Uvsx protein mutants and uses thereof

Abstract

The present disclosure provides a UvsX protein mutant having a mutation at one or more of the amino acids corresponding to position 35, 39, 52, 56, 166, 169, 197, 198, 200, 211, 212, 214, 318 of SEQ ID NO: 4, relative to a wild-type UvsX protein. The UvsX protein mutant of the present disclosure has high yield and improved activity compared to the wild-type UvsX protein.

Description

Technical Field

[0001] This disclosure pertains to the field of bioengineering, specifically relating to UvsX protein mutants and their applications. Background Technology

[0002] Recombinase polymerase amplification (RPA) technology is widely used for the rapid detection of bacteria and viruses in various industries, including medical diagnostics, environmental monitoring, food safety, and agriculture.

[0003] RPA, as an emerging nucleic acid amplification technology, has been widely used in molecular biology. Despite its many advantages, several challenges remain for further exploration: RPA is temperature-sensitive, typically operating at 37-42°C, which may limit its application in off-site environments, especially without temperature-controlled equipment. This presents challenges for point-of-care diagnostics. Compared to traditional PCR, which usually uses polymerase reaction buffers, RPA uses specific reaction buffer systems containing special components (such as single-stranded binding proteins and polymerases). Furthermore, the operational process is more complex, involving the regulation of multiple enzymes and components, requiring more experimental steps, increasing complexity and time consumption. Reagent costs are also relatively high, currently limiting its large-scale application.

[0004] Furthermore, the high price, insufficient activity, and instability of current RPA enzymes limit the development of RPA technology. There is an urgent need to develop heterologous expression systems and performance evaluation systems to obtain high-performance UvsX.

[0005] T4UvsX protein is an important recombinase, primarily used for DNA repair and recombination. This protein has wide applications in genetic engineering, biocatalysis, and gene editing. While T4UvsX protein shows significant promise in these fields, existing production methods face the following technical challenges:

[0006] (1) Low protein yield: Existing recombinant protein production technologies (such as expression in E. coli) often encounter the problem of low expression levels, which directly affects the efficiency of protein extraction and purification. Although improvements can be made by optimizing expression conditions and host strains, it is still difficult to meet the needs of large-scale production.

[0007] (2) Poor protein stability: T4UvsX protein often exhibits poor stability after expression. Especially under high temperature, high salt or other extreme conditions, the protein is prone to denaturation or degradation, which negatively impacts protein storage and application. In existing technologies, protein stability improvement is mainly achieved through chemical modification or ligand binding, but these methods have limited effectiveness and are difficult to adapt to the requirements of different environments.

[0008] (3) Insufficient protein activity: Although T4UvsX protein is important in DNA repair, existing protein activity testing and optimization methods still have shortcomings. Most existing activity enhancement technologies rely on external regulation of the enzyme or optimization under specific conditions, resulting in limited room for activity improvement, especially in practical applications where it is difficult to consistently improve its effectiveness.

[0009] Therefore, there is an urgent need in this field to develop a T4UvsX protein with better performance. Summary of the Invention

[0010] To address the shortcomings of existing technologies, this disclosure solves the problems of low yield and insufficient activity of T4UvsX protein by systematically improving the expression system, mutant screening, and stability optimization techniques. This disclosure provides T4UvsX protein mutants, which can be widely used in biopharmaceutical, enzyme engineering, genetic engineering, and molecular biology research, particularly in enzyme preparation production, gene repair, DNA recombination, and vaccine development.

[0011] One aspect of this disclosure provides a UvsX protein mutant, relative to the wild-type UvsX protein, wherein the UvsX protein mutant has a mutation at one or more of the following amino acids corresponding to SEQ ID NO:4: positions 35, 39, 52, 56, 166, 169, 197, 198, 200, 211, 212, 214, and 318.

[0012] In some embodiments, the UvsX protein mutant includes one or more mutations selected from K35G, M39L, Q52P, L56T, M166K, M166R, A169R, Y197R, E198N, E198R, E198K, E198W, Q200R, Q200K, G211K, T212R, P214L, and T318E.

[0013] In some embodiments, the UvsX protein mutant includes the K35G / E198N mutation. In some embodiments, the UvsX protein mutant includes the K35G / E198R mutation. In some embodiments, the UvsX protein mutant includes the K35G / E198K mutation.

