Restriction endonuclease mutant as well as preparation method and application thereof

Abstract

The invention discloses a restriction enzyme Eco31I mutant as well as a preparation method and application thereof, and relates to the technical field of biology. The restriction enzyme Eco31I mutant provided by the invention can be efficiently expressed in a methylation host, has excellent enzyme digestion efficiency, can be applied to linearization of large fragments, and has the advantages of short cutting time, few byproducts and the like.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present disclosure claims priority to a Chinese patent application with application number 202311519732.7 filed with the Chinese Patent Office on November 14, 2023, entitled “Restriction endonuclease mutants, preparation methods and applications thereof”, the entire contents of which are incorporated by reference into the present disclosure. Technical Field

[0003] The invention relates to the field of biotechnology, and in particular to an Eco31I restriction endonuclease mutant and a preparation method and application thereof. Background Art

[0004] Restriction endonucleases are a type of enzyme that can recognize specific nucleotide sequences in double-stranded DNA molecules and cut the phosphodiester bonds in the DNA chain at specific locations. They are referred to as restriction enzymes. Different restriction endonucleases recognize different DNA sequences. They can cut DNA within the recognition sequence or at a position adjacent to the recognition sequence. After enzyme cutting, different types of products will be formed, such as blunt-end or sticky-end products.

[0005] As we all know, restriction endonucleases are most commonly used in molecular cloning experiments. But in addition to cloning, it can also be used in vaccine research and development, gene sequencing, SNP identification, ddPCR and other fields. According to the complexity of the structure, the mode of action and the difference in cofactors, restriction endonucleases can be roughly divided into four categories: type I enzymes, type II enzymes, type III enzymes and type IV enzymes. Among them, the characteristic of type II restriction endonucleases is that they only have the function of recognition and cutting, and have no modification activity; the recognition sequence is mostly a short palindromic sequence (generally 4-8bp), which cuts the site within or adjacent to the recognition sequence to produce a DNA product with a 3' hydroxyl and 5' phosphate group, and requires Mg for action. 2+ , are the most commonly used type of restriction enzymes in molecular cloning.

[0006] Type II restriction endonucleases are further subdivided into subcategories such as IIP, IIA, IIB, IIC, and IIS. Eco31I restriction endonuclease belongs to type IIS. It is an isoschizomer with BsaI, Bso31I, and BspTNI, and they are all widely used restriction endonucleases. Eco31I can specifically recognize a six-base nucleic acid sequence (5'GGTCTCN^NNNN^3') site. An outstanding feature of this type of enzyme is that it has no requirements for the sequence of the cleavage site. The seamless cloning technology derived from its characteristics can seamlessly assemble multiple DNA fragments into a compatible vector in any order. In addition, Eco31I has important application value not only in molecular cloning, but also in other directions in the biological field, such as the preparation of in vitro transcription templates for mRNA vaccines.

[0007] Different from the preparation process of other types of enzymes, when a single recombinant expression restriction enzyme is expressed in a prokaryotic expression system, since the host's own DNA will not be modified by the methylase, the restriction enzyme produced cuts the host DNA, causing the death of the host cell. In response to this phenomenon, relevant scientists introduced methyltransferase genes in the prokaryotic expression system. The specific modification of the methyltransferase on the recognition sequence can prevent the cutting of the restriction enzyme. The restriction enzyme-methyltransferase gene is often closely linked. Bacteria regulate the expression of both, so that their own DNA is first protected by methylation, and then the restriction enzyme is expressed after the protection is completed, while the invading heterologous DNA is not protected and is degraded by the restriction enzyme. That is, the intracellular methyltransferase-related methyltransferase methylates the same specific sequence recognized by its paired restriction enzyme, and makes the modified DNA have the characteristics of resisting the cutting of its restriction enzyme, protecting the host DNA and degrading the unmethylated foreign DNA.

[0008] However, the regulation of restriction enzymes and methyltransferases by bacteria is often unbalanced. This imbalance often leads to low or even no expression of wild-type Eco31I, resulting in cumbersome separation and purification procedures, low protein yields, and other problems. As a result, the current commercially available Eco31I restriction enzyme is not only expensive, but also limits its wide application and in-depth research. At the same time, the above-mentioned series of problems also indirectly affect the enzyme activity of Eco31I, making the enzyme cleavage rate of wild-type Eco31I slow, often requiring about 1 hour to completely cleave 1μg of plasmid. This long-term enzyme cleavage is often prone to enzyme star activity, leading to the introduction of undesirable cleavage results.

[0009] Therefore, it is of great significance to provide an Eco31I restriction endonuclease with high expression level, simple separation and purification procedure, high protein yield, low price and good enzyme cutting efficiency and a preparation method thereof.

[0010] In view of this, the present invention is proposed. Summary of the invention

[0011] The object of the present invention is to provide an Eco31I restriction endonuclease mutant and a preparation method and application thereof.

[0012] In order to achieve the above object, according to a first aspect of the present invention, a method for identifying and cutting DNA is provided, the method comprising the following steps:

[0013] (a) adding a DNA fragment and an Eco31I restriction endonuclease to a cleavage system to produce a reaction mixture, wherein the Eco31I restriction endonuclease comprises an amino acid substitution at position 283 corresponding to SEQ ID NO: 1 or at other Eco31I restriction endonuclease equivalent sites; and

[0014] (b) incubating the reaction mixture under conditions suitable for cleavage of the DNA fragments to produce DNA cleavage products of the fragments.

