1 enzyme by optimizing and improving the activity of Bxb1 enzyme.

[0005] To solve the above technical problems, the present invention provides a method for improving the recombination efficiency of Bxb1 enzyme by optimization, comprising the following steps: (1) selecting mutation sites that affect the activity of Bxb1 enzyme based on the structure of Bxb1 enzyme; (2) performing targeted mutagenesis on the mutation sites selected in step (1) to obtain different Bxb1 enzyme mutants; and (3) establishing a fluorescence assay in host cells to evaluate the recombination efficiency of different Bxb1 enzyme mutants in step (2).

[0006] Specifically, the mutation sites affecting the enzyme activity of Bxb1 in step (1) are Bxb1(L82W), Bxb1(D13R), and Bxb1(T15R).

[0007] Specifically, the method for obtaining different Bxb1 enzyme mutants in step (2) is: designing two overlapping primers, performing circular PCR on the plasmid of the wild-type Bxb1 enzyme to obtain the Bxb1 enzyme mutant plasmid, and verifying whether the corresponding mutation is successfully introduced by sequencing.

[0008] Specifically, the host cell in step (3) is CHO-S.ATTP-GFP.A12.

[0009] Specifically, the CHO-S.ATTP-GFP.A12 contains a CMV promoter, an attP recombination site, followed by a GFP gene and PolyA. The CHO-S.ATTP-GFP.A12 can express the GFP (green fluorescent protein) gene and emit green light.

[0010] Specifically, the method for evaluating the recombination efficiency in step (3) is:

[0011] Co-transfecting two plasmids into the host cell, one is a Bxb1 enzyme mutant plasmid and the other is a donor plasmid;

[0012] The attB recombination site of the donor plasmid is followed by the RFP (red fluorescent protein) gene and polyA, and then a complete BFP (blue fluorescent protein) gene expression cassette;

[0013] After electroporation, the transfected host cells will express the BFP gene and emit blue light;

[0014] Only the successfully recombined host cells will emit red light because there is a promoter in front of the RFP gene, which enables the RFP gene to be expressed normally;

[0015] Recombination efficiency (%) = ratio of red fluorescent cells / ratio of blue fluorescent cells*100%.

[0016] Specifically, the molar ratio of the mixed plasmid consisting of the Bxb1 enzyme mutant plasmid and the donor plasmid during electroporation is (2-10):1.

[0017] Specifically, the volume of the mixed plasmids during electroporation cannot exceed 10% of the total volume of the transfection system.

[0018] Beneficial effects of the present invention:

[0019] The present invention successfully generated three Bxb1 enzyme mutants through targeted mutagenesis and fluorescence assay evaluation: Bxb1(L82W), Bxb1(D13R), and Bxb1(T15R). Compared to the wild-type Bxb1 enzyme, these three Bxb1 enzyme mutants showed improved recombination efficiency in cell lines. The recombination efficiency of the Bxb1(L82W) mutant was 1.5 times that of the wild-type, the recombination efficiency of the Bxb1(D13R) mutant was 1.8 times that of the wild-type, and the recombination efficiency of the Bxb1(T15R) mutant was 1.8 times that of the wild-type (see Table 2). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the present invention. 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 any creative work.

[0021] Figure 1 It is the structure prediction of Bxb1 serine protease;

[0022] Figure 2 It is the selection of mutation sites for Bxb1 serine protease;

[0023] Figure 3 It is a fluorescent system for evaluating the recombination efficiency of Bxb1 enzyme mutants;

[0024] Figure 4 The plasmid map of the wild-type Bxb1 enzyme (or the plasmid map of a Bxb1 enzyme mutant, where the mutation site is at the D13R site);

[0025] Figure 5 This is the sequencing result of the Bxb1 enzyme D13R mutant plasmid;

[0026] Figure 6 This is the plasmid map of the Bxb1 enzyme D13R mutant plasmid. DETAILED DESCRIPTION