[0014] In some embodiments, the wild-type UvsX protein has the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:4, or an amino acid sequence having undergone one or more amino acid additions, deletions, substitutions, or modifications to the amino acid sequence shown in SEQ ID NO:4.

[0015] In some embodiments, the UvsX protein mutant has one or more of the amino acid sequences shown in SEQ ID NO:5-11, 41-46, 72-74.

[0016] Another aspect of this disclosure provides a nucleic acid molecule encoding the UvsX protein mutant described in this disclosure.

[0017] Another aspect of this disclosure provides a recombinant expression vector containing the nucleic acid molecules described herein.

[0018] In some embodiments, the recombinant expression vector comprises a plasmid with a TIR mutation.

[0019] In some embodiments, the recombinant expression vector comprises a pET plasmid with a TIR mutation.

[0020] In some embodiments, the recombinant expression vector comprises a pET28a plasmid with a TIR mutation.

[0021] Another aspect of this disclosure provides a host cell containing the UvsX protein mutant described in this disclosure, the nucleic acid molecule, or the recombinant expression vector described in this disclosure.

[0022] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0023] In some embodiments, the prokaryotic cells may be selected from Escherichia coli or Bacillus subtilis, such as Escherichia coli BL21, T7E, C41, Arctic, etc. In some specific embodiments, the host cell is Escherichia coli BL21(DE3) strain.

[0024] In some embodiments, the eukaryotic cells may be selected from yeast cells, insect cells, plant cells, animal cells, etc., such as yeast cells, CHO cells, 293 cells, Vero cells, or NSO cells.

[0025] Another aspect of this disclosure provides a composition comprising (i) the UvsX protein mutant described in this disclosure, and (ii) one or more reagents selected from the group consisting of buffers, primers, probes, dyes, detection agents, target nucleic acids, and cell lysis agents.

[0026] In some embodiments, the composition is provided in the form of a kit.

[0027] Another aspect of this disclosure provides a method for preparing the UvsX protein mutant of this disclosure, wherein the method includes the following steps: culturing the host cells of this disclosure; and optionally, isolating the UvsX protein mutant from the host cells or from a growth medium or supernatant.

[0028] Another aspect of this disclosure provides the application of the UvsX protein mutant, the nucleic acid molecule, the recombinant expression vector, the host cell, or the composition described herein in enzyme preparation production, gene repair, DNA recombination, or vaccine preparation.

[0029] In some embodiments, the UvsX protein mutant is used for recombinase polymerase amplification reactions.

[0030] Another aspect of this disclosure provides a method for generating DNA molecules, including the step of using the UvsX protein mutant described in this disclosure.

[0031] In some embodiments, the UvsX protein mutant is incubated together with template DNA and primers.

[0032] In some embodiments, the method is carried out via a recombinase polymerase amplification reaction. Attached Figure Description

[0033] Figure 1 The expression of recombinase UvsX in different pET28a mutant plasmids is shown.

[0034] Figure 2 The standard curve for phosphorus standard determination is shown.

[0035] Figure 3 The effect of virtual saturation mutants on RPA response is shown.

[0036] Figure 4 The effect of consensus mutants on RPA response was shown.

[0037] Figure 5 The effect of the two-point mutant on the RPA response was shown. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in numerous publications.

[0039] definition

[0040] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure pertains. For purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0041] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0042] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0043] The term "recombinase T4UvsX protein" used in this article refers to the UvsX protein of T4 bacteriophage obtained from host cells such as Escherichia coli through genetic engineering. UvsX is a DNA repair and recombinase in T4 bacteriophage, which is usually involved in DNA repair and homologous recombination processes.

[0044] The term "mutant activity assay" as used in this article refers to testing changes in the enzyme activity or other biological functions of the UvsX protein under different conditions after gene mutation or engineering treatment.

[0045] The term "substitution" as used herein for amino acids refers to the replacement of at least one amino acid residue in an amino acid sequence with another different "substituted" amino acid residue. The term "insertion" as used herein for amino acids refers to the incorporation of at least one additional amino acid into an amino acid sequence. While inserts typically consist of one or two inserted amino acid residues, larger "peptide inserts" can also be prepared, for example, inserts of about three to five or even up to about ten, fifteen, or twenty amino acid residues. As disclosed above, the inserted residues can be naturally occurring or non-naturally occurring. The term "deletion" as used herein for amino acids refers to the removal of at least one amino acid residue from an amino acid sequence.

