UvsX protein mutant and application thereof

Abstract

The invention provides a UvsX protein mutant, and compared with a wild type UvsX protein, the UvsX protein mutant has mutation at one or more of the 35th amino acid, the 39th amino acid, the 52th amino acid, the 56th amino acid, the 166th amino acid, the 169th amino acid, the 197th amino acid, the 198th amino acid, the 200th amino acid, the 211th amino acid, the 212th amino acid, the 214th amino acid and the 318th amino acid corresponding to SEQ ID NO: 4. Compared with a wild type UvsX protein, the UvsX protein mutant disclosed by the invention is high in yield and improved in activity.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and specifically relates to UvsX protein mutants and applications thereof. Background Art

[0002] Recombinase Polymerase Amplification (RPA) technology is widely used in the rapid detection of bacteria and viruses in different industries, including medical diagnosis, environmental monitoring, food safety and agriculture.

[0003] As an emerging nucleic acid amplification technology, RPA has been widely used in the field of molecular biology. Despite its many advantages, there are still some problems for the following exploration directions: RPA is temperature sensitive and usually reacts at 37-42°C, which may limit its application in environments outside the laboratory, especially in the absence of constant temperature equipment. Therefore, there are some problems in instant diagnosis. Compared with traditional PCR, which usually uses polymerase reaction buffer, RPA uses a specific reaction buffer system, which includes some special components (such as single-stranded binding proteins and polymerases). In addition, the operation process is relatively complicated, involving the regulation of multiple enzymes and multiple components, requiring more experimental steps, increasing the complexity of the operation and the time-consuming experiment. The cost of reagents is also relatively high, which currently limits its large-scale promotion and application.

[0004] In addition, the current high price, insufficient activity and stability of RPA enzymes limit the development of RPA technology. It is urgent to develop its heterologous expression system and performance evaluation system to obtain high-performance UvsX.

[0005] T4UvsX protein is an important recombinase, mainly used for DNA repair and recombination. This protein is widely used in genetic engineering, biocatalysis and gene editing. Although T4UvsX protein has significant application prospects in the above fields, the existing production methods face the following technical difficulties:

[0006] (1) Low protein yield: Existing recombinant protein production technologies (such as expression in E. coli) often encounter the problem of low expression, 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 has poor stability after expression. Especially under high temperature, high salt or other extreme conditions, the protein is prone to denaturation or degradation, which has a negative impact on the storage and application of the protein. In the prior art, the improvement of protein stability is mainly achieved through chemical modification or ligand binding, but these methods have limited effects and are difficult to adapt to the requirements of different environments.

[0008] (3) Insufficient protein activity: Although the activity of T4UvsX protein in DNA repair is important, the existing protein activity testing and optimization methods still have defects. Most of the 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 stably improve the performance.

[0009] Therefore, there is an urgent need in the art to develop T4UvsX proteins with better performance. Summary of the invention

[0010] In view of the deficiencies in the prior art, the present disclosure solves the problems of low yield and insufficient activity of T4UvsX protein by systematically improving the expression system, mutant screening and stability optimization technology. The present disclosure provides T4UvsX protein mutants, which can be widely used in the fields of biopharmaceuticals, enzyme engineering, genetic engineering and molecular biology research, especially in enzyme preparation production, gene repair, DNA recombination, vaccine development, etc.

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

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

[0013] In some embodiments, the UvsX protein mutant comprises a K35G / E198N mutation. In some embodiments, the UvsX protein mutant comprises a K35G / E198R mutation. In some embodiments, the UvsX protein mutant comprises a 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 a sequence identity of 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% compared to SEQ ID NO: 4, or an amino acid sequence in which one or more amino acids are added, deleted, substituted, or modified.

[0015] In some embodiments, the UvsX protein mutant has an amino acid sequence shown in one or more of SEQ ID NOs: 5-11, 41-46, 72-74.

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

[0017] Another aspect of the present disclosure provides a recombinant expression vector containing the nucleic acid molecule of the present disclosure.

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

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

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

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

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

[0023] In some embodiments, the prokaryotic cell can 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 cell can 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, etc.

