Production method of restriction enzyme PvuI

Abstract

The invention provides a production method of restriction enzyme PvuI. The production method is characterized by comprising the step of carrying out methylation protection on a PvuI recognition sequence by adopting methyltransferase M.XorII. According to the method, methylation protection is carried out on a PvuI recognition sequence by adopting methyltransferase M.XorII, so that a PvuI recognition sequence methylation protection positive strain is successfully constructed, host DNA (Deoxyribose Nucleic Acid) is prevented from being cut by expressed restriction enzyme PvuI, and the methylation protection positive strain can be successfully applied to production of the restriction enzyme PvuI. The PvuI restriction endonuclease can be prepared on a large scale by using a restriction-modification system.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a production method of restriction endonuclease PvuI. Background Art

[0002] There is a "restriction-modification" system in bacteria, that is, their own DNA is modified by methyltransferase, so as to avoid the cutting of their own DNA fragments by the generated restriction endonuclease, so as to achieve the purpose of resisting foreign phage infection. Therefore, a single recombinant expression restriction endonuclease in a prokaryotic expression system will cause the host to die due to the cutting of genomic DNA. The specific modification of the recognition sequence by methyltransferase can prevent the cutting of restriction endonuclease. Restriction-modification genes are often closely linked. Bacteria regulate the expression of both, and the intracellular related methyltransferase methylates the same specific sequence recognized by the paired restriction endonuclease, and makes the modified DNA have the characteristics of resisting the cutting of its restriction endonuclease, protecting the host DNA and degrading the unmethylated foreign DNA.

[0003] PvuI is a type II restriction endonuclease derived from Proteus vulgaris, with a recognition sequence of 5'-CGATCG-3', and is widely used in genetic engineering research. The traditional preparation method is to directly separate and purify PvuI restriction endonuclease from bacteria, which has a long preparation cycle, low yield, and the activity and stability of the extracted PvuI restriction endonuclease are not high.

[0004] In EP0374771A1, only the PvuI restriction endonuclease gene recombinant expression plasmid was constructed for expression, and no methylation protection enzyme plasmid was constructed. However, the PvuI expressed alone can cause the host to die due to the cutting of genomic DNA, so the expression strain cannot be replicated on a large scale. Therefore, it is particularly critical to find a suitable PvuI methylation modification enzyme to carry out methylation protection on the DNA sequence recognized by PvuI in the expression of PvuI restriction endonuclease. Summary of the invention

[0005] The present invention aims to overcome the above-mentioned defects and provides a methyltransferase M.XorII which can be used for methylating the recognition sequence of restriction endonuclease PvuI when it is expressed. M.XorII recognizes and methylates the recognition sequence 5'-CGATCG-3' of PvuI, thereby being able to methylate the recognition sequence of restriction endonuclease PvuI, thereby preventing the expressed restriction endonuclease PvuI from cutting the host DNA and causing the host to be unable to grow normally.

[0006] The invention provides a production method of restriction endonuclease PvuI, which is characterized by comprising the step of using methyltransferase M.XorII to methylate and protect the PvuI recognition sequence.

[0007] The scheme for methylation protection can be selected from: M.XorII methyltransferase methylates the PvuI recognition sequence, or other methyltransferases such as M.Afa22MI can be used to achieve the above purpose.

[0008] Furthermore, the present invention provides a method for producing a restriction endonuclease PvuI, which is also characterized in that: an expression plasmid containing a restriction endonuclease target gene is used to co-transform with a methylation protection plasmid containing a methylation transfer gene.

[0009] Furthermore, the present invention provides a method for producing a restriction endonuclease PvuI, which is also characterized in that the specific production method is as follows: S1. Construction of pACYC184-M.XorII methylation protection enzyme plasmid; S2. Construction of pBAD-PvuI expression plasmid; S3. After the successfully constructed pBAD-PvuI expression plasmid and pACYC184-M.XorII methylation protection enzyme plasmid were co-transformed into competent cells, the positive clones screened out were the PvuI expression strains; S4. Induce the expression of restriction endonuclease PvuI; S5. Purification of restriction endonuclease PvuI.