[0046] The mutants or fragments thereof disclosed herein may contain conserved amino acid substitutions at one or more amino acid residues, such as essential or non-essential amino acid residues. A “conserved amino acid substitution” is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, in this document, essential or non-essential amino acid residues in the mutants are preferably replaced with another amino acid residue from the same side chain family.

[0047] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are included, but those from the reference sequence are excluded.

[0048] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0049] In this disclosure, a recombinant expression vector for UvsX is constructed, and a high-performance RPA enzyme is obtained at a low cost through induced expression. Subsequently, the expression vector sequence is optimized, and high-yielding engineered bacteria are constructed and screened to increase protein production. Simultaneously, an enzyme performance evaluation system is established, which detects the inorganic phosphorus (PO4) generated from ATP hydrolysis. 3- The content of UvsX was used to evaluate the heterologous expression effect and the activity of the target UvsX. Furthermore, point mutations were performed using a semi-rational design method involving alanine scanning, saturation mutagenesis, and homologous protein sequence alignment to improve enzyme activity, resulting in mutants with enhanced activity.

[0050] The following embodiments are provided to aid in understanding this disclosure. However, it should be understood that these embodiments are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of this disclosure is set forth in the claims. It should be understood that any modifications and changes may be made without departing from the spirit of this disclosure.

[0051] Example

[0052] Example 1: Expression vector sequence optimization to increase protein yield

[0053] The recombinase was expressed in three different pET28a plasmids or their variants (TIR, T7, and dtHis) to construct a UvsX recombinant expression vector that helps to improve the expression level of the recombinase UvsX. Among them, TIR and T7 were derived from the literature Shilling PJ, Mirzadeh K, Cumming AJ, et al. Improved Designs for Pet Expression Plasmids Increase Protein Production Yield in Escherichia Coli[J]. Communications Biology, 2020, 3(1), and dtHis was wild-type pET28a.

[0054] 1. Construction of recombinant plasmids

[0055] (1) Synthesis of target gene

[0056] The gene sequence encoding UvsX (SEQ ID NO:4) was synthesized by Suzhou Genewise Biotechnology Co., Ltd., and then inserted into the NdeⅠ and XhoⅠ sites of the pET-28a(+) plasmid, thereby constructing the pET-UvsX recombinant plasmid.

[0057] (2) Extraction of pET-UvsX recombinant plasmid

[0058] A small number of T1 single colony strains carrying the pET-UvsX recombinant plasmid were selected from plate preservation and transferred to 5 mL of LB liquid medium containing 0.1 mM kanamycin (Table 1). The culture was carried out at 37℃ and 220 rpm for 12 h. After that, the bacterial cells were collected by centrifugation and plasmid extraction was performed. The plasmid extraction method was performed according to the instructions of the plasmid extraction kit (TruGold).

[0059] (3) Construction of E. coli BL21

[0060] In a clean bench, 10 μL of pET-UvsX recombinant plasmid was aspirated and mixed thoroughly with 50 μL of E. coli BL21 competent cells. The pET-UvsX recombinant plasmid was then introduced into E. coli BL21 competent cells and placed on ice for 30 min.

[0061] Quickly transfer to a 42°C water bath for 45 seconds, then transfer to ice and cool for 2 minutes.

[0062] Add 500 μL of LB liquid culture medium to the transformation system in a clean bench and incubate at 37℃ and 220 rpm for 1 h.

[0063] Centrifuge at 8000x g for 1 min, discard 450 μL of supernatant in a clean bench, resuspend the bacterial cells on a plate containing kanamycin, and incubate at 37°C for 12 h.

[0064] Single clones were selected and cultured in liquid LB medium at 37°C and 220 rpm for 12 hours for bacterial preservation.

[0065] (4) Directed mutagenesis and screening of pET-28a(+) vector sequence

[0066] use The Fast multisite mutagenesis system was used to perform site-directed mutagenesis on the pET-28a(+) vector sequence. The multisite mutagenesis system used plasmid pET-UvsX as a template, and agarose gel migration assays were used to detect the success of PCR.

[0067] PCR product purification and DNA gel recovery were performed according to the Solarbio-polyacrylamide gel DNA recovery kit instructions.

[0068] Add 0.6 μL of DMT enzyme to 30 μL of the purified product and incubate at 37°C for 1 h to perform template nitration; use Visual Green qPCR SuperMix cyclization;

[0069] The circularized plasmid was introduced into E. coli T1 competent cells, and the transformation system was evenly spread on LB agar plates containing kanamycin and incubated at 37°C for 12 h.