[0015] In order to achieve the above object, according to a second aspect of the present invention, a method for preparing Eco31I restriction endonuclease is provided, the method comprising the following steps:

[0016] (a) selecting an Eco31I restriction endonuclease in which an amino acid at position 283 of SEQ ID NO: 1 or other Eco31I restriction endonuclease equivalent sites is substituted for purification; and

[0017] (b) Obtain the above-mentioned Eco31I restriction endonuclease.

[0018] In order to achieve the above object, according to the third aspect of the present invention, an Eco31I restriction endonuclease is provided, wherein the Eco31I restriction endonuclease comprises an amino acid substitution at position 283 of SEQ ID NO: 1 or at other Eco31I restriction endonuclease equivalent sites.

[0019] In order to achieve the above object, according to the fourth aspect of the present invention, a conjugate is provided, which contains the above Eco31I restriction endonuclease and a marker for purification.

[0020] In order to achieve the above object, according to the fifth aspect of the present invention, an isolated nucleic acid is provided, which encodes the amino acid of the above Eco31I restriction endonuclease.

[0021] In order to achieve the above object, according to the sixth aspect of the present invention, a recombinant vector is provided, which contains the above isolated nucleic acid.

[0022] In order to achieve the above object, according to the seventh aspect of the present invention, a host cell is provided, which contains the above vector.

[0023] In order to achieve the above object, according to the eighth aspect of the present invention, a kit is provided, which comprises the above-mentioned Eco31I restriction endonuclease.

[0024] In order to achieve the above object, according to the ninth aspect of the present invention, there is provided the use of the above Eco31I restriction endonuclease in DNA cleavage.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The optimized Eco31I restriction endonuclease mutant of the present invention overcomes the problem that the conventional Eco31I restriction endonuclease cannot be expressed in a methylated host, can be efficiently expressed in a methylated host, has excellent enzyme cutting efficiency, can be applied to the linearization of large fragments, and has the advantages of short cutting time, few by-products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 A schematic diagram of the ion exchange chromatography purification results of the Eco31IM1 sample purified in Example 1 of the present application is shown, wherein the lane before desalting is the combined sample after elution by the Ni affinity chromatography column, the lane after desalting is the combined sample after the desalting step by the gel filtration chromatography column, the loading lane is the combined sample before purification by the ion exchange chromatography column, lanes 1-2 are the impurity protein samples after linear elution using a high salt buffer, and lanes 3-8 are the mutant Eco31IM1 target proteins after linear elution using a high salt buffer;

[0029] Figure 2 The electrophoretic diagram of the enzyme activity assay after gradient dilution of the purified Eco31IM1 and the standard in Example 2 of the present application is shown;

[0030] Figure 3 The electrophoresis diagram of the enzyme cleavage efficiency determination after gradient dilution of the purified Eco31IM1 and the standard in Example 3 of the present application is shown, M represents the Marker band, Yin represents the negative control of the restriction endonuclease, lanes 1-10 are the enzyme cleavage results after 5 minutes of cutting with different gradients of wild type (standard) and mutant Eco31I (Eco31IM1), and lanes 11-20 are the enzyme cleavage results after 15 minutes of cutting with different gradients of wild type and mutant Eco31I. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0032] As mentioned in the background technology, although the existing commercial restriction endonuclease Eco31I also has a certain cutting efficiency, the imbalance of the protein expression host on the regulation of restriction enzymes and methyltransferases leads to low or no expression of the commercial enzyme Eco31I, high cost and selling price, and a long cutting time, and poor practical application effect. Therefore, the present application intends to protect an Eco31I restriction endonuclease with high cutting efficiency and suitable for industrial production.

[0033] The inventors of the present application analyzed the function, protein structure and key sites of the Eco31I restriction endonuclease sequence, and designed a large number of mutations for stability modification, and finally obtained the following Eco31I restriction endonuclease mutant that is efficiently expressed in a methylated host. The restriction enzyme mutant can complete fragment cleavage within 5 minutes, can be applied to the linearization of large fragments, and has the advantages of short cleavage time and few by-products.

[0034] In a first typical embodiment of the present invention, a method for identifying and cutting DNA is provided, the method comprising the following steps: (a) adding a DNA fragment and an Eco31I restriction endonuclease to a cutting system to produce a reaction mixture, wherein the Eco31I restriction endonuclease comprises an amino acid substitution at position 283 corresponding to SEQ ID NO: 1 or other Eco31I restriction endonuclease equivalent sites; and (b) incubating the reaction mixture under conditions suitable for DNA fragment cutting to produce a DNA cutting product of the fragment.

[0035]

[0036] In one or more preferred embodiments, the DNA fragment is a double-stranded nucleic acid. Any double-stranded nucleic acid containing DNA is applicable to the present application.

[0037] In one or more preferred embodiments, the double-stranded nucleic acid comprises: linear double-stranded DNA or circular double-stranded DNA.

[0038] In one or more preferred embodiments, at least one strand of the double-stranded nucleic acid is cleaved during the cleavage process to form a DNA cleavage product.

[0039] In one or more preferred embodiments, the DNA cleavage product contains a nick at at least one end.

[0040] In order to maintain the activity of restriction endonuclease Eco31I and provide a suitable environment for its reaction, in one or more preferred embodiments, the reaction mixture comprises a cutting system, a DNA fragment and Eco31I restriction endonuclease.

[0041] In one or more preferred embodiments, the cleavage system comprises Tris-HCl, Mg 2+ 、Na + , K + , serum protein, EDTA, DTT, and glycerol.

