CN120005871A - Restriction endonuclease mutant and its preparation method and application - Google Patents

Abstract

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

Description

Restriction endonuclease mutant and preparation method and application thereof

Cross Reference to Related Applications

The present disclosure claims priority from chinese patent application No. 202311519732.7, entitled "restriction enzyme mutant, its preparation method and use" filed on day 14 11, 2023, the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to the technical field of biology, in particular to an Eco31I restriction enzyme mutant and a preparation method and application thereof.

Background

Restriction enzymes are a class of enzymes, abbreviated as restriction enzymes, that are capable of recognizing a specific nucleotide sequence in a double-stranded DNA molecule and cleaving a phosphodiester bond in the DNA strand at a specific position. Different restriction enzymes recognize different DNA sequences which can cleave DNA within or adjacent to the recognition sequence, and which, after cleavage, form different types of products, such as blunt-ended or cohesive-ended products.

Restriction enzymes are known to be most commonly used in molecular cloning experiments. But besides cloning, it can also be applied to the fields of vaccine development and production, gene sequencing, SNP identification, ddPCR and the like. Restriction enzymes can be broadly classified into four types, i.e., type I enzyme, type II enzyme, type III enzyme and type IV enzyme, depending on the complexity of the structure, the mode of action and the cofactor. The type II restriction enzyme is characterized by having only the function of recognition and cutting and no modification activity, the recognition sequence is mostly a short palindromic sequence (generally 4-8 bp), and the recognition sequence is cut at a site positioned in or adjacent to the recognition sequence to generate a DNA product with 3 '-hydroxyl and 5' -phosphate groups, and Mg ²⁺ is needed when the restriction enzyme acts, so that the restriction enzyme is the most commonly used restriction enzyme in molecular cloning.

Type II restriction enzymes are further subdivided into IIP, IIA, IIB, IIC, IIS or other subcategories. The Eco31I restriction enzyme belongs to IIS type, and is an isoschizomer with BsaI and Bso31I, bspTNI, and is a restriction enzyme with wider application. The Eco31I can specifically recognize six-base nucleic acid sequence (5 'GGTCTCN NNNNN 3') sites, and one outstanding characteristic of the enzyme is that the enzyme has no requirement on the sequence of the cleavage site. By utilizing the seamless cloning technology derived from the characteristics, a plurality of DNA fragments can be assembled on a compatible carrier in any order. In addition, the Eco31I not only has important application value in molecular cloning, but also has other directions in the biological field, such as the preparation of an mRNA vaccine in-vitro transcription template and the like.

Unlike the preparation of other types of enzymes, when a single recombinant expression restriction enzyme is used in a prokaryotic expression system, the restriction enzyme generated cleaves the host DNA and causes the death of the host cell because the host DNA itself is not modified by the methylase. In response to this phenomenon, related scientists have introduced methyltransferase genes into prokaryotic expression systems. Specific modifications of the methyltransferase on the recognition sequence may prevent cleavage by the restriction enzyme. The restriction enzyme-methyltransferase genes are often closely linked, and bacteria regulate the expression of both genes so that the self DNA is first protected by methylation and then the restriction enzyme is expressed after the protection is completed, and the invaded heterologous DNA is not protected and is degraded by the restriction enzyme. That is, intracellular related methyltransferases methylate the same specific sequence recognized by its cognate restriction enzyme and confers to the modified DNA a property that resists cleavage by its restriction enzyme, protecting the host DNA from degradation of unmethylated foreign DNA.

However, bacterial regulation of restriction and methyltransferases is often unbalanced. The imbalance often causes low or even no expression of the wild type Eco31I, which causes the problems of complicated separation and purification procedures, low protein yield and the like, so that the currently marketed Eco31I restriction enzyme is high in price, and the wide application and the deep research of the enzyme are limited. Meanwhile, a series of problems mentioned above also indirectly affect the enzyme activity of the Eco31I, so that the enzyme digestion rate of the wild type Eco31I is slow, about 1 hour is often required to completely enzyme-cleave 1 μg of plasmid, and the enzyme digestion for a long time is often easy to generate the condition of enzyme star activity, so that an unexpected cleavage result is introduced.

Therefore, the Eco31I restriction endonuclease with high expression, simple separation and purification procedures, high protein yield, low price and good digestion efficiency and the preparation method thereof are provided.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide an Eco31I restriction enzyme mutant, a preparation method and application thereof.

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

(a) Adding a DNA fragment and an Eco31I restriction enzyme comprising an amino acid substitution at position 283 corresponding to SEQ ID NO. 1 or other equivalent positions of the Eco31I restriction enzyme to the cleavage system to produce a reaction mixture, and

(B) Incubating the reaction mixture under conditions suitable for cleavage of the DNA fragments to produce DNA cleavage products of the fragments.