[0010] Furthermore, the present invention provides a method for producing a restriction endonuclease PvuI, which is also characterized in that: the specific method for constructing the pACYC184-M.XorII methylation protection enzyme plasmid is to optimize the DNA sequence of M.XorII for Escherichia coli codons, amplify the PCR product with synthetic primers and seamlessly clone it into the pACYC184 vector cut with XbaI / KpnI, transform competent cells, and construct the pACYC184-M.XorII methylation protection plasmid.

[0011] Furthermore, the present invention provides a method for producing a restriction endonuclease PvuI, which is also characterized in that: the specific method for constructing the pBAD-PvuI expression plasmid is to add a tag to the C-terminus of the PvuI amino acid sequence, optimize the Escherichia coli codon, amplify the PCR product with synthetic primers and seamlessly clone it into the pBAD vector cut with BamHI / BsaI, transform competent cells, and construct the pBAD-PvuI expression plasmid.

[0012] Furthermore, the present invention provides a method for producing a restriction endonuclease PvuI, which is also characterized in that: after the successfully constructed pBAD-PvuI expression plasmid and the pACYC184-M.XorII methylation protection enzyme plasmid are co-transformed into competent cells, the positive clones screened out are the PvuI expression strains. The specific method is to co-transform the successfully constructed pBAD-PvuI expression plasmid and the pACYC184-M.XorII methylation protection enzyme plasmid into competent cells, and spread them on kanamycin and chloramphenicol double-resistant LB plates to screen positive clones. The positive clones contain two plasmids, pBAD-PvuI and pACYC184-M.XorII. After sequencing verification, the two plasmids are sequenced correctly, and the positive clones with correct sequencing are the PvuI expression strains.

[0013] Furthermore, the present invention provides a method for producing a restriction endonuclease PvuI, which is characterized in that: the specific method of inducing the expression of the restriction endonuclease PvuI is to dilute the constructed PvuI expression strain with a sterile LB liquid culture medium, apply the diluted bacterial solution on a kanamycin and chloramphenicol double-resistant LB plate, and culture it at 37°C overnight; pick a single clone on the plate and inoculate it in an LB liquid culture medium containing kanamycin and chloramphenicol, and place it in a 37°C constant temperature shaker to culture until the bacterial solution concentration OD 600 The expanded culture solution was transferred to LB liquid medium containing kanamycin and chloramphenicol, placed in a 37°C constant temperature shaker, and cultured until the concentration of the bacterial solution OD 600 Not less than 1.2; add inducer and continue induction culture at 37℃.

[0014] Furthermore, the present invention provides a method for producing a restriction endonuclease PvuI, which is also characterized in that: the specific method for purifying the restriction endonuclease PvuI is to obtain a purified protein by subjecting the bacteria after induction culture of the PvuI expression strain to high-pressure lysis, centrifugation, and column chromatography.

[0015] Function and effect of the present invention: The invention provides a new methylation protection method for the expression of restriction endonuclease PvuI, which uses methyltransferase M.XorII to methylate the PvuI recognition sequence, successfully constructs a PvuI recognition sequence methylation protection positive strain, avoids the expressed restriction endonuclease PvuI from cutting host DNA, and the methylation protection positive strain can be successfully applied to the production of restriction endonuclease PvuI. The PvuI restriction endonuclease can be prepared on a large scale using the "restriction-modification" system.

[0016] The expression of a normal restriction endonuclease requires the simultaneous construction of an expression plasmid containing a restriction endonuclease target gene and a methylation protection plasmid containing a methylation transfer gene. After each of the two is successfully constructed, the methylation protection plasmid containing the methylation transfer gene is first transformed into ER2566 competent cells, and then the ER2566 competent cells containing the methylation protection plasmid containing the methylation transfer gene are prepared. Finally, the expression plasmid containing the restriction endonuclease target gene is transferred into the ER2566 competent cells containing the methylation protection plasmid containing the methylation transfer gene to induce the expression of the restriction endonuclease.