[0042] The Eco31I restriction endonuclease of the present application has good cleavage activity. To further provide conditions for a suitable cleavage reaction, in one or more preferred embodiments, the conditions suitable for DNA fragment cleavage include: reaction temperature and reaction time;

[0043] In one or more preferred embodiments, the reaction temperature of the cutting is 25-65°C; preferably, the reaction temperature is 25-40°C; further preferably, the reaction temperature is 37°C.

[0044] In one or more preferred embodiments, the reaction temperature of cleavage can be 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C.

[0045] In one or more preferred embodiments, the reaction time of cutting is 5-720 min; preferably, the reaction time is 5-15 min; further preferably, the reaction time is 5 min.

[0046] In one or more preferred embodiments, the reaction time of cutting can be 5 min, 15 min, 30 min, 60 min, 120 min, 180 min, 360 min, or 720 min.

[0047] In a second typical embodiment of the present invention, a method for preparing an Eco31I restriction endonuclease is provided, the method comprising the following steps: (a) selecting an Eco31I restriction endonuclease in which an amino acid substitution occurs at position 283 of SEQ ID NO:1 or at other Eco31I restriction endonuclease equivalent sites for purification; and (b) obtaining the above-mentioned Eco31I restriction endonuclease. Wherein, due to the characteristics of the restriction endonuclease itself, it will cut the unmodified host DNA, causing the death of the host cell. Therefore, any protein preparation method that can prevent the restriction endonuclease from cutting the host DNA when expressed is applicable to the present application.

[0048] In one or more preferred embodiments, the purification step further comprises: (1) constructing a recombinant expression plasmid of the methyltransferase; (2) transferring the plasmid obtained in step (1) into a non-methylation protection strain to obtain an Eco31I specific protection strain; (3) constructing an expression vector of a gene corresponding to the Eco31I restriction endonuclease as described in the second aspect, transforming the specific protection strain obtained in step (2) to obtain a mutant restriction endonuclease Eco31I recombinant strain; (4) transferring the mutant restriction endonuclease obtained in step (3) into a non-methylation protection strain; (5) transferring the plasmid obtained in step (1) into a non-methylation protection strain to obtain an Eco31I specific protection strain; (6) transferring the plasmid obtained in step (1) into a non-methylation protection strain to obtain an Eco31I specific protection strain; (7) transferring the plasmid obtained in step (1) into a non-methylation protection strain to obtain an Eco31I specific protection strain; (8) transferring the plasmid obtained in step (1) into a non-methylation protection strain to obtain an Eco31I specific protection strain; (9) transferring the plasmid obtained in step (1) into a non-methylation protection strain to obtain an Eco31I specific protection strain; (10) Inducing and culturing the recombinant strain of restriction endonuclease Eco31I; (5) collecting the cells of the mutant Eco31I recombinant strain induced and cultured in step (4), and lysing them at 3-6°C and 60-90MPa to obtain a lysate; (6) centrifuging the lysate obtained in step (5) at 5000-8000rpm for 3-5min, collecting the supernatant to obtain a crude product; (7) purifying the crude product obtained in step (6) by a Ni affinity chromatography column and an ion exchange chromatography column in sequence to obtain a mutant restriction endonuclease Eco31I.

[0049] In one or more preferred embodiments, the amino acid sequence of the methyltransferase in the above step (1) is as shown in SEQ ID NO:2; preferably, the nucleotide sequence of the methyltransferase in the above step (1) is as shown in SEQ ID NO:3.

[0050]

[0051]

[0052] In one or more preferred embodiments, the method for constructing the recombinant expression plasmid in step (1) above includes obtaining the methyltransferase gene, seamlessly cloning the methyltransferase gene fragment into the expression vector, and obtaining the recombinant expression plasmid of the methyltransferase.

[0053] In one or more preferred embodiments, the expression vector is pK1.

[0054] In one or more preferred embodiments, the method for obtaining the methyltransferase gene includes any one or a combination of at least two of PCR amplification, gene synthesis or DNA library construction.

[0055] In one or more preferred embodiments, the method for screening protective strains in the above step (2) includes the following steps: transforming the plasmid pK1-BsmAIM into competent cells BL21 (DE3), extracting the pK1-BsmAIM plasmid after cultivation and digesting it with restriction endonuclease Eco31I, and screening methylation protective strains so that the pK1-BsmAIM plasmid is completely protected by methyltransferase in the bacteria.

[0056] In one or more preferred embodiments, the construction of the expression vector in the above step (3) includes the following steps: histidine-tagged mutant restriction endonuclease Eco31I gene fragment is connected to the expression vector.

[0057] In the present invention, the mutant restriction endonuclease Eco31I gene fragment can be obtained by gene synthesis, or by performing point mutations one by one on a vector containing the wild-type restriction endonuclease Eco31I gene fragment.

[0058] In one or more preferred embodiments, the expression vector comprises pCold1.

[0059] In one or more preferred embodiments, the induction culture conditions in the above step (4) are as follows: inoculating the mutant restriction endonuclease Eco31I recombinant strain in step (3) into LB medium, 37°C, culture speed 180-250rpm, culture to OD600 0.45-0.75, then adding the inducer, 15°C, 130rpm, and culturing for 6-10h.

[0060] In one or more preferred embodiments, the culture rotation speed is 180-250 rpm, for example, it can be 180 rpm, 190 rpm, 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm or 250 rpm, preferably 220 rpm.

[0061] In one or more preferred embodiments, the culture is performed to an OD600 of 0.45-0.75, for example, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7 or 0.75, preferably 0.7.

[0062] In one or more preferred embodiments, the culture is for 6-10 h, for example, 6 h, 7 h, 8 h, 9 h or 10 h, preferably 8 h.