[0025] Another aspect of the present disclosure provides a composition comprising (i) the UvsX protein mutant described in the present disclosure, and (ii) one or more reagents selected from the following: a buffer, a primer, a probe, a dye, a detection agent, a target nucleic acid and a cell lysis agent.

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

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

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

[0029] In some embodiments, the UvsX protein mutant is used in a recombinase polymerase amplification reaction.

[0030] Yet another aspect of the present disclosure provides a method for producing a DNA molecule, comprising the step of using the UvsX protein mutant described in the present disclosure.

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

[0032] In some embodiments, the method is performed by a recombinase polymerase amplification reaction. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0034] Figure 2 A standard curve graph for the phosphorus standard assay is shown.

[0035] Figure 3 The effects of virtual saturation mutants on the RPA response are shown.

[0036] Figure 4 The effects of the consensus mutants on the RPA response are shown.

[0037] Figure 5 The effects of the double point mutants on the RPA response are shown. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical scheme and advantages of the present disclosure more clear, the present disclosure is further described in detail below in conjunction with the accompanying drawings and embodiments. The specific embodiments described herein are only used to explain the present disclosure and are not intended to constitute any limitation to the present disclosure. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many 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 belongs. For the purpose 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 dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

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

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

[0044] The term "mutant activity test" used herein refers to testing the changes in the enzyme activity or other biological functions of the UvsX protein under different conditions after subjecting the UvsX protein to 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 by another different "replacement" 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. Although inserts typically consist of the insertion of 1 or 2 amino acid residues, larger "peptide inserts" can also be prepared, for example, inserting about 3 to 5 or even up to about 10, 15 or 20 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] Mutants disclosed herein or fragments thereof may comprise conservative amino acid substitutions at one or more amino acid residues, such as at essential or non-essential amino acid residues. "Conservative amino acid substitutions" are amino acid residues replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been 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), non-polar 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 article, the essential or non-essential amino acid residues in the mutant are preferably replaced with another amino acid residue from the same side chain family.

[0047] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide or amino acid, gaps present in the target sequence are not counted. Likewise, since target sequence nucleotides or amino acids are counted and nucleotides or amino acids from the reference sequence are not counted, gaps present in the reference sequence are not counted.

[0048] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain 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 sequence identity percentage. The comparison of the sequence and the determination of the sequence identity percentage between the two sequences can be completed using software that is easy to use and download online. Suitable software programs can be obtained from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite that can be obtained from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. Government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used for comparing nucleic acid sequences, and BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0049] In the present disclosure, a recombinant expression vector of UvsX was constructed, and high-performance RPA enzyme was obtained at a low cost by inducing expression. Then, the expression vector sequence was optimized, and high-yield engineering bacteria were constructed and screened to increase protein production. At the same time, an enzyme performance evaluation system was established to detect the inorganic phosphorus (PO4 3- ) content to evaluate the heterologous expression effect of UvsX and the activity of the target UvsX. Furthermore, point mutations were performed by semi-rational design methods such as alanine scanning, saturation mutation, and homologous protein sequence alignment to improve the activity of the enzyme. Mutants with improved activity were obtained.

[0050] Examples are provided below to help understand the present disclosure. However, it should be understood that these examples are only used to illustrate the present disclosure and do not constitute any limitation. The actual protection scope of the present disclosure is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present disclosure.

[0051] Example

[0052] Example 1: Optimizing expression vector sequences to increase protein yield

[0053] The recombinant enzyme was expressed in three different pET28a plasmids or their variants (TIR, T7 and dtHis) to construct a UvsX recombinant expression vector that helps to increase the expression of the recombinant enzyme UvsX. Among them, TIR and T7 are 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 is the wild-type pET28a.

[0054] 1. Construction of recombinant plasmid

[0055] (1) Target gene synthesis

[0056] The gene sequence encoding UvsX (SEQ ID NO: 4) was synthesized by Suzhou Jinweizhi 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 amount of T1 single colony strain carrying the pET-UvsX recombinant plasmid stored on the plate was picked and transferred to 5 mL LB liquid culture medium containing 0.1 mM kanamycin (Table 1), cultured at 37 ° C and 220 rpm for 12 h, and then centrifuged to collect the bacteria for plasmid extraction. The plasmid extraction method was operated according to the instructions of the plasmid extraction kit (Quanshijin).