[0017] However, in the research of the present invention, it was found that the co-transformation method is also applicable to the production process of the present invention. In the process of the present invention, the pACYC184-M.XorII methylation protection enzyme plasmid and the pBAD-PvuI expression plasmid are first constructed respectively, and then the pBAD-PvuI expression plasmid and the pACYC184-M.XorII methylation protection enzyme plasmid are co-transformed into ER2566 competent cells. There is no need to first transform ER2566 with the pACYC184-M.XorII methylation protection enzyme plasmid and then prepare ER2566+pACYC184-M.XorII competent cells, which simplifies the co-transformation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the plasmid map of pACYC184-M.XorII; Figure 2 is the plasmid map of pBAD-PvuI; Figure 3 This is the result of SDS-PAGE gel electrophoresis after PvuI purification; Figure 4 The results of the activity test of PvuI in this patent and PvuI from a well-known enzyme supplier are compared. DETAILED DESCRIPTION

[0019] The present invention is capable of various modifications and various embodiments, and therefore specific embodiments are illustrated and described in the accompanying drawings. However, this is not intended to limit the present invention to specific implementations, but should be understood to include all modifications, equivalents, and even substitutes that fall within the concept and technical scope of the present invention.

[0020] 1. Construction of pACYC184-M.XorII methylation protection plasmid: Optimize the DNA sequence of M.XorII in E. coli, amplify the PCR product with synthetic primers and seamlessly clone it into the pACYC184 vector digested with XbaI / KpnI, transform DH5α competent cells, and construct pACYC184-M.XorII methylation protection plasmid.

[0021] Among them, the amino acid sequence of M.XorII (SEQ ID NO.1) MTRPIGIDLFAGAGGLSLGFEQAGFDLVAAVDIDPIHCAAHKFNFPKCATVCKSVVDVTGDELRRIAGIGKRDIDIVIGGAPCQGFSLIGKRALDDSRNQLVHHYV RVVMELKPKYFVFENVKGLTVGKHRQFLKEVIEAFQNGGYDVVTDYRVLNAADYGVPQDRRRLILMGARKGLPLPAYPEPTGRTTVGDAIGDIPDAESFPELWERD WVKAKYGKPSTYAAYLRGKKDDPTDFGYRRQFDPMLLTGSLLTDHTELSRQRFAAIAPDDVEPVSRFKKLALNGICNTLRAGTASDRGAFTSPRPIHPTVPRVITVREAARLHSYPDWFRFHATKWHGFRQIGNSVPPLLARAVGGQIMKALRKKPEKPSEMLALGDQSLIGISMTDAAAMFGVSSTVIARRSRPVDRPAPRRHEERELVTA Nucleotide sequence after M.XorII codon optimization for E. coli (SEQ ID NO.2) pACYC184-M.XorII methylation protection enzyme plasmid full-length nucleotide sequence (SEQ ID NO.3) 2. Construction of pBAD-PvuI expression plasmid: Add 6xHis tag to the C-terminus of PvuI amino acid sequence, optimize E. coli codon, amplify PCR product with synthetic primers and seamlessly clone it into pBAD vector digested with BamHI / BsaI, transform DH5α competent cells, and construct pBAD-PvuI expression plasmid.