[0070] Single clones were selected and cultured in LB liquid medium at 37°C and 220 rpm for 12 h. DNA sequencing was then used to confirm whether the mutation was successful.

[0071] The successfully mutated plasmid was introduced into E. coli BL21 competent cells. The transformation system was evenly spread on LB agar plates containing kanamycin and incubated at 37°C for 12 h. Single colonies were picked and incubated in LB liquid medium at 37°C and 220 rpm for 12 h to preserve the bacteria.

[0072] Table 1 Preparation of LB medium

[0073] Components LB liquid culture medium / g / L LB plate culture medium / g / L peptone 10 10 yeast powder 5 5 Sodium chloride 10 10 Agar powder - 15

[0074] 2. Heterologous expression and purification of recombinant proteins

[0075] The pET-UvsX recombinant plasmid was introduced into E. coli BL21 competent cells and the protein was expressed in E. coli BL21(DE3) strain.

[0076] Take 10 μL of bacterial culture and inoculate it into 5 mL of LB liquid medium containing 0.1 mM kanamycin. Incubate overnight at 37°C and 220 rpm for 12 h. The next day, take 1 mL of the culture and transfer it to 80 mL of new LB medium containing 0.1 mM kanamycin. Continue incubation until the OD600 of the bacterial culture reaches 0.8. Then, add IPTG to a final concentration of 0.05 mM and continue incubation at 16°C and 160 rpm for 20 h.

[0077] The culture was then centrifuged at 8000×g at 4℃ for 5 min to collect the bacteria. The supernatant was discarded, and the bacterial pellet was stored in a centrifuge tube. The cells were resuspended in 10 mL of lysis buffer and stored on ice. Following the instructions of the cell disruptor, the bacterial culture was poured into the cell disruptor and disrupted three times. The resulting bacterial culture was balanced with lysis buffer and centrifuged at 10000×g at 4℃ for 60 min. The pellet was discarded, and the supernatant was filtered through a 0.45 μm microporous membrane. The protein in the supernatant was then purified using a metal affinity chromatography column on a Ni-NTA column. After equilibrating the nickel medium with Buffer I, the sample was loaded, washed with Buffer I containing 60 mM imidazole, and then eluted with Buffer I containing 500 mM imidazole.

[0078] All purification processes were performed at 4°C. The eluted protein was dialyzed in storage buffer and concentrated to less than 1 mL. The composition of the buffers used is shown in Tables 2 to 7 below.

[0079] The purified proteins were analyzed by 12% sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE), and their concentrations were determined using Solarbio's BCA protein assay kit.

[0080] Table 2 Preparation of Sterilization Solution

[0081]

[0082]

[0083] Table 3. Preparation of Buffer-I

[0084]

[0085] Table 4. Preparation of Buffer-A

[0086]

[0087] Table 5 Preparation of washing buffer

[0088] Components concentration Washing buffer Washing buffer Buffer-I 50mM 188mL 470mL Buffer-A (pH 7.2) 1mM 12mL 30mL

[0089] Table 6 Preparation of Elution Buffer

[0090] Components concentration Elution buffer Elution buffer Buffer-I 50mM 100mL 250mL Buffer-A (pH 7.2) 1mM 100mL 250mL

[0091] Table 7 Preparation of enzyme storage solution

[0092] Components concentration composition Sodium chloride 300mM 17.5328g Tris-HCl (pH 8) 20mM 2.4228g DTT 1mM 0.1543g EDTA 0.1mM 0.0292g distilled water - Adjust the volume to 1L

[0093] In this example, the recombinase was expressed in three different pET28a mutant plasmids (TIR, T7, and dtHis), and the results are as follows: Figure 2 As shown, the results indicate that the expression level of UvsX TIR is 1.43 times that of WT(UvsX dtHis) protein, while the expression level of UvsX T7 is 0.98 times that of WT(UvsX dtHis). Therefore, this disclosure uses the TIR mutation, demonstrating that mutation of the translation initiation region of the vector pET-28a(+) helps to increase the expression level of the recombinase UvsX.