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

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

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

[0062] In the clean bench, take 500 μL of LB liquid culture medium and add it to the transformation system, and culture it at 37°C and 220 rpm for 1 hour;

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

[0064] A single clone was picked and cultured in liquid LB medium at 37°C and 220 rpm for 12 h to maintain the bacteria.

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

[0066] use The Fast multi-site mutagenesis system was used to perform site-directed mutagenesis on the pET-28a(+) vector sequence. The multi-site mutagenesis system used plasmid pET-UvsX as a template and an agarose gel migration assay was used to detect whether the PCR was successful;

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

[0068] To 30 μL of purified product, add 0.6 μL of DMT enzyme and incubate at 37°C for 1 h for template nitration; 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 plate medium containing kanamycin and cultured at 37°C for 12 hours;

[0070] Pick a single clone, culture it in LB liquid medium at 37°C and 220 rpm for 12 h, and confirm whether the mutation is successful by DNA sequencing;

[0071] The successfully mutated plasmid was introduced into E. coli BL21 competent cells, and the transformation system was evenly spread on LB plate culture medium containing kanamycin, cultured at 37°C for 12 hours, and a single clone was picked and cultured in LB liquid culture medium at 37°C and 220 rpm for 12 hours to maintain the bacteria.

[0072] Table 1 Preparation of LB medium

[0073] Components LB liquid medium / g / L LB plate 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 the E. coli BL21 (DE3) strain.

[0076] Take 10 μL of the bacterial solution and inoculate it into 5 mL LB liquid medium containing 0.1 mM kanamycin and culture it overnight at 37°C and 220 rpm for 12 hours. On the second day, take 1 mL of the culture solution and transfer it into a new 80 mL LB medium containing 0.1 mM kanamycin. When the OD600 of the bacterial solution reaches 0.8, add IPTG with a final concentration of 0.05 mM and continue to culture it in an incubator at 16°C and 160 rpm for 20 hours.

[0077] Then the culture was centrifuged at 8000×g for 5 minutes at 4°C, the supernatant was discarded, and the bacterial pellet was stored in a centrifuge tube. The cells were suspended with 10mL of bacterial disruption solution and stored on ice. According to the operating instructions of the cell disruptor, the bacterial solution was poured into the cell disruptor and disrupted three times. The resulting bacterial solution was balanced with lysis solution and centrifuged at 10000×g for 60 minutes at 4°C. The pellet was discarded, the supernatant was filtered with a 0.45μm microporous filter membrane, and the protein in the supernatant was purified using a metal affinity chromatography column on a Ni-NTA column. After balancing the nickel medium with Buffer I, the sample was loaded, then washed with Buffer I containing 60mM imidazole, and then eluted with Buffer I containing 500mM imidazole.

[0078] All purification processes were run at 4°C. The eluted protein was dialyzed against storage buffer and concentrated to less than 1 mL. The buffer compositions used are shown in Tables 2 to 7 below.

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

[0080] Table 2 Preparation of lysis 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 Wash buffer Wash 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 - Make up to 1L

[0093] In this example, the recombinase was expressed in three different pET28a mutant plasmids (TIR, T7 and dtHis). Figure 2 As shown, the results show 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, the present disclosure selects TIR mutation, indicating 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] According to the RPA amplification reaction principle, UvsX recombinase, as an ATP hydrolase, needs to consume ATP as an energy source to drive the synthesis of DNA chains. As the amplification reaction proceeds, ATP is continuously converted into ADP+Pi, so the amount of ATP conversion can be used to reflect the activity of the enzyme.

[0096] To evaluate the enzyme activity, malachite green-phosphomolybdic acid spectrophotometry was used to detect the generated phosphate (PO4 3- ) content. First, the phosphate in the reaction solution reacts with ammonium molybdate, and then reacts with the color developer. The absorbance is detected by a spectrophotometer and substituted into the standard curve to calculate the total phosphorus content, thereby evaluating the ATPase activity of the enzyme.