In order to achieve the above object, according to a second aspect of the present invention, there is provided a method for producing an Eco31I restriction enzyme, comprising the steps of:

(a) Selecting an Eco31I restriction enzyme with substitution of amino acid at position 283 of SEQ ID NO. 1 or other Eco31I restriction enzyme equivalent site for purification, and

(B) The above-mentioned Eco31I restriction enzyme was obtained.

In order to achieve the above object, according to a third aspect of the present invention, there is provided an Eco31I restriction enzyme comprising an amino acid substitution at position 283 of SEQ ID NO. 1 or at the equivalent position of other Eco31I restriction enzyme.

In order to achieve the above object, according to a fourth aspect of the present invention, there is provided a conjugate comprising the above Eco31I restriction enzyme and a label for purification.

In order to achieve the above object, according to a fifth aspect of the present invention, there is provided an isolated nucleic acid encoding an amino acid of the above Eco31I restriction enzyme.

In order to achieve the above object, according to a sixth aspect of the present invention, there is provided a recombinant vector containing the above isolated nucleic acid.

In order to achieve the above object, according to a seventh aspect of the present invention, there is provided a host cell containing the vector described above.

In order to achieve the above object, according to an eighth aspect of the present invention, there is provided a kit comprising the above Eco31I restriction enzyme.

In order to achieve the above object, according to a ninth aspect of the present invention, there is provided the use of the above-mentioned Eco31I restriction enzyme in DNA cleavage.

Compared with the prior art, the invention has the following beneficial effects:

The optimized Eco31I restriction enzyme mutant breaks through the problem that the conventional Eco31I restriction enzyme cannot be expressed in a methylation host, can be efficiently expressed in the methylation host, has excellent digestion efficiency, can be applied to linearization of large fragments, and has the advantages of short cutting time, few byproducts and the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram showing the purification results of an ion exchange chromatography column for the Eco31IM1 sample purified in example 1 of the present application, wherein the lanes before desalting are the pooled samples after eluting with a Ni affinity chromatography column, the lanes after desalting are the pooled samples after desalting with a gel filtration chromatography column, the lanes before loading are the pooled samples before purifying with an ion exchange chromatography column, lanes 1-2 are the hybrid protein samples after linear elution with a high salt buffer, and lanes 3-8 are the mutant Eco31IM1 target proteins after linear elution with a high salt buffer;

FIG. 2 shows an electrophoretogram of the enzyme cleavage activity assay after gradient dilution of purified Eco31IM1 with standard in example 2 of the application;

FIG. 3 shows an electrophoresis chart of the cleavage efficiency measurement after gradient dilution of purified Eco31IM1 and a standard in example 3 of the present application, M represents a Marker band, and yin represents a negative control of an unlimited endonuclease, lanes 1 to 10 are cleavage results after 5min of cleavage by wild-type (standard) and mutant Eco31I (Eco 31IM 1) having different gradients, and lanes 11 to 20 are cleavage results after 15min of cleavage by wild-type and mutant Eco31I having different gradients.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

As mentioned in the background art, the existing commercial restriction endonuclease Eco31I also has certain cutting efficiency, but the unbalance of the protein expression host to the regulation and control of restriction enzyme and methyltransferase leads to low or no expression of the commercial enzyme Eco31I, the cost and the selling price are high, the cutting time is long, and the practical application effect is poor. Therefore, the application aims to protect the Eco31I restriction enzyme which has higher cutting efficiency and is suitable for industrial production.

The inventor of the application analyzes the function, the protein structure and the key site of the Eco31I restriction endonuclease, designs a large number of mutations to carry out stability transformation, and finally obtains the Eco31I restriction endonuclease mutant which is expressed in a methylation host with high efficiency, and the restriction endonuclease mutant can complete fragment cutting within 5min, can be applied to linearization of large fragments, and has the advantages of short cutting time, few byproducts and the like.

In a first exemplary embodiment of the present invention, a method for identifying and cleaving DNA is provided, comprising the steps of (a) adding a DNA fragment and an Eco31I restriction enzyme to a cleavage system to produce a reaction mixture, said Eco31I restriction enzyme comprising an amino acid substitution at position 283 corresponding to SEQ ID NO. 1 or other Eco31I restriction enzyme equivalent site, and (b) incubating said reaction mixture under conditions suitable for cleavage of the DNA fragment to produce a DNA cleavage product of said fragment.

In one or more preferred embodiments, the DNA fragments described above are double-stranded nucleic acids. Any double-stranded nucleic acid comprising DNA is suitable for use in the present application.

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

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.