[0027] The following is a clear and complete description of the technical solutions of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0028] Example 1. Based on the Bxb1 enzyme structure, the mutation sites that affect the enzyme activity were selected

[0029] 1) Bxb1 enzyme structure simulation

[0030] The structure of large serine proteases is primarily divided into two parts: the N-terminus and the C-terminus. The C-terminus contains the CC, ZD, and RD domains, responsible for DNA recognition, binding, and intersubunit interactions, respectively. The N-terminus contains the E helix and DNA catalytic domain. To modify the Bxb1 enzyme without affecting its DNA binding specificity, the catalytic domain was primarily targeted. Table 1 below summarizes the structural information of serine proteases published to date:

[0031] Table 1. Summary of published structures of serine proteases

[0032]

[0033] As shown in Table 1, the first three are large serine proteases and the last three are small serine proteases. There is no direct structural information for Bxb1. Although the sequence homology of this family is not high, about 15% to 20%, their catalytic domains are very similar based on sequence analysis and the resolved structures. Therefore, the structure of the catalytic domain of Bxb1 enzyme can be predicted based on the existing structure.

[0034] The structure of the catalytic domain of Bxb1 enzyme was predicted using Swiss-Model and Alphafold2. When using Swiss-Model, TP901 and yδ-resolvase were selected as the initial models respectively. TP901 is a tetrameric structure of a large serine protease, which has a higher similarity to the Bxb1 sequence, but there is no DNA in the structure. Although yδ-resolvase is a small serine protease, its resolved structure is a tetrameric structure in the activated state bound to DNA, so these two models were preferred. Alphafold2 was also used for structural prediction, so a total of three structures were obtained. After comparing them, it was found that except for the positions of the two loops, which were quite different, the alignment of other positions was relatively good (such as Figure 1 ), the spatial positions of the same amino acids in the three structures are basically the same.

[0035] 2) Selection of Bxb1 enzyme mutation sites

[0036] Next, based on the predicted structure and homologous family protein sequence alignment information, the mutation sites were selected. The mutation sites mainly include the following two categories: the first category is the alignment of the sequences of the homologous family catalytic domain (such as Figure 2 ), Figure 2The arrows in the middle mark the four completely conserved amino acids in the catalytic center, namely R at position 8, S at position 10, R at position 82, and R at position 85. Relatively conserved points near the catalytic center were selected for directed mutagenesis. The principle of directed mutagenesis is: if only one amino acid is different, it will be mutated to that different amino acid, or mutated to an amino acid with the same side chain properties. In this way, a total of 12 mutations were selected (such as Figure 2 At the same time, through literature research, it was found that some arginine residues near the active center of the enzyme may interact with the DNA chain. This interaction between the two may, to a certain extent, play a role in stabilizing the catalytic intermediate. Therefore, the amino acid at the corresponding site near the original arginine residue and close to the DNA was mutated to R, thereby enhancing the interaction with DNA.

[0037] Example 2. Different Bxb1 enzyme mutants were obtained by targeted mutagenesis of the selected sites through plasmid circular PCR

[0038] Plasmid map containing wild-type Bxb1 enzyme (eg Figure 4 , SEQ ID NO: 1). Taking the Bxb1 enzyme D13R mutant plasmid as an example, two overlapping primers were designed (upstream primer sequence: gagccgagtgacaAGGgccaccacaagtccc, SEQ ID NO: 2; downstream primer sequence: gggacttgtggtggcCCTtgtcactcggctc, SEQ ID NO: 3). PCR was performed on the wild-type Bxb1 enzyme plasmid. Circular PCR was used to obtain the Bxb1 enzyme mutant plasmid (SEQ ID NO: 4), and sequencing was used to verify the successful introduction of the mutation. Figure 5 The sequencing results in [ 15 ] confirmed the successful acquisition of the Bxb1 enzyme D13R mutant plasmid. Other Bxb1 enzyme mutant plasmids were also obtained using similar experimental procedures as described above.