[0097] The specific method is as follows:

[0098] At 37°C, the absorbance of the supernatant after staining with 1×SafeGreen dye was detected by Eppendorf BioPhotometer D30 UV spectrophotometer, and the total phosphorus content was substituted into the standard curve to evaluate the enzyme performance. The reaction volume for determining the standard curve was 1 mL. 80 μL of color developer (as shown in Table 9) and 40 μL of 1% polyvinyl alcohol solution (w / v) were added to 2 mL EP tubes containing the phosphorus standard concentrations as shown in Table 8, and then filled to 1 mL with distilled water. After mixing, color was developed in a water bath at 37°C for 20 minutes, and then the UV spectrophotometer was used to measure three readings at a wavelength of 620 nm to draw the standard curve, as shown in FIG. Figure 1 shown.

[0099] When determining the activity of UvsX, the enzyme activity reaction system is 50 μL, containing 3 μg UvsX, 0.3 mM ATP, 25 μL buffer (as shown in Table 10), and distilled water is added to 50 μL. After reacting in a 37°C water bath for 10 minutes, it is immediately placed on ice to terminate the reaction, and then the color developer and 1% polyvinyl alcohol solution (w / v) are added to make up to 1 mL, mixed and colored in a 37°C water bath for 20 minutes, and then the absorbance at 620 nm is measured using a UV spectrophotometer. Each parallel sample is tested three times to eliminate accidental errors. Finally, the generated phosphate content is substituted into the standard curve to evaluate its ATPase activity. Two parallel samples are set for each sample.

[0100] Table 8 Preparation of different phosphorus standard concentration systems

[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 color developer

[0103] Components Volume ratio Ammonium heptamolybdate solution 40 Concentrated sulfuric acid 32 Malachite green solution 34

[0104] Table 10 Preparation of ATPase activity assay buffer for 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, the plasmid of which was purchased from Quanshijin. The primer sequences and the amplified nucleotide sequences are shown in Table 11 below.

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

[0109] After the reaction, an equal volume of DNA extraction phenol was added to the reaction solution to extract nucleic acid. After centrifugation at 12000×g for 7 minutes, the supernatant was taken and the extracted product was separated on a 2.0% (w / v) agarose gel and stained with 1×Safe Green dye. Each group of experiments 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 Make up 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:

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

[0123] Example 4

[0124] In order to improve the activity of T4UvsX protein, the key amino acid residues of the wild type (SEQ ID NO: 4) of UvsX protein were mutated. The disclosed embodiment uses virtual saturation mutation design technology to predict the key amino acid residues near the active site of T4UvsX protein and perform virtual mutations. 12 mutants with the lowest binding mutation free energy were selected from the 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, T212R. The amino acid sequences of the mutants are shown in Table 14 below. The primers used for the mutation sites are shown in Table 15 below. The purified mutants were obtained according to the method of 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 of Example 2. The detection results are shown in Table 16.

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

[0135]

[0136] According to Table 16, it can be seen that the mutants Q200R, Q200K, E198N and E198R have better enzyme activities than the wild type, and M166K and Y197R are comparable to the wild type.

[0137] RPA reaction was performed on each mutant according to the method of Example 3. The test results are as follows Figure 3 shown.

[0138] Figure 3 Agarose gel electrophoresis showed that the mutants Q200R, Q200K, E198K, E198N and E198R all had RPA activity. Compared with the wild type, the RPA activity of E198K was increased by 39%, the RPA activity of E198N was increased by 34%, and the RPA activity of E198R was increased by 26%.

[0139] Example 5

[0140] This example uses a consensus mutation design method to mutate the conserved sequences and important functional regions of the T4UvsX protein. Homologous sequences of the UvsX protein family were collected from the database for comparison to identify sites that are highly conserved during evolution. Usually, highly conserved sites are important in function, and mutations may significantly affect protein function. These conserved sites were analyzed, and the most common amino acids were selected as mutation targets to improve the structural stability of the UvsX protein. Six candidate mutation sites with a homology comparison conservation greater than 50% were selected for further testing.