In one or more preferred embodiments, the DNA cleavage product described above contains a nick at least one end.

In order to maintain the restriction enzyme Eco31I activity and provide a suitable environment for its reaction, in one or more preferred embodiments, the above reaction mixture comprises a cleavage system, a DNA fragment and an Eco31I restriction enzyme.

In one or more preferred embodiments, the cleavage system comprises at least one member of the group consisting of Tris-HCl, mg ²⁺、Na⁺、K⁺, serum proteins, EDTA, DTT, glycerol.

The Eco31I restriction enzyme of the application has better cleavage activity, and in order to further provide suitable conditions for the cleavage reaction, in one or more preferred embodiments, the above conditions suitable for the cleavage of DNA fragments include reaction temperature and reaction time;

In one or more preferred embodiments, the cleavage reaction temperature is 25-65 ℃, preferably 25-40 ℃, and more preferably 37 ℃.

In one or more preferred embodiments, the reaction temperature of the cleavage may be 25 ℃,30 ℃, 35 ℃, 37 ℃, 40 ℃, 45 ℃,50 ℃, 55 ℃, 60 ℃, 65 ℃.

In one or more preferred embodiments, the reaction time for cleavage is from 5 to 720 minutes, preferably from 5 to 15 minutes, and more preferably 5 minutes.

In one or more preferred embodiments, the reaction time for the cleavage may be 5min, 15min, 30min, 60min, 120min, 180min, 360min, 720min.

In a second exemplary embodiment of the present application, there is provided a method for producing an Eco31I restriction enzyme, comprising the steps of (a) selecting an Eco31I restriction enzyme in which an amino acid at position 283 of SEQ ID NO. 1 or other Eco31I restriction enzyme equivalent site is substituted for purification, and (b) obtaining the above-mentioned Eco31I restriction enzyme. Among them, due to the nature of the restriction enzyme, it cleaves unmodified host DNA, causing host cell death. Thus, any method of preparing a protein that prevents cleavage of host DNA upon expression of a restriction enzyme is suitable for use in the present application.

In one or more preferred embodiments, the purification step further comprises (1) constructing a recombinant expression plasmid of methyltransferase, (2) transferring the plasmid obtained in the step (1) into a non-methylation protective strain to obtain an Eco31I specific protective strain, (3) constructing an expression vector of a gene corresponding to the Eco31I restriction enzyme according to the second aspect, transforming the specific protective strain obtained in the step (2) to obtain a mutant restriction enzyme Eco31I recombinant strain, (4) inducing and culturing the mutant restriction enzyme Eco31I recombinant strain obtained in the step (3), (5) collecting thalli obtained by inducing and culturing the mutant Eco31I recombinant strain in the step (4), cracking at 3-6 ℃ under 60-90MPa to obtain a cracking solution, (6) centrifuging the cracking solution obtained in the step (5) at 5000-5000 rpm for 3-5min, collecting supernatant to obtain a crude product, (7) purifying the crude product obtained in the step (6) sequentially by a Ni affinity chromatography column and an ion exchange chromatography column to obtain the mutant restriction enzyme Eco 31.

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

In one or more preferred embodiments, the construction method of the recombinant expression plasmid in the step (1) includes obtaining a methyltransferase gene, and seamlessly cloning a methyltransferase gene fragment into an expression vector to obtain the recombinant expression plasmid of methyltransferase.

In one or more preferred embodiments, the above expression vector is pK1.

In one or more preferred embodiments, the methyltransferase gene is obtained by any one or a combination of at least two of PCR amplification, gene synthesis, and DNA library construction.

In one or more preferred embodiments, the method of screening the protective strain in the above step (2) comprises the steps of transforming plasmid pK1-BsmAIM into competent cell BL21 (DE 3), extracting plasmid pK1-BsmAIM after culturing and digesting with restriction enzyme Eco31I, and screening the methylation protective strain so that plasmid pK1-BsmAIM is completely protected by methyltransferase in the cell.

In one or more preferred embodiments, the construction of the expression vector in step (3) above includes the step of histidine-tagging the mutant restriction enzyme Eco31I gene fragment and then ligating the fragment to the expression vector.

In the present invention, the mutant restriction enzyme Eco31I gene fragment may be obtained by gene synthesis, or may be obtained by point-by-point mutation of a vector comprising the wild-type restriction enzyme Eco31I gene fragment.

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

In one or more preferred embodiments, the conditions for the induction culture in the above step (4) are that the recombinant strain of the mutant restriction enzyme Eco31I in the step (3) is inoculated into LB medium, cultured at 37℃and a culture speed of 180-250rpm until OD600 is 0.45-0.75, and then an inducer is added, at 15℃and 130rpm, and cultured for 6-10 hours.