[0039] Example 3. Evaluation of recombination efficiency (%)

[0040] A fluorescence assay was designed in CHO-S.ATTP-GFP.A12 host cells to evaluate the recombination efficiency of Bxb1 enzyme mutants (e.g. Figure 3 The principle is as follows: CHO-S.ATTP-GFP.A12 host cells have a CMV promoter, attP recombination site, followed by GFP and PolyA, so the host cells express GFP (green fluorescent protein) and emit green light. Two plasmids are co-transfected into the host cells, one of which is a Bxb1 enzyme mutant plasmid (such as Figure 4 , SEQ ID NO: 4), and the other is a donor plasmid (e.g. Figure 6, SEQ ID NO: 5). Following the attB recombination site of the donor plasmid is the RFP (red fluorescent protein) gene and polyA, followed by a complete BFP (blue fluorescent protein) gene expression cassette. After co-transfection, transfected host cells will express BFP and emit blue light, so BFP expression can be used to evaluate transfection efficiency. Only host cells that have successfully recombined will normally express RFP and emit red light because the promoter is located in front of RFP. Therefore, RFP expression can be used to evaluate recombination efficiency. The specific recombination efficiency (%) calculation formula is:

[0041] Recombination efficiency (%) = ratio of red fluorescent cells / ratio of blue fluorescent cells*100 (Formula 1).

[0042] Based on the above principles, two days after transient transfection of CHO-S.ATTP-GFP.A12 cells, FACS analysis was performed to obtain the ratio of red to blue fluorescent cells, and the recombination efficiency was calculated using Formula 1. The process for transient transfection of the CHO-S.ATTP-GFP.A12 cell line using Bxb1 serine enzyme mutants (such as Bxb1(L82W), Bxb1(D13R), and Bxb1(T15R)) is the same as the traditional cell transient transfection method except for the differences in host cells, Bxb1 enzyme plasmid, and donor plasmid. The specific steps are as follows:

[0043] 1. Preparation of CHO-S.ATTP-GFP.A12 Host Cells

[0044] 1.1 Prepare different cell numbers according to different electroporation volumes.

[0045] 1.2 Centrifuge the cells at room temperature: 800 rpm for 5 minutes and remove the supernatant. Then wash the cells with PBS solution containing 2% FBS (resuspend and centrifuge, then remove the supernatant).

[0046] 1.3 Resuspend the cells in Resuspension Buffer R provided in the electroporation kit to a final density of 2.0 × 10 7 cells / mL. Gently pipette the cells to obtain a single-cell suspension.

[0047] 2. Prepare the Plasmid Mix

[0048] Transfer an appropriate amount of plasmid to a sterile 1.5 mL EP tube. The molar ratio of the Bxb1 enzyme mutant plasmid (SEQ ID NO: 4) to the donor plasmid (SEQ ID NO: 5) is 9:1.

[0049] 3. Electroporation

[0050] 3.1 Gently mix the prepared CHO-S.ATTP-GFP.A12 host cells and the plasmid mixture. The volume of the plasmid mixture should not exceed 10% of the total volume of the transfection system. Avoid creating bubbles during this step.

[0051] 3.2 Perform transfection according to the Neon electroporator instructions and recommended parameters.

[0052] 4. Confirmation of target protein expression

[0053] Flow cytometry analysis was performed 48 hours after transfection to determine the ratio of red fluorescent and blue fluorescent cells.

[0054] The FACS assay steps are as follows:

[0055] 1) Prepare transfected CHO-S.ATTP-GFP.A12 cells: Count cells, 2E5 cells per well

[0056] 2) Cell plating: Add the prepared cells to a 96-well plate according to the detection order, with 2E5 cells per well, and centrifuge at 1500 rpm (200×g) for 4 minutes.