[0141] The 6 consensus mutation design sites are: K35G, M39L, P214L, Q52P, T318E, L56T. The amino acid sequences of the mutants are shown in Table 17 below. The primers used for the mutation sites are shown in Table 18 below. The purified mutants were obtained according to the method of Example 1.

[0142] Table 17

[0143]

[0144] Table 18

[0145]

[0146]

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

[0148] Table 19 ATPase activity data of consensus mutations

[0149]

[0150] As can be seen from Table 19, the mutants K35G, M39L, P214L, Q52P, T318E, and L56T have better enzyme activities than the wild type, especially K35G, M39L, and P214L.

[0151] RPA reaction was performed on each mutant according to the method of Example 3. The test results are as follows Figure 4 shown.

[0152] Figure 4 Agarose gel electrophoresis showed that mutants K35G, M39L, Q52P, and L56T all had RPA activity, among which the RPA activity of K35G was increased by 28% compared with the wild type.

[0153] Example 6

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

[0155] Table 20

[0156]

[0157]

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

[0159] Table 21 ATPase activity data of double point mutations

[0160]

[0161] It can be seen from Table 21 that the mutant K35G / E198R has better enzyme activity than the wild type.

[0162] RPA reaction was performed on each mutant according to the method of Example 3. The test results are as follows Figure 5 shown.

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

[0164] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments, and all technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. A UvsX protein mutant, characterized in that: Relative to the wild-type UvsX protein, the UvsX protein mutant has a mutation at one or more of the amino acids 35, 39, 52, 56, 166, 197, 198, 200, 214, and 318 corresponding to SEQ ID NO:

4.

2. The UvsX protein mutant according to claim 1, characterized in that The UvsX protein mutants include one or more mutations of K35G, M39L, Q52P, L56T, M166K, Y197R, E198N, E198R, E198K, Q200R, Q200K, P214L, T318E; Preferably, the UvsX protein mutants include Q200R, Q200K, E198K, E198N, E198R, K35G, M39L, Q52P, L56T, K35G / E198N mutation, K35G / E198R mutation or K35G / E198K mutation; Preferably, 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% sequence identity compared to SEQ ID NO: 4, or an amino acid sequence shown in SEQ ID NO: 4 in which one or more amino acids are added, deleted, substituted or modified; Preferably, the UvsX protein mutant has an amino acid sequence shown in one or more of SEQ ID NOs: 5-11, 41-46, 72-74.

3. A nucleic acid molecule encoding the UvsX protein mutant according to claim 1 or 2.

4. A recombinant expression vector containing the nucleic acid molecule according to claim 3.

5. The recombinant expression vector according to claim 4, characterized in that: The recombinant expression vector comprises a plasmid having a TIR mutation; Preferably, the recombinant expression vector comprises a pET plasmid having a TIR mutation.

6. A host cell, characterized in that The host cell contains the UvsX protein mutant according to claim 1 or 2, the nucleic acid molecule according to claim 3, or the recombinant expression vector according to claim 4 or 5.

7. A composition comprising (i) the UvsX protein mutant of claim 1 or 2, and (ii) one or more reagents selected from the group consisting of a buffer, a primer, a probe, a dye, a detection agent, a target nucleic acid, and a cell lysing agent; Preferably, the composition is in the form of a kit.

8. A method for preparing the UvsX protein mutant according to claim 1 or 2, wherein: The method comprises the following steps: Cultivating the host cell according to claim 6; and Optionally, the UvsX protein mutant is isolated from the host cell or from the growth medium or supernatant.

9. Use of the UvsX protein mutant according to claim 1 or 2, the nucleic acid molecule according to claim 3, the recombinant expression vector according to claim 4 or 5, the host cell according to claim 6 or the composition according to claim 7 in enzyme preparation production, gene repair, target gene detection, DNA recombination or vaccine preparation; Preferably, the UvsX protein mutant is used for recombinase polymerase amplification reaction.

10. A method for producing a DNA molecule, comprising the step of using the UvsX protein mutant according to claim 1 or 2; Preferably, the UvsX protein mutant is incubated with template DNA and primers; Preferably, the method is performed by a recombinase polymerase amplification reaction.

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