In one or more preferred embodiments, the above-mentioned culture rotation speed is 180-250rpm, and may be, for example, 180rpm, 190rpm, 200rpm, 210rpm, 220rpm, 230rpm, 240rpm or 250rpm, preferably 220rpm.

In one or more preferred embodiments, the culture is carried out 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.

In one or more preferred embodiments, the above-mentioned culture is carried out for 6 to 10 hours, for example, 6 hours, 7 hours, 8 hours, 9 hours or 10 hours, preferably 8 hours.

In one or more preferred embodiments, the inducer comprises isopropyl- β -D-thiogalactoside (IPTG), preferably the final concentration of the inducer is 0.15-0.3mM, and more preferably the final concentration of the inducer is 0.25mM.

In one or more preferred embodiments, the lysing in step (5) above comprises the step of resuspending the cells with a lysis buffer at a ratio of 10:1 to give a resuspended suspension, and collecting the supernatant to give the lysate. Wherein the lysis buffer solution contains Tris-HCl with a final concentration of 15-25 mmol/L, EDTA with a final concentration of 0.05-0.15 mmol/L, DTT with a final concentration of 0.5-1.5 mmol/L, 10% Glycerol and NaCl with a final concentration of 300-500 mmol/L, and has a working pH value of 7.5+/-0.1 and a working temperature of 25 ℃.

In one or more preferred embodiments, the operation of the Ni affinity chromatography column in step (7) above comprises adding the above crude product to the above Ni affinity chromatography column, washing the Ni affinity chromatography column with a concentration gradient of imidazole eluent, and collecting the eluent containing mutant Eco 31I.

In one or more preferred embodiments, the eluent comprises final concentration of 10-25 mmol/LTris, 300-500 mmol/L NaCl and 0.5-1.5 mmol/L imidazole, and the working pH value is 7.5+ -0.1, and the working temperature is 25 ℃.

In one or more preferred embodiments, the operation of the ion exchange chromatography column in step (7) above comprises passing through an anion exchange chromatography column and a cation exchange chromatography column.

In one or more preferred embodiments, the operation of the above-mentioned anion exchange chromatography column comprises diluting the eluate of the mutant restriction enzyme Eco31I subjected to Ni affinity chromatography with a low-salt buffer, adding the diluted eluate to the anion exchange chromatography column, washing the above-mentioned anion exchange chromatography column with a NaCl solution having a concentration gradient, and collecting the eluate containing the mutant restriction enzyme Eco 31I.

In one or more preferred embodiments, the low-salt buffer solution comprises Tris with a final concentration of 10-25 mmol/L, DTT with a final concentration of 1-5 mmol/L, EDTA with a final concentration of 0.1-0.5 mmol/L and NaCl with a final concentration of 25-50 mmol/L, a working pH of 7.0+ -0.1 and a working temperature of 25 ℃.

In one or more preferred embodiments, the anion exchange chromatography column described above comprises a Q anion exchange chromatography column.

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

In one or more preferred embodiments, the operation of the cation exchange chromatography column comprises desalting the mutant restriction enzyme Eco31I passing through the anion exchange chromatography column by a gel filtration chromatography column, respectively using 3-4 times of high-salt buffer and low-salt buffer as balance buffers, balancing the cation exchange chromatography column, adding a sample into the cation exchange chromatography column, then performing linear elution by using 5-10 times of elution buffer, and collecting the eluent containing the mutant Eco 31I.

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

In a third exemplary embodiment of the present invention, an Eco31I restriction enzyme is provided, wherein said Eco31I restriction enzyme comprises an amino acid substitution at position 283 of SEQ ID NO. 1 or at the equivalent position of other Eco31I restriction enzymes.

In one or more preferred embodiments, the amino acid substitution at position 283 of SEQ ID NO.1 or at the equivalent of another Eco31I restriction enzyme is Q283-.

According to the invention, the 283 th amino acid in the amino acid sequence of the wild type restriction enzyme Eco31I is subjected to deletion mutation to obtain the mutant restriction enzyme Eco31I, so that the expression quantity of the mutant restriction enzyme Eco31I is increased, the purification difficulty is reduced, the yield is improved, the cutting efficiency of the restriction enzyme is further improved, and the mutant restriction enzyme can be applied to linearization of large fragments.

In the present invention, the equivalent site refers to a site having similar functions and activities in two enzymes. In general, a spatial structure model of a target protein is firstly constructed according to the amino acid sequence of an enzyme, the spatial structures of two enzymes are rotated to enable the two enzymes to overlap each other as much as possible, and two amino acids which are in the same spatial position after being spatially compounded to a certain overlapping distance standard between the two different enzymes are called equivalent sites.