[0063] In one or more preferred embodiments, the inducer includes isopropyl-β-D-thiogalactoside (IPTG); preferably, the final concentration of the inducer is 0.15-0.3 mM; further preferably, the final concentration of the inducer is 0.25 mM.

[0064] In one or more preferred embodiments, the lysis in the above step (5) includes the following steps: resuspending the above bacteria with a lysis buffer to obtain a resuspension, wherein the ratio of the above lysis buffer to the bacteria is 10:1, collecting the supernatant to obtain the above lysate. The above lysis buffer contains Tris-HCl with a final concentration of 15 to 25 mmol / L, EDTA with a final concentration of 0.05 to 0.15 mmol / L, DTT with a final concentration of 0.5 to 1.5 mmol / L, 10% Glycerol and NaCl with a final concentration of 300 to 500 mmol / L, the working pH is 7.5±0.1, and the working temperature is 25°C.

[0065] In one or more preferred embodiments, the operation of the Ni affinity chromatography column in the above step (7) includes: adding the above crude product to the above Ni affinity chromatography column, washing the Ni affinity chromatography column with an imidazole eluent with a concentration gradient, and collecting the eluate containing the mutant Eco31I.

[0066] In one or more preferred embodiments, the eluent comprises final concentrations of 10-25 mmol / L Tris, 300-500 mmol / L NaCl and 0.5-1.5 mmol / L imidazole, a working pH of 7.5±0.1, and a working temperature of 25°C.

[0067] In one or more preferred embodiments, the operation of the ion exchange chromatography column in the above step (7) includes: an anion exchange chromatography column operation and a cation exchange chromatography column operation.

[0068] In one or more preferred embodiments, the operation of the anion exchange chromatography column includes: diluting the eluate of the mutant restriction endonuclease Eco31I after Ni affinity chromatography with a low-salt buffer, adding it to the anion exchange chromatography column, washing the anion exchange chromatography column with a concentration gradient NaCl solution, and collecting the eluate containing the mutant restriction endonuclease Eco31I.

[0069] In one or more preferred embodiments, the low-salt buffer comprises a final concentration of 10-25 mmol / L Tris, 1-5 mmol / L DTT, 0.1-0.5 mmol / L EDTA and 25-50 mmol / L NaCl, a working pH of 7.0±0.1, and a working temperature of 25°C.

[0070] In one or more preferred embodiments, the anion exchange chromatography column comprises a Q anion exchange chromatography column.

[0071] In one or more preferred embodiments, the particle size of the Q anion exchange chromatography column is 20-90 μm.

[0072] In one or more preferred embodiments, the operation of the above-mentioned cation exchange chromatography column includes: desalting the mutant restriction endonuclease Eco31I that has passed through the anion exchange chromatography column through a gel filtration chromatography column, using 3-4 times the column bed volume of high salt buffer and low salt buffer as equilibration buffers, respectively, after equilibrating the above-mentioned cation exchange chromatography column, adding the sample to the cation exchange chromatography column, and then using 5 to 10 times the column bed volume of elution buffer for linear elution, and collecting the eluate containing the mutant Eco31I.

[0073] In one or more preferred embodiments, the high salt buffer comprises a final concentration of 10-25 mmol / L Tris, 1-5 mmol / L DTT, 0.1-0.5 mmol / L EDTA and 0.5-1 mol / L NaCl, a working pH of 7.0±0.1, and a working temperature of 25°C.

[0074] In a third typical embodiment of the present invention, an Eco31I restriction endonuclease is provided, wherein the Eco31I restriction endonuclease comprises an amino acid substitution at position 283 of SEQ ID NO: 1 or at other Eco31I restriction endonuclease equivalent sites.

[0075] In one or more preferred embodiments, the amino acid at position 283 of SEQ ID NO: 1 or other Eco31I restriction endonuclease equivalent sites is substituted with Q283-.

[0076] In the present invention, a mutant restriction endonuclease Eco31I is obtained by performing a deletion point mutation on the 283rd amino acid in the amino acid sequence of the wild-type restriction endonuclease Eco31I, thereby increasing its expression level, thereby reducing the difficulty of purification, increasing the yield, and further increasing the cutting efficiency of the restriction enzyme, which can be applied to the linearization of large fragments.

[0077] In the present invention, an equivalent site refers to a position with similar functions and activities in two enzymes. Generally, a target protein spatial structure model is first constructed based on the amino acid sequence of the enzyme, and the spatial structures of the two enzymes are rotated so that their positions overlap as much as possible. Two amino acids in the same spatial position after the two enzymes are spatially compounded to a certain overlapping distance standard are called equivalent sites.

[0078] In one or more preferred embodiments, the Eco31I restriction endonuclease has a changed cleavage efficiency or a changed expression level relative to the Eco31I restriction endonuclease shown in SEQ ID NO: 1.

[0079] In one or more preferred embodiments, the Eco31I restriction endonuclease further comprises an amino acid substitution at any one or more of the following positions: at position 138, 181, 237, 240, 255, 256, 259, 262, 264, 271, 274, 278, 311, 326, 333, 334, 337, 340, 345, 361, 372, 457, 459, 460, 472 or 475 of SEQ ID NO: 1; further preferably, the Eco31I restriction endonuclease further comprises a combination of any one or more of the following mutations: Y138F, Y181F, H237A, N240A, N255A, L256P, Y259A, D262A, R264D, D271A, W274C, D278A, D311A, F326A, C333S, N334D, K337A, R340A, D345A, S361A, S361P, K372A, R457A, I459V, R460A, K472E, or R475A.