[0094] Example 2: Establishment of a method for testing protein ATPase activity

[0095] Based on the principle of RPA amplification, UvsX recombinase, as an ATP hydrolase, requires ATP as an energy source to drive DNA strand synthesis. As the amplification reaction proceeds, ATP is continuously converted into ADP+Pi, therefore the amount of ATP converted can be used to reflect enzyme activity.

[0096] To assess enzyme activity, the malachite green-phosphomolybdic acid spectrophotometric method was used in this experiment to detect the generated phosphate (PO4) ions. 3- The total phosphorus content is determined by first reacting phosphate ions with ammonium molybdate in the reaction solution, followed by a colorimetric reaction with a colorimetric reagent. The absorbance is measured using a spectrophotometer, and the total phosphorus content is calculated by substituting it into a standard curve. This method is then used to assess the ATPase activity of the enzyme.

[0097] The specific method is as follows:

[0098] At 37℃, the absorbance of the supernatant after staining with 1×SafeGreen dye was measured using an Eppendorf BioPhotometer D30 UV spectrophotometer. The absorbance was then substituted into the standard curve to determine the total phosphorus content, which was used to evaluate enzyme performance. The total reaction volume for the standard curve was 1 mL. 80 μL of colorimetric reagent (as shown in Table 9) and 40 μL of 1% polyvinyl alcohol solution (w / v) were added to 2 mL EP tubes containing phosphorus standard concentrations as shown in Table 8, and then the volume was brought to 1 mL with distilled water. After mixing, the mixture was incubated in a 37℃ water bath for 20 min. Three readings were then taken at a wavelength of 620 nm using a UV spectrophotometer to plot the standard curve. Figure 1 As shown.

[0099] For the determination of UvsX activity, the enzyme activity reaction system consisted of 50 μL containing 3 μg UvsX, 0.3 mM ATP, 25 μL buffer (as shown in Table 10), and distilled water to a final volume of 50 μL. The reaction was initiated in a 37°C water bath for 10 min, then immediately placed on ice to terminate the reaction. Next, the chromogenic reagent and 1% polyvinyl alcohol solution (w / v) were added to a final volume of 1 mL. After mixing, the mixture was incubated in a 37°C water bath for 20 min. The absorbance at 620 nm was then measured using a UV spectrophotometer. Each parallel sample was tested three times to eliminate random errors. Finally, the phosphate content generated was calculated using the standard curve to evaluate the ATPase activity. Two parallel samples were prepared for each sample.

[0100] Table 8. Preparation of systems with different phosphorus standard concentrations

[0101] Phosphorus standard concentration - system / μg / mL Distilled water / μL Phosphorus standard / μL 0 880 0 0.05 860 20 0.10 840 40 0.15 820 60 0.20 800 80 0.25 780 100

[0102] Table 9 Preparation of colorimetric reagents

[0103] Components volume ratio Ammonium heptamolybdate solution 40 concentrated sulfuric acid 32 Malachite green solution 34

[0104] Table 10 Preparation of buffer for ATPase activity assay of recombinant enzyme UvsX

[0105] Components concentration Tris-HCl (pH 8) 60mM <![CDATA[CH3COOK]]> 80mM DTT 4mM

[0106] Example 3: RPA reaction

[0107] The RPA detection system used to detect pUC 19 had its plasmid purchased from TransGold. Primer sequences and amplified nucleotide sequences are shown in Table 11 below.

[0108] The reaction volume was 50 μL. The components of all reaction buffers are shown in Table 12, and the reaction conditions are shown in Table 13. The reaction was carried out at 41 °C for 30 minutes. After mixing, the reaction was performed in 1.5 mL EP tubes in a water bath.

[0109] After the reaction was complete, an equal volume of DNA extraction phenol was added to extract nucleic acids. After centrifugation at 12000×g for 7 minutes, the supernatant was collected, and the extracted products were separated on a 2.0% (w / v) agarose gel and stained with 1×Safe Green dye. Each experiment was repeated three times in parallel.