In one or more preferred embodiments, the above-described Eco31I restriction enzyme has an altered cleavage efficiency or expression level relative to the Eco31I restriction enzyme shown in SEQ ID NO. 1.

In one or more preferred embodiments, the Eco31I restriction enzyme described above further comprises amino acid substitutions at any one or more of positions 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, and further preferably the Eco31I restriction enzyme described above further comprises a combination ：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 of any one or more of the following mutations.

In the invention, higher cutting activity can be obtained after N334D mutation, new cutting activity is added after R264D, K337A or R340A mutation, new cutting activity is added after D311A or D345A mutation, Y138F, Y181F, L256P, W274 333S, S361P, I459V or K472E mutation still has stronger cutting activity after H237A, N240A, N A, Y259A, D A, D271A, D278A, F326 38361 372A, R457A, R A or R475A mutation, and the cutting activity is hardly changed after mutation.

In one or more preferred embodiments, the Eco31I restriction enzyme described above further comprises any one of the following combinations of mutations (a) K82R and V270M, (b) Y138F and L256P, (C) Y181F and V260D, (D) I214T and Y259C, (e) F326S and L350F, (F) T9A, C S and V419L, (g) D278G, I289V and F363L, (h) S19P, K R, V270M and N334D.

In the invention, the mutant still has stronger cutting activity after the combined mutation.

The invention also includes mutant Eco31I restriction enzymes having one or more of the above amino acid substitutions, wherein the remainder of the Eco31I restriction enzyme 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 enzyme, preferably the wild-type Eco31I restriction enzyme has the sequence of SEQ ID NO. 1.

In a fourth exemplary embodiment of the invention, there is provided a conjugate comprising an Eco31I restriction enzyme mutant as described in any of the preceding examples and a label for purification.

In one or more preferred embodiments, the labels for purification include purification tags, including but not limited to His Tag (HIS-Tag), glutathione-thiol transferase Tag (GST-Tag), maltose binding protein Tag (MBP-Tag), transcription termination/anti-termination protein Tag (NusA-Tag), small ubiquitin related modifier (SUMO).

In a fifth exemplary embodiment of the invention, an isolated nucleic acid encoding an Eco31I restriction enzyme as described in any of the preceding examples is provided. In the translation process, each host cell for protein expression has a certain preference for codons, and in order to obtain a protein product with higher expression level in a specific host cell, the nucleotide sequence for encoding the protein can be subjected to codon optimization.

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

In a sixth exemplary embodiment of the invention, a recombinant vector is provided comprising an isolated nucleic acid as described in any of the preceding examples.

In one or more preferred embodiments, the vectors described above contain the elements necessary for transcription and translation of the inserted coding sequence.

In a seventh exemplary embodiment of the invention, a host cell is provided comprising a vector as described in any of the preceding examples.

In one or more preferred embodiments, the host cell is a prokaryotic cell, preferably E.coli, and more preferably competent cell BL21 (DE 3).

Based on the disclosure of the amino acid sequence of the Eco31I restriction enzyme mutant, those skilled in the art will easily recognize that the Eco31I restriction enzyme mutant is prepared by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, the Eco31I restriction enzyme mutant is obtained by separating and purifying a culture product of recombinant cells capable of recombinantly expressing the Eco31I restriction enzyme mutant as described in any one of the above embodiments, which is easy to implement for those skilled in the art, and based on this, the Eco31I restriction enzyme mutant of the disclosure is prepared by any technique, which is within the scope of protection of the disclosure.

In an eighth exemplary embodiment of the present invention, there is provided a kit comprising the Eco31I restriction enzyme described in any of the foregoing examples.

The kit includes any form capable of carrying the Eco31I restriction enzyme, and may be, but is not limited to, a reagent tube, a kit, a reagent strip, a reagent bottle, etc.

In one or more preferred embodiments, the above-described kit further comprises at least one of a buffer, a preservation solution, and a diluent.

In a ninth exemplary embodiment of the present invention, there is provided the use of the Eco31I restriction enzyme described in any of the foregoing examples in DNA cleavage.

In one or more preferred embodiments, the use of DNA cleavage includes molecular cloning, vaccine development and production, gene sequencing, SNP identification, ddPCR, and the like.

The features and capabilities of the present application are described in further detail below in connection with particular examples, which should not be construed as limiting the scope of the application as claimed.

EXAMPLE 1 construction of Eco31I restriction endonuclease mutant Eco31IM1

1.1 Obtaining methylation-protected Strain

1.1.1 Methyltransferase BsmAIM Gene Synthesis

The amino acid sequence and nucleotide sequence (SEQ ID NO:2 and SEQ ID NO: 3) of BsmAIM gene were obtained via UniProt and NCBI website, and after codon optimization based on E.coli, the nucleotide sequence was sent to gene company for synthesis, thus obtaining methyltransferase BsmAIM amplification template.