[0080] In the present invention, higher cleavage activity can be obtained after N334D mutation, incision activity is added after R264D, K337A or R340A mutation, nicking activity is added after D311A or D345A mutation, strong cleavage activity is still obtained after Y138F, Y181F, L256P, W274C, C333S, S361P, I459V or K472E mutation, and there is almost no change in cleavage activity after H237A, N240A, N255A, Y259A, D262A, D271A, D278A, F326A, S361A, K372A, R457A, R460A or R475A mutation.

[0081] In one or more preferred embodiments, the above-mentioned Eco31I restriction endonuclease also includes any one of the following mutation combinations: (a) K82R and V270M; (b) Y138F and L256P; (c) Y181F and V260D; (d) I214T and Y259C; (e) F326S and L350F; (f) T9A, C333S and V419L; (g) D278G, I289V and F363L; (h) S19P, K82R, V270M and N334D.

[0082] In the present invention, after the above combined mutation, the mutant still has strong cleavage activity.

[0083] The present invention also includes a mutant Eco31I restriction endonuclease having one or more of the above-mentioned amino acid point substitutions, wherein the remainder of the Eco31I restriction endonuclease is at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to the wild-type Eco31I restriction endonuclease; preferably, the above-mentioned wild-type Eco31I restriction endonuclease sequence is SEQ ID NO: 1.

[0084] In a fourth typical embodiment of the present invention, a conjugate is provided, which contains the Eco31I restriction endonuclease mutant described in any of the above embodiments and a marker for purification.

[0085] In one or more preferred embodiments, the markers used for purification include: purification tags, including but not limited to His tag (HIS-Tag), glutathione sulfhydryl transferase tag (GST-Tag), maltose binding protein tag (MBP-Tag), transcription termination / anti-termination protein tag (NusA-Tag), and small ubiquitin-related modifier (SUMO).

[0086] A fifth typical embodiment of the present invention provides an isolated nucleic acid encoding the Eco31I restriction endonuclease described in any of the preceding embodiments. Each host cell for protein expression has a certain preference for codons during the translation process. In order to obtain a protein product with a higher expression level in a specific host cell, the nucleotide sequence encoding the protein can be codon optimized.

[0087] Nucleic acids are typically RNA or DNA, and nucleic acid molecules can be single-stranded or double-stranded. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence. DNA nucleic acids are used when they are linked to a vector.

[0088] In a sixth typical embodiment of the present invention, a recombinant vector is provided, which contains the isolated nucleic acid described in any of the above embodiments.

[0089] In one or more preferred embodiments, the above-mentioned vectors contain the necessary elements for transcription and translation of the inserted coding sequences.

[0090] In a seventh typical embodiment of the present invention, a host cell is provided, which contains the vector described in any of the above embodiments.

[0091] In one or more preferred embodiments, the host cell is a prokaryotic cell, preferably Escherichia coli, and more preferably a competent cell BL21 (DE3).

[0092] Based on the disclosure of the amino acid sequence of the Eco31I restriction endonuclease mutant in the present application, it is easy for those skilled in the art to conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the Eco31I restriction endonuclease mutant, for example, separating and purifying the Eco31I restriction endonuclease mutant from the culture product of a recombinant cell that can recombinantly express the Eco31I restriction endonuclease mutant as described in any of the above embodiments. This is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the Eco31I restriction endonuclease mutant disclosed in the present invention, it belongs to the protection scope of the present disclosure.

[0093] In an eighth typical embodiment of the present invention, a kit is provided, comprising the Eco31I restriction endonuclease described in any of the aforementioned embodiments.

[0094] The above-mentioned kit includes any form that can carry the above-mentioned Eco31I restriction endonuclease, which can be but is not limited to a reagent tube, a reagent kit, a reagent strip, a reagent bottle, etc.

[0095] In one or more preferred embodiments, the kit further comprises: at least one of a buffer solution, a preservative solution and a diluent.

[0096] In a ninth typical embodiment of the present invention, there is provided use of the Eco31I restriction endonuclease described in any of the above embodiments in DNA cleavage.

[0097] In one or more preferred embodiments, the applications of DNA cutting include: molecular cloning, vaccine research and development and production, gene sequencing, SNP identification, ddPCR, etc.

[0098] The features and performance of the present invention are further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0099] Example 1 Construction of Eco31I restriction endonuclease mutant Eco31IM1

[0100] 1.1 Obtaining methylation-protected strains

[0101] 1.1.1. Synthesis of methyltransferase BsmAIM gene

[0102] The amino acid sequence and nucleotide sequence (SEQ ID NO: 2 and SEQ ID NO: 3) of the BsmAIM gene were obtained through UniProt and NCBI websites. After codon optimization based on Escherichia coli, the nucleotide sequence was sent to a gene company for synthesis to obtain a methyltransferase BsmAIM amplification template.

[0103] 1.1.2 Construction of recombinant expression strain of methyltransferase

[0104] The synthesized fragment was used as a PCR template, and the corresponding NdeI and EcoRI restriction site sequences and the 5' end sequence or 3' end sequence of the gene were added to the primer sequence of PCR amplification. The target sequence was amplified using Q5 high-fidelity polymerase (NEB, item number M0491L). After the amplification, agarose gel electrophoresis and gel cutting recovery were performed to obtain a BsmAIM gene fragment consistent with the theoretical value. The vector pK1 was double-digested with EcoRI and NdeI (NEB, item numbers R3101V and R0111S, respectively), the enzyme digestion products were subjected to agarose gel electrophoresis, and the linearized vector fragment was recovered by gel cutting. The target gene BsmAIM was connected to the vector pK1 according to the instructions using a seamless cloning kit (Biyuntian, item number D7010M), and the connected recombinant plasmid was transformed into competent cells BL21 (DE3), and the cells were cultured under the corresponding antibiotic selection conditions to obtain the recombinant expression strain of BsmAIM. The correctness of the sequence was confirmed by sequencing.