[0110] Table 11 Primers and amplification sequences used in the experiment

[0111]

[0112] Table 12 Preparation of 2×RPA Buffer

[0113]

[0114]

[0115] Table 13 Preparation of Each Reagent for RPA Amplification Reaction

[0116] Components Volume / μL 2×RPA Buffer 25 Bsu DNA polymerase (1.5 μg / μL) 1 UvsX wild-type or mutant (3 μg / μL) 2 UvsY (1 μg / μL) 1 gp32 single-chain binding protein (4 μg / μL) 2.5 Creatine kinase (CK) (2 μg / μL, purchased from Yisheng Biotechnology) 2.5 Forward primer pUC19lacZα-2F (10 μM) 1.5 Reverse primer pUC19lacZα-2R (10 μM) 1.5 <![CDATA[Amplification template pUC19 (SEQ ID NO:1, 10 6 copies / μL)]]> 1 <![CDATA[Mg 2+ (280mM)]]> 2.5 distilled water Adjust the volume to 50 μL

[0117] In Table 13, the amino acid sequence of Bsu is as follows:

[0118] DQSLEDINVKTVTDVTSDILVSPSAFVVEQIGDNYHEEPILGFSIVNETGAYFIPKDIAVESEVFKEWVENDEQKKWVFDSKRAVVALRWQGIELKGAEFDTLLAAYIINPGNSYDDVASVAKDYGLHIVSSDESVYGKGAKRAVPSEDVLSEHLGRKALAIQSLREKLVQELENNDQLELFEELEMPLALILGEMESTGVKVDVDRLKRMGEELGAKLKEYEEKIHEIAGEPFNINSPKQLGVILFEKIGLPVVKKTKTGYSTSADVLEKLADKHDIVDYILQYRQIGKLQSTYIEGLLKVTRPDSHKVHTRFNQALTQTGRLSSTDPNLQNIPIRLEEGRKIRQAFVPSEKDWLIFAADYSQIELRVLAHISKDENLIEAFTNDMDIHTKTAMDVFHVAKDEVTSAMRRQAKAVNFGIVYGISDYGLSQNLGITRKEAGAFIDRYLESFQGVKAYMEDSVQEAKQKGYVTTLMHRRRYIPELTSRNFNIRSFAERTAMNTPIQGSAADIIKKAMIDMAAKLKEKQLKARLLLQVHDELIFEAPKEEIEILEKLVPEVMEHALALDVPLKVDFASGPSWYDAK(SEQ ID NO:62);

[0119] The amino acid sequence of UvsY is as follows:

[0120] MRLEDLQEELKKDVFIDSTKLQYEAANNVMLYSKWLNKHSSIKKEMLRIEAQKKVALKARLDYYS GRGDGDEFSMDRYEKSEMKTVLSADKDVLKVDTSLQYWGILLDFCSGALDAIKSRGFAIKHIQDMRAF EAGK(SEQID NO:63);

[0121] The amino acid sequence of the gp32 single-chain binding protein is as follows:

[0122] MFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKYRFGKKIWDKINAMIA VDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDLDAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDL(SEQ ID NO:64).

[0123] Example 4

[0124] To enhance the activity of the T4UvsX protein, key amino acid residues of the wild-type UvsX protein (SEQ ID NO:4) were mutated. This disclosure utilizes a virtual saturation mutagenesis design technique to predict key amino acid residues near the active site of the T4UvsX protein and perform virtual mutations. Twelve mutants with the lowest binding free energy were selected from a mutant library for further testing.

[0125] The 12 virtual saturation mutation design sites are: Q200R, E198R, Q200K, E198N, Y197R, M166K, E198K, G211K, M166R, A169R, E198W, and T212R. The amino acid sequences of each mutant are shown in Table 14 below, and the primers used for the mutation sites are shown in Table 15 below. The purified mutants were obtained according to the method in Example 1.

[0126] Table 14

[0127]

[0128]

[0129]

[0130] Table 15

[0131]

[0132]

[0133] The ATPase activity of each mutant was detected according to the method in Example 2, and the results are shown in Table 16.

[0134] Table 16 ATPase activity data from virtual saturation mutations

[0135]

[0136] As shown in Table 16, mutants Q200R, Q200K, E198N, and E198R have better enzyme activities than wild-type, while M166K and Y197R are comparable to wild-type.

[0137] RPA reactions were performed on each mutant according to the method in Example 3, and the results are as follows: Figure 3 As shown.

[0138] Figure 3 Agarose gel electrophoresis showed that mutants Q200R, Q200K, E198K, E198N, and E198R all had RPA activity. Among them, compared with wild type, E198K had 39% more RPA activity, E198N had 34% more RPA activity, and E198R had 26% more RPA activity.