1.1.2 Construction of recombinant expression strains of methyltransferases

The synthesized fragment is used as a PCR template, ndeI and EcoRI enzyme cutting site sequences corresponding to a vector and a gene 5 'end sequence or a gene 3' end sequence are added into a primer sequence for PCR amplification, a target sequence is amplified by using Q5 high-fidelity polymerase (NEB, product number M0491L), agarose gel electrophoresis and gel cutting recovery are carried out after the amplification is finished, and a BsmAIM gene fragment with the same size as a theoretical value is obtained. Vector pK1 was subjected to EcoRI and NdeI double digestion (NEB, cat# R3101V and R0111S respectively), the digested products were subjected to agarose gel electrophoresis, linearized vector fragments were recovered by gel cutting, the target gene BsmAIM was ligated to vector pK1 according to the instructions using a seamless cloning kit (Biyun, cat# D7010M), the ligated recombinant plasmid was transformed into competent cell BL21 (DE 3), and cell culture was performed under the corresponding antibiotic selection conditions to obtain BsmAIM recombinant expression strain, and the sequence accuracy was confirmed by sequencing.

1.1.3 Screening of methylation-protected strains

Culturing the correct recombinant expression strain by using a culture medium of the corresponding antibiotics, and collecting bacterial liquid to extract plasmids after the bacterial liquid is turbid for about 16 hours. In a 25. Mu.L reaction system, 1. Mu.L of Eco31I restriction enzyme (Thermo, cat. No. ER 0292) was added to the reaction system, and the mixture was incubated with 500ng of recombinant expression plasmid at 37℃for 1 hour, and then the digestion of the substrate was detected by agarose gel electrophoresis, confirming that the recombinant expression plasmid was completely resistant to cleavage by Eco31I, and that the genomic DNA of the default host bacteria was also protected by BsmAIM methyltransferase expressed by the recombinant plasmid, and the strain completely resistant to cleavage by Eco31I was prepared as competent cell preservation and later designated (pK 1-BsmAIM-BL21 (DE 3)).

Wherein, 25. Mu.L reaction system for screening methylation recombinant expression plasmid is shown in the following table:

1.2 expression purification of mutant Eco31IM1

1.2.1 Molecular cloning protocols

1.2.1.1 Gene Synthesis

And (3) obtaining a wild type Eco31I restriction enzyme sequence (SEQ ID NO: 1) on NCBI, sending the sequence to a gene company for synthesis after codon optimization based on escherichia coli, and adding a 6His tag to obtain a wild type restriction enzyme Eco31I amplification template.

1.2.1.2 Construction of mutant Eco31IM1

The wild type restriction enzyme Eco31I gene is used as a template, and a primer is designed to construct a mutant Eco31I plasmid. The primer sequence is shown as SEQ ID NO. 4-7, F and W-R and W-F and R are used for amplifying Eco31I genes respectively, after gel cutting and recovery, F and R are used for fusion PCR amplification mutant templates, and electrophoresis gel cutting and recovery are carried out. The mutant template and the enzyme-digested vector pCold I are subjected to seamless connection by using endonucleases NdeI and KpnI (NEB, product numbers are R0111S and R3142V respectively), a seamless cloning kit (Biyun, product number D7010M) is used for carrying out seamless connection after electrophoresis recovery, the connection product is transformed into methylation competent cells (pK 1-BsmAIM-BL21 (DE 3)), and the methylation competent cells are coated on a plate containing chloramphenicol antibiotics for screening culture to obtain a recombinant expression strain of the mutant restriction enzyme Eco31IM1 (SEQ ID NO: 8).

1.2.2 Induction of expression of strains

The recombinant strain obtained is subjected to amplification culture by three steps of activation, secondary activation, fermentation and cooling induction.

Activating, namely adding 100 mu L of glycerinum into 3mL of LB culture medium containing resistance, wherein the activating condition is that the temperature is 37 ℃, the rpm is 250, the time is 4-6 hours, adding 10 mu L of activated bacterial liquid into 500mL of LB liquid culture medium, preparing a bottle of seeds, adding antibiotics with corresponding resistance, shaking uniformly, putting into a shaking table (the temperature is 37 ℃, the rpm is 130), and culturing overnight.

Fermentation, namely adding 5mL of the secondary activated bacterial liquid into 500mL of culture medium, and culturing for 3-4h by a shaking table under the conditions of 37 ℃ and 220 rpm.

And (3) cooling and inducing, namely, inducing at 15 ℃ at 130rpm for 6-10h and 0.15-0.30mM IPTG.