[0105] 1.1.3 Screening of methylation-protected strains

[0106] The correct recombinant expression strain was cultured in a medium containing the corresponding antibiotics. After the bacterial solution became turbid after about 16 hours of culture, the bacterial solution was collected to extract the plasmid. In a 25 μL reaction system, 1 μL of Eco31I restriction endonuclease (Thermo, catalog number ER0292) was added to 500 ng of the recombinant expression plasmid and incubated at 37°C for 1 hour. Then, agarose gel electrophoresis was used to detect the digestion of the substrate to confirm that the recombinant expression plasmid could completely resist the enzyme digestion of Eco31I. The host bacterial genomic DNA was also protected by the BsmAIM methyltransferase expressed by the recombinant plasmid by default. The strain that could completely resist the enzyme digestion of Eco31I was prepared into competent cells and stored for standby use, named (pK1-BsmAIM-BL21(DE3)).

[0107] Among them, the 25 μL reaction system for screening methylation recombinant expression plasmids is shown in the following table:

[0108]

[0109]

[0110] 1.2 Expression and purification of mutant Eco31IM1

[0111] 1.2.1 Molecular cloning protocol

[0112] 1.2.1.1 Gene synthesis

[0113] The wild-type Eco31I restriction endonuclease sequence (SEQ ID NO: 1) was obtained from NCBI, and after codon optimization based on Escherichia coli, it was sent to a gene company for synthesis, and a 6His tag was added to obtain a wild-type restriction endonuclease Eco31I amplification template.

[0114] 1.2.1.2 Construction of mutant Eco31IM1

[0115] Using the wild-type restriction endonuclease Eco31I gene as a template, primers were designed to construct mutant Eco31I plasmids. The primer sequences are shown in SEQ ID NO: 4-7. First, F and WR and WF and R were used to amplify the Eco31I gene respectively. After gel cutting and recovery, F and R were used to amplify the mutant template by fusion PCR, and electrophoresis gel cutting and recovery were also performed. The expression vector pCold I was double-digested with endonucleases NdeI and KpnI (NEB, item numbers R0111S and R3142V, respectively), and the mutant template was seamlessly connected to the restriction vector pCold using a seamless cloning kit (Biyuntian, item number D7010M) after electrophoresis recovery. The connection product was transformed into a methylation competent cell (pK1-BsmAIM-BL21 (DE3)), and was coated on a plate containing chloramphenicol antibiotics for screening and culture to obtain a recombinant expression strain of the mutant restriction enzyme Eco31IM1 (SEQ ID NO: 8).

[0116]

[0117]

[0118]

[0119] 1.2.2 Induced expression of strains

[0120] The recombinant strain obtained above was cultured in three steps: activation and secondary activation, fermentation, and cooling induction.

[0121] Activation: Add 100 μL of glycerol bacteria to 3 ml of LB medium containing resistance, activation conditions: 37°C, 250 rpm, 4-6 h; take 10 μL of activated bacterial solution and add it to 500 mL of LB liquid medium, prepare a bottle of seeds, add the corresponding resistance antibiotics, shake evenly, and place it in a shaker (37°C, 130 rpm) for overnight culture.

[0122] Fermentation: Take 5 mL of the secondary activated bacterial solution and add it to 500 mL of the culture medium. Incubate on a shaker at (37°C, 220 rpm) for 3-4 hours.

[0123] Cooling induction: 15°C, 130rpm, 6-10h, 0.15-0.30mM IPTG for induction.

[0124] Collect bacteria: Use a 500mL centrifuge cup, 5000-8000rpm, 3-5min, 4℃ to collect bacteria.

[0125] 1.2.3 Purification of target protein

[0126] The obtained bacteria were lysed, subjected to Ni affinity chromatography column and ion exchange chromatography column to obtain mutant restriction endonuclease Eco31IM1.

[0127] Lysis: resuspend the cells with lysis buffer, lyse at 3-6°C and 600 bar to obtain a resuspension. The ratio of lysis buffer to cells is 10:1, and the supernatant is collected to obtain the lysate.

[0128] The lysis buffer contains Tris-HCl with a final concentration of 40 to 50 mmol / L, EDTA with a final concentration of 0.05 to 0.15 mmol / L, DTT with a final concentration of 0.5 to 1.5 mmol / L, 10% Glycerol, and NaCl with a working pH of 7.5 ± 0.1, and a working temperature of 25 ° C. Specifically, the lysis buffer is added to the bacteria, and a stirrer is placed on a magnetic stirrer and stirred for 15 to 25 minutes to resuspend the bacteria. The obtained resuspension is homogenized and crushed, and the supernatant is collected by solid-liquid separation to obtain the lysate. The mixed solution after crushing is centrifuged at 12000 to 13500 rpm and 4 ° C for 20 to 30 minutes, and the supernatant is collected to obtain a lysate containing the target protein mutant Eco31IM1.

[0129] Ni affinity chromatography column: The operation of the Ni affinity chromatography column includes adding the crude product to the Ni affinity chromatography column, washing the Ni affinity chromatography column with an imidazole eluent of a concentration gradient, and collecting the eluent containing the mutant Eco31IM1.