[0139] Example 5

[0140] This embodiment employs a consensus mutation design approach, targeting conserved sequences and key functional regions of the T4UvsX protein for mutation. Homologous sequences from the UvsX protein family were collected from databases for alignment to identify highly conserved sites during evolution. These highly conserved sites are typically functionally important, and mutations can significantly impact protein function. Analysis of these conserved sites was performed, selecting the most common amino acids as mutation targets to improve the structural stability of the UvsX protein. Six candidate mutation sites with homology conservatism greater than 50% were selected for further testing.

[0141] The six consensus mutation design sites are: K35G, M39L, P214L, Q52P, T318E, and L56T. The amino acid sequences of each mutant are shown in Table 17 below, and the primers used for the mutation sites are shown in Table 18 below. Each purified mutant was obtained according to the method in Example 1.

[0142] Table 17

[0143]

[0144] Table 18

[0145]

[0146]

[0147] For each mutant, ATPase activity was detected according to the method in Example 2, and the results are shown in Table 19.

[0148] Table 19 ATPase activity data for consensus mutations

[0149]

[0150] As shown in Table 19, mutants K35G, M39L, P214L, Q52P, T318E, and L56T have better enzyme activities than wild-type mutants, especially K35G, M39L, and P214L.

[0151] RPA reactions were performed on each mutant according to the method in Example 3, and the results are as follows: Figure 4 As shown.

[0152] Figure 4 Agarose gel electrophoresis showed that mutants K35G, M39L, Q52P, and L56T all had RPA activity. Among them, K35G had 28% higher RPA activity than wild-type.

[0153] Example 6

[0154] This embodiment is used to investigate whether the combination of the two mutation strategies in Examples 4 and 5 will produce an additive, synergistic, or antagonistic effect on homologous recombination. The mutation sites that increase RPA activity were combined to obtain three double mutation sites (K35G / E198N, K35G / E198R, and K35G / E198K, with amino acid sequences shown in Table 20 below).

[0155] Table 20

[0156]

[0157]

[0158] The ATPase activity of each mutant was detected according to the method in Example 2, and the results are shown in Table 21.

[0159] Table 21 ATPase activity data for double-point mutations

[0160]

[0161] As can be seen from Table 21, the mutant K35G / E198R has superior enzyme activity compared to the wild type.

[0162] RPA reactions were performed on each mutant according to the method in Example 3, and the results are as follows: Figure 5 As shown.

[0163] according to Figure 5 The agarose gel electrophoresis results showed that mutants K35G / E198N, K35G / E198R, and K35G / E198K all possessed RPA activity. Furthermore, compared to the wild type, the RPA activity of K35G / E198R was increased by 43.4%, that of K35G / E198N by 27.6%, and that of K35G / E198K by 15.6%.

[0164] The technical solutions disclosed herein are not limited to the specific embodiments described above. Any technical modifications made based on the technical solutions disclosed herein shall fall within the protection scope of this disclosure.

Claims

1. A UvsX protein mutant, characterized in that, The amino acid sequence of the UvsX protein mutant is shown in SEQ ID NO: 41 or 60.

2. A nucleic acid molecule encoding the UvsX protein mutant of claim 1.

3. A recombinant expression vector containing the nucleic acid molecule of claim 2.

4. A host cell, characterized in that, The host cell contains the UvsX protein mutant of claim 1, the nucleic acid molecule of claim 2, or the recombinant expression vector of claim 3, and the host cell is not a plant cell.

5. A composition comprising (i) the UvsX protein mutant of claim 1, and (ii) one or more reagents selected from the group consisting of buffers, primers, probes, dyes, target nucleic acids, and cell lysis agents.

6. The composition according to claim 5, characterized in that, The composition is in the form of a kit.

7. A method for preparing the UvsX protein mutant of claim 1, wherein, The method includes the following steps: Cultivating the host cells as described in claim 4; and The UvsX protein mutant is isolated from the host cell or from the growth medium or supernatant.

8. The use of the UvsX protein mutant of claim 1, the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3, the host cell of claim 4, or the composition of claim 5 or 6 in enzyme preparation production, target gene detection for non-diagnostic purposes, or DNA recombination.

9. The application according to claim 8, characterized in that, The UvsX protein mutant was used for recombinase polymerase amplification reaction.

10. A method for generating DNA molecules, comprising the step of using the UvsX protein mutant of claim 1.

11. The method according to claim 10, characterized in that, The UvsX protein mutant was incubated with template DNA and primers.

12. The method according to claim 10, characterized in that, The method is implemented via a recombinase polymerase amplification reaction.

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