The cells were collected by centrifugation at 500mL in a centrifuge cup at 5000-5000 rpm for 3-5min at 4 ℃.

1.2.3 Purification of the protein of interest

The bacterial cells obtained above were subjected to lysis, ni affinity chromatography and ion exchange chromatography to obtain mutant restriction enzyme Eco31IM1.

And (3) cracking, namely re-suspending the thalli by using a cracking buffer solution, and cracking at 3-6 ℃ under 600bar to obtain a re-suspension. The ratio of the lysis buffer to the thalli is 10:1, and the supernatant is collected to obtain the lysis buffer.

Wherein the lysis buffer solution contains Tris-HCl with a final concentration of 40-50 mmol/L, EDTA with a final concentration of 0.05-0.15 mmol/L, DTT with a final concentration of 0.5-1.5 mmol/L, 10% glycidol, naCl with a working pH value of 7.5+/-0.1 and a working temperature of 25 ℃. Specifically, a lysis buffer solution is added into the thalli, a stirrer is placed on a magnetic stirrer to be stirred for 15-25 min, so that the thalli are resuspended. Homogenizing and crushing the obtained heavy suspension, and carrying out solid-liquid separation to collect supernatant to obtain the lysate. Centrifuging the crushed mixed solution at 12000-13500 rpm and at 4 ℃ for 20-30 min, and collecting the supernatant to obtain the lysate containing the target protein mutant Eco31IM 1.

Ni affinity chromatography column the operation of the Ni affinity chromatography column comprises adding the crude product to the Ni affinity chromatography column, washing the Ni affinity chromatography column with imidazole eluent with concentration gradient, and collecting eluent containing mutant Eco31IM 1.

The loading buffer solution comprises 10-25 mmol/L Tris, 300-500 mmol/L NaCl and 5-10 mmol/L imidazole, the working pH value is 7.5+/-0.1, the working temperature is 25 ℃, the eluent comprises 10-25 mmol/L Tris, 300-500 mmol/L NaCl and 300-500 mmol/L imidazole, the working pH value is 7.5+/-0.1, and the working temperature is 25 ℃. Balancing the chromatographic column with a loading buffer solution for 3-5 column bed volumes, adding the collected lysate containing the target protein into the balanced chromatographic column containing Ni affinity chromatographic packing, collecting the flow-through liquid, flushing the balancing column with the loading buffer solution with 5-15 times of column volume, washing with imidazole eluent with concentration gradient, and collecting eluent containing mutant Eco31I, wherein the concentration of imidazole in the eluent is sequentially 50mmol/L, 100mmol/L, 200mmol/L and 300 mmol/L. The collected eluent is subjected to SDS-PAGE protein gel, so that the concentration gradient of the eluted target protein in imidazole is judged.

The operation of the ion exchange chromatographic column comprises the steps of desalting the eluent of the mutant restriction enzyme Eco31I passing through the Ni affinity chromatographic column by using a low-salt buffer solution through a gel filtration chromatographic column, adding the desalted eluent into a cation exchange chromatographic column, washing the cation exchange chromatographic column by using a NaCl solution with a concentration gradient, and collecting the eluent containing the mutant restriction enzyme Eco 31I.

The low-salt buffer solution comprises 10-25 mmol/L Tris, 1-5 mmol/L DTT, 0.1-0.5 mmol/EDTA, 25-50 mmol/L NaCl, and has a working pH value of 7.0+/-0.1 and a working temperature of 25 ℃. The high-salt buffer solution comprises 10-25 mmol/L Tris, 1-5 mmol/LDTT, 0.1-0.5 mmol/L EDTA and 0.5-1 mol/L NaCl, wherein the working pH value is 7.0+/-0.1, and the working temperature is 25 ℃. The crude product obtained above was desalted by passing through a gel filtration column. And respectively balancing the cation exchange chromatographic column by using a high-salt balancing buffer solution and a low-salt buffer solution which are 3-4 times of the volume of the column bed as balancing buffer solutions, and then adding the sample into the cation exchange chromatographic column. Ion exchange achieves the aim of separation and purification by exchanging charged solute molecules with exchangeable ions in an ion exchange chromatography medium. And then, carrying out linear elution by using an elution buffer solution containing 0% -100% buffer B with the volume of 5-10 times of the bed volume. Specifically, the NaCl concentration in the high-salt buffer solution with a linear gradient is 0.5-1 mol/L.

The purified eluate was collected and the purified samples were run on SDS-PAGE protein gels with the gel run as shown in FIG. 1.

1.3 Experimental results

As can be seen from the results of FIG. 1, after purification by cation exchange chromatography column, the mutant Eco31IM1 protein has no other impurity protein residue, and the purity can reach more than 95% (1-8: linear elution is performed by using high salt buffer, wherein 1-2 is impurity protein, and 3-8 is mutant target protein band).