[0057] 3) Discard the supernatant and wash the cells three times with 2% FBS-PBS, centrifuging at 1500 rpm (200 x g) for 3 minutes each time.

[0058] 4) Take another new 96-well plate (mark it), cover it with a filter membrane, resuspend the washed cells in 100 μl of 2% FBS-containing PBS into the new 96-well plate covered with the filter membrane, and send it to the FACS reader.

[0059] 5. Calculation of recombination efficiency (%)

[0060] The red fluorescent cell ratio and blue fluorescent cell ratio (Table 2) were obtained from the FACS test results in step 4 and were substituted into Formula 1 to calculate the recombination efficiency of Bxb1 enzyme mutants (e.g., Bxb1(L82W), Bxb1(D13R), Bxb1(T15R)).

[0061] Based on the above experimental methods, a total of 50 mutations were made, and finally three mutants Bxb1(L82W), Bxb1(D13R), and Bxb1(T15R) were screened. Compared with the wild-type Bxb1, the recombination efficiency was increased by about two times (see Table 2).

[0062] Table 2. Recombination efficiency of Bxb1 serine protease mutants

[0063]

[0064]

[0065] In order to obtain more convincing results, the recombination efficiency of three mutant types, Bxb1(L82W), Bxb1(D13R), and Bxb1(T15R), was repeatedly measured, and the same results were obtained.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for improving the recombination efficiency of Bxb1 enzyme by optimization, characterized in that: At least the following steps are included: (1) Based on the Bxb1 enzyme structure, a mutation site affecting the Bxb1 enzyme activity was selected, wherein the mutation site was Bxb1(D13R) or Bxb1(L82W); (2) performing directed mutagenesis on the mutation sites selected in step (1) to obtain different Bxb1 enzyme mutants, wherein the method for obtaining different Bxb1 enzyme mutants is as follows: designing two overlapping primers, performing circular PCR on the plasmid of the wild-type Bxb1 enzyme, obtaining Bxb1 enzyme mutant plasmids, and verifying whether the corresponding mutation is successfully introduced by sequencing; The sequence of the wild-type Bxb1 enzyme plasmid is shown in SEQ ID NO: 1, the sequences of the two overlapping primers are shown in SEQ ID NO: 2 and SEQ ID NO: 3, the Bxb1 enzyme D13R mutant plasmid is shown in SEQ ID NO. 4, the donor plasmid is shown in SEQ ID NO. 5, and the Bxb1 enzyme L82W mutant plasmid is shown in SEQ ID NO. 6; (3) Establishing a fluorescence assay in a host cell to evaluate the recombination efficiency of different Bxb1 enzyme mutants in step (2), wherein the host cell is CHO-S.ATTP-GFP.A12. The CHO-S.ATTP-GFP.A12 host cell has a CMV promoter, an attP recombination site, followed by a GFP gene and PolyA. The CHO-S.ATTP-GFP.A12 host cell can express the GFP (green fluorescent protein) gene and emit green light; The evaluation methods are: Co-transfecting two plasmids into the host cell, one is a Bxb1 enzyme mutant plasmid and the other is a donor plasmid; The attB recombination site of the donor plasmid is followed by the RFP (red fluorescent protein) gene and polyA, and then a complete BFP (blue fluorescent protein) gene expression cassette; After electroporation, the transfected host cells will express the BFP gene and emit blue light; Only the successfully recombined host cells will emit red light because there is a promoter in front of the RFP gene, which allows the RFP gene to be expressed normally; Recombination efficiency (%) = ratio of red fluorescent cells / ratio of blue fluorescent cells*100%.

2. The method according to claim 1, characterized in that The molar ratio of the mixed plasmid composed of the Bxb1 enzyme mutant plasmid and the donor plasmid during electrotransformation is (2-10):

1.

3. The method according to claim 2, characterized in that The volume of the mixed plasmids during electroporation should not exceed 10% of the total volume of the transfection system.

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