[0130] The loading buffer includes Tris at a final concentration of 10 to 25 mmol / L, NaCl at 300 to 500 mmol / L, and imidazole at 5 to 10 mmol / L, a working pH of 7.5 ± 0.1, and a working temperature of 25°C. The eluent includes Tris at a final concentration of 10 to 25 mmol / L, NaCl at 300 to 500 mmol / L, and imidazole at 300 to 500 mmol / L, a working pH of 7.5 ± 0.1, and a working temperature of 25°C. The chromatography column was balanced with loading buffer for 3 to 5 column volumes, and the collected lysate containing the target protein was added to the above-mentioned balanced chromatography column containing Ni affinity chromatography filler, and the flow-through was collected; the balanced column was washed with 5 to 15 column volumes of loading buffer; the eluent was washed with an imidazole eluent with a concentration gradient, and the imidazole concentration in the eluent was 50mmol / L, 100mmol / L, 200mmol / L, and 300mmol / L, and the eluent containing the mutant Eco31I was collected. The collected eluent was run on SDS-PAGE protein gel to determine in which imidazole concentration gradient the eluted target protein was.

[0131] Ion exchange chromatography column: The operation includes desalting the eluate of the mutant restriction endonuclease Eco31I that has passed through the Ni affinity chromatography column with a low-salt buffer through a gel filtration chromatography column, then adding it to a cation exchange chromatography column, washing the cation exchange chromatography column with a concentration gradient NaCl solution, and collecting the eluate containing the mutant restriction endonuclease Eco31I.

[0132] The low-salt buffer comprises a final concentration of 10-25 mmol / L Tris, 1-5 mmol / L DTT and 0.1-0.5 mmol / EDTA, 25-50 mmol / L NaCl, a working pH of 7.0±0.1, and a working temperature of 25°C. The high-salt buffer comprises a final concentration of 10-25 mmol / L Tris, 1-5 mmol / L DTT, 0.1-0.5 mmol / L EDTA and 0.5-1 mol / L NaCl, a working pH of 7.0±0.1, and a working temperature of 25°C. The crude product obtained above is desalted through a gel filtration chromatography column. The cation exchange chromatography column is balanced with a high-salt equilibrium buffer and a low-salt buffer of 3-4 times the column bed volume as equilibrium buffers, and then the sample is added to the cation exchange chromatography column. Ion exchange achieves the purpose of separation and purification by exchanging charged solute molecules with exchangeable ions in the ion exchange chromatography medium. Then, linear elution is performed with 5 to 10 times the column volume of an elution buffer containing 0% to 100% Buffer B. Specifically, the NaCl concentration in the linear gradient high salt buffer is 0.5 to 1 mol / L.

[0133] Collect the purified eluate and run the purified sample on SDS-PAGE protein gel. The gel results are as follows: Figure 1 shown.

[0134] 1.3 Experimental Results

[0135] Depend on Figure 1 The results showed that after purification by cation exchange chromatography, no other impurity proteins remained in the mutant Eco31IM1 protein, and the purity could reach more than 95% (1-8: linear elution using high salt buffer, where 1-2 was impurity protein and 3-8 was mutant target protein band).

[0136] Example 2 Activity identification of mutant Eco31IM1

[0137] 2.1 Principles of restriction endonuclease activity identification

[0138] Taking the plasmid / DNA fragment containing the restriction endonuclease recognition sequence corresponding to Eco31I (5'GGTCTCN^NNNN^3') as the substrate, the purified Eco31I restriction enzyme was reacted with a certain amount of substrate in a certain reaction buffer for a certain period of time, and then the reaction system was subjected to agarose gel electrophoresis to observe the digestion of the substrate plasmid / DNA fragment to qualitatively and quantitatively determine the enzyme activity.

[0139] 2.2 Restriction endonuclease activity identification steps

[0140] The enzyme activity determination method used in this example is the 2-fold gradient dilution method, that is, the mutant Eco31IM1 and the control sample (Thermo, product number ER0292, activity of 10U / μL) after purification and dialyzation in Example 1 are gradiently diluted (1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64), and then 1 μL of the diluted enzyme test sample and the control sample are added to the reaction system of the same volume and the same amount of substrate, and incubated with the substrate pUC19 plasmid (containing an Eco31I restriction site) at 37°C for 1 h in a 50 μL reaction system under buffer conditions, and then the enzyme cleavage of the substrate is detected by 1% agarose gel electrophoresis; the electrophoresis result is the activity determination result.

[0141] The 50 μL enzyme activity assay reaction system is as follows:

[0142] system concentration Dosage 10X Buffer G 10X 5μL pUC19 plasmid / 1 μg Eco31I With the gradient dilution 1μL Nuclease-free water / Make up to 50 μL Total volume / 50μL

[0143] 2.3 Experimental Results

[0144] like Figure 2As shown, the pUC19 plasmid contains an Eco31I restriction site. When the mutant (experimental sample) and the wild-type Eco31I (control sample, Thermo) were diluted 4 times and subjected to restriction reaction, the plasmid was completely cut into a linear state. Therefore, the activity of the mutant Eco31I (Eco31IM1) is equivalent to that of the wild-type. The enzyme activity of the Eco31IM1 mutant purified in Example 1 was calibrated to be 10U / μL.