EXAMPLE 2 identification of mutant Eco31IM1 Activity

2.1 Principle of identification of restriction Endonuclease Activity

Taking a plasmid/DNA fragment containing a restriction enzyme recognition sequence (5 'GGTCTCN NNNNN 3') corresponding to the Eco31I as a substrate, enabling the purified Eco31I restriction enzyme to act with a certain amount of the substrate in a certain reaction buffer for a certain time, performing agarose gel electrophoresis on a reaction system, and observing the digestion condition of the substrate plasmid/DNA fragment to perform qualitative and quantitative analysis on the enzyme activity.

2.2 Restriction Endonuclease Activity identification step

The enzyme activity determination method used in this example was a 2-fold gradient dilution method, namely, after subjecting the purified and dialyzed mutant Eco31IM1 and control sample (Thermo, cat. No. ER0292, activity 10U/. Mu.L) of example 1 to gradient dilution (1/2, 1/4, 1/8, 1/16, 1/32, 1/64), respectively, adding the diluted 1. Mu.L enzyme test sample and control sample to a reaction system of the same volume and the same substrate amount, incubating with the substrate pUC19 plasmid (containing a cleavage site of Eco 31I) at 37℃for 1h under a buffer condition in the 50. Mu.L reaction system, and detecting the cleavage of the substrate by 1% agarose gel electrophoresis, wherein the electrophoresis result is the activity determination result.

The 50. Mu.L enzyme activity assay reaction system was as follows:

System of	Concentration of	Dosage of
			10X Buffer G	10X	5μL
PUC19 plasmid	/	1μg
			Eco31I	With gradient dilution	1μL
Nuclease-free water	/	Supplement to 50 mu L
			Total volume of	/	50μL

2.3 Experimental results

As shown in FIG. 2, pUC19 plasmid contains an enzyme cleavage site of Eco31I, and when the mutant type (experimental sample) and the wild type Eco31I (control sample, thermo) were diluted 4-fold respectively and then subjected to enzyme cleavage reaction, the plasmid was completely cleaved into a linear state, thus indicating that the activity of the mutant type Eco31I (Eco 31IM 1) was equivalent to that of the wild type, and the enzyme activity of the Eco31IM1 mutant obtained by purification in example 1 was 10U/. Mu.L.

Example 3Eco31IM1 cleavage efficiency detection comparative experiment

3.1 Experimental procedure

After the calibration of the activity, the wild-type endonuclease Eco31I (Thermo, cat. No. ER 0292) and the mutant endonuclease Eco31I (Eco 31IM 1) were subjected to the same gradient dilution (1/2, 1/4, 1/8, 1/16, 1/32) from 10U/. Mu.L, and 1. Mu.L of each diluted enzyme and a set of restriction enzymes (see Table for specific information of each experimental group) were incubated with 0.5. Mu.g of plasmid pUC19 in a 20. Mu.L reaction system for 5min and 15min, respectively, and the cleavage of the substrate was detected by 1% agarose gel electrophoresis.

The 20. Mu.L enzyme activity assay reaction system was as follows:

3.2 experimental results

As a result, FIG. 3 shows that the gradient dilution set of the wild-type and mutant Eco31I in lanes 1-10 is the cleavage result after 5min of the reaction, and the gradient dilution set of the wild-type and mutant Eco31I in lanes 11-20 is the cleavage result after 15min of the reaction. When the reaction time is 5min, a small amount of non-linearized plasmid still exists after the wild type group (control group) is diluted by 4 times of enzyme amount, the mutant group can still completely linearize the plasmid after being diluted by 8 times, when the reaction time is 15min, the wild type group (control group) still exists after being diluted by 8 times of enzyme amount, the mutant group can still completely linearize the plasmid after being diluted by 16 times, which means that DNA can be completely linearized within 5min after being diluted by 8 times of Eco31I mutant, DNA linearization can not be guaranteed to be completed within 15min after being diluted by 8 times of wild type enzyme, and the enzyme cutting efficiency of the mutant group is higher than that of the wild type group by more than 2 times.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

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 other Eco31I restriction endonuclease equivalent sites 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 at position 283 of SEQ ID NO: 1 or other Eco31I restriction endonuclease equivalent sites is substituted with Q283-. 5. The method, preparation method or Eco31I restriction endonuclease according to claim 4, characterized in that the Eco31I restriction endonuclease has a changed cutting efficiency or a changed expression amount 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 it contains the Eco31I restriction endonuclease according to any one of claims 1 to 6 and a marker for purification. 8. An isolated nucleic acid, a recombinant vector or a 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.