[0145] Example 3 Comparative experiment on Eco31IM1 enzyme cutting efficiency detection

[0146] 3.1 Experimental procedures

[0147] After activity calibration, the wild-type endonuclease Eco31I (Thermo, catalog number ER0292) and the mutant endonuclease Eco31I (Eco31IM1) were diluted in the same gradient (1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32) starting from 10U / μL. 1μL of enzyme in each group and a group without adding restriction enzyme (specific information of each experimental group is shown in the table below) were taken and incubated with 0.5μg of plasmid pUC19 in a 20μL reaction system for 5min and 15min respectively, and the enzyme cleavage of the substrate was detected by 1% agarose gel electrophoresis.

[0148]

[0149] The 20 μL enzyme activity assay reaction system is as follows:

[0150]

[0151]

[0152] 3.2 Experimental Results

[0153] The results are as follows Figure 3 The gradient dilution groups of wild-type and mutant Eco31I in lanes 1-10 are the results of enzyme digestion after 5 minutes of reaction, and the gradient dilution groups of wild-type and mutant Eco31I in lanes 11-20 are the results of enzyme digestion after 15 minutes of reaction. When the reaction time is 5 minutes, there is still a small amount of unlinearized plasmid in the wild-type group (control group) after diluting the enzyme volume 4 times; while the mutant group can still completely linearize the plasmid after diluting 8 times; when the reaction time is 15 minutes, there is still a small amount of unlinearized plasmid in the wild-type group (control group) after diluting the enzyme volume 8 times; while the mutant group can still completely linearize the plasmid after diluting 16 times, indicating that the Eco31I mutant can complete DNA linearization within 5 minutes after diluting 8 times, while the wild-type enzyme cannot guarantee DNA linearization within 15 minutes after diluting 8 times, and the enzyme digestion efficiency of the mutant group is more than 2 times higher than that of the wild-type group.

[0154] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for identifying and cutting DNA, comprising: (a) adding a DNA fragment and an Eco31I restriction endonuclease to a cleavage system to produce a reaction mixture, wherein the Eco31I restriction endonuclease comprises an amino acid substitution corresponding to position 283 of SEQ ID NO: 1 or other Eco31I restriction endonuclease equivalent sites; and (b) incubating the reaction mixture under conditions suitable for cleavage of the DNA fragments to produce DNA cleavage products of the fragments; Preferably, the DNA fragment is a double-stranded nucleic acid; Preferably, the double-stranded nucleic acid comprises linear double-stranded DNA or circular double-stranded DNA; Preferably, at least one strand of the double-stranded nucleic acid is cleaved during the cleavage process to form a DNA cleavage product; Preferably, the DNA cleavage product contains a nick at at least one end; Preferably, the conditions suitable for DNA fragment cleavage include: reaction temperature and reaction time; Preferably, the reaction temperature is 25-65°C, preferably 25-40°C, and more preferably 37°C; Preferably, the reaction time is 5-720 min, preferably 5-15 min, and more preferably 5 min.

2. A method for preparing Eco31I restriction endonuclease, comprising: (a) selecting an Eco31I restriction endonuclease in which an amino acid at position 283 of SEQ ID NO: 1 or at another Eco31I restriction endonuclease equivalent site is substituted for purification; and (b) obtaining the Eco31I restriction endonuclease.

3. An Eco31I restriction endonuclease, characterized in that The Eco31I restriction endonuclease comprises an amino acid substitution at position 283 of SEQ ID NO: 1 or at other Eco31I restriction endonuclease equivalent sites.

4. The method, preparation method or Eco31I restriction endonuclease according to any one of claims 1 to 3, characterized in that: The amino acid substitution at position 283 of SEQ ID NO: 1 or other Eco31I restriction endonuclease equivalent sites is Q283-.

5. The method, preparation method or Eco31I restriction endonuclease according to claim 4, characterized in that: The Eco31I restriction endonuclease has a changed cleavage efficiency or a changed expression level relative to the Eco31I restriction endonuclease shown in SEQ ID NO:

1.

6. The method, preparation method or Eco31I restriction endonuclease according to claim 4, characterized in that: The Eco31I restriction endonuclease further comprises an amino acid substitution at any one or more of the following positions: at position 138, 181, 237, 240, 255, 256, 259, 262, 264, 271, 274, 278, 311, 326, 333, 334, 337, 340, 345, 361, 372, 457, 459, 460, 472 or 475 of SEQ ID NO: 1; Preferably, the Eco31I restriction endonuclease further comprises a combination of any one or more of the following mutations: Y138F, Y181F, H237A, N240A, N255A, L256P, Y259A, D262A, R264D, D271A, W274C, D278A, D311A, F326A, C333S, N334D, K337A, R340A, D345A, S361A, S361P, K372A, R457A, I459V, R460A, K472E or R475A; Preferably, the Eco31I restriction endonuclease further comprises any one of the following mutation combinations: (a) K82R and V270M; (b) Y138F and L256P; (c) Y181F and V260D; (d) I214T and Y259C; (e) F326S and L350F; (f) T9A, C333S, and V419L; (g) D278G, I289V and F363L; (h) S19P, K82R, V270M and N334D.

7. A conjugate, characterized in that: The invention comprises the Eco31I restriction endonuclease according to any one of claims 1 to 6 and a marker for purification.

8. An isolated nucleic acid, recombinant vector or host cell, characterized in that: The isolated nucleic acid encodes the Eco31I restriction endonuclease according to any one of claims 1 to 6, the recombinant vector contains the isolated nucleic acid, and the host cell contains the recombinant vector.

9. A kit, characterized in that: It comprises the Eco31I restriction endonuclease according to any one of claims 1 to 6.

10. Use of the Eco31I restriction endonuclease according to any one of claims 1 to 6 in DNA cleavage.

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