[0022] PvuI amino acid sequence after adding 6xHis tag (SEQ ID NO.4) MKKNRYESIIEGIFLDKYVDGNDIVEFNRTDIISKSAELDINLPKNIGDVIYSFKYRASLPVSITKKAQNGKEWVIKNIGRSLYCFQQVNYSRILPDMMLSTIKIPDSTPTIVAEHAFNDEQALLTRVRYNRL IDIFTGAVCYSLQNHLRTTVPSVGQIETDEIYVGVDRLGRQFIFPVQAKGGKDELGIVQIEQDFLLCKHKYPNLICRPIATQFISNDKIAIFEFVLENNEVKKLQEKHYLLVGKGQISVDELSNYNFHHHHHH Nucleotide sequence after PvuI codon optimization in E. coli (SEQ ID NO.5) ATGAAAAAAAACCGCTACGAATCTATCATTGAAGGCATTTTCCTGGACAAATACGTAGACGGCAACGACATCGTGGAGTTCAACCGCACTGATATTATCAGCAAGTCTGCTGAACTGGACATCAACCTGCCGAAGAACATCGGCGATGTCATCTACTCCTTCAAGTATCGCGCGTCCCTGCCAGTAAGCATTACGAAAAAGGCGCAAAACGGCAAAGAATGGGTGATCAAAAACATCGGCCGCTCCCTGTACTGTTTCCAGCAGGTTAACTACAGCCGTATCCTGCCGGACATGATGCTGAGCACCATCAAAATTCCGGACAGCACCCCGACCATCGTCGCTGAGCATGCCTTTAATGACGAACAGGCACTGCTGACCCGTGTACGTTACAACCGCCTGATCGACATTTTCACTGGTGCTGTGTGTTACTCCCTGCAGAACCACCTGCGTACCACTGTCCCGTCCGTAGGTCAGATCGAAACCGACGAAATCTATGTTGGCGTTGATCGTCTGGGCCGTCAGTTCATTTTTCCGGTTCAAGCGAAGGGTGGTAAAGACGAACTGGGTATCGTTCAGATCGAACAGGATTTCCTGCTGTGTAAACACAAATACCCGAACCTGATCTGCCGTCCAATCGCAACCCAGTTTATCTCCAACGACAAAATCGCTATCTTCGAGTTTGTTCTGGAGAACAACGAGGTGAAAAAACTGCAGGAAAAACACTACCTGCTGGTTGGCAAAGGTCAGATTAGCGTTGACGAACTGAGCAACTACAACTTTCATCACCACCATCATCAT Full-length nucleotide sequence of pBAD-PvuI expression plasmid (SEQ ID NO.6) 3. The successfully constructed pBAD-PvuI expression plasmid and pACYC184-M.XorII methylation protection enzyme plasmid were co-transformed into ER2566 competent cells, and the positive clones were screened on kanamycin and chloramphenicol double-resistant LB plates. The positive clones contained two plasmids, pBAD-PvuI and pACYC184-M.XorII. After sequencing verification, both plasmids were sequenced correctly. The positive clones with correct sequencing were the PvuI expression strains.

[0023] 4. Induced expression of restriction endonuclease PvuI: Take 1 μl of the constructed PvuI expression strain and dilute it to 500 μl with sterile LB liquid culture medium, then take 100 μl of the diluted bacterial solution and spread it on a kanamycin and chloramphenicol double-resistant LB plate, and culture it at 37°C overnight; pick a single clone on the plate and inoculate it into 50 ml of LB liquid culture medium containing kanamycin and chloramphenicol, place it in a 37°C constant temperature shaker at 220 rpm, and culture it for 4-6 h to make the bacterial solution concentration OD600 = 0.8; transfer 10 ml of the expanded culture bacterial solution to 1 L LB liquid culture medium containing kanamycin and chloramphenicol, place it in a 37°C constant temperature shaker at 220 rpm, and culture it for 3-4 h to make the bacterial solution concentration OD600 = 1.2; add 1 ml of 20% inducer L-arabinose, and continue to induce culture at 37°C for 4 h.

[0024] 5. Purification of restriction endonuclease PvuI: (1) Collect the cells of the PvuI expression strain after induction culture, and lyse them under high pressure at 3-6°C and 800-850Pa to obtain a lysate; (2) Centrifuging the lysate obtained in step (1) at 2-8°C, 10,000 rpm, for 40-60 min, collecting the supernatant to obtain a crude product; (3) The crude product obtained in step (2) is purified by Ni affinity chromatography column, ion exchange chromatography column and heparin affinity chromatography column in sequence to finally obtain a purified PvuI protein sample. The results of SDS-PAGE gel electrophoresis after PvuI purification are as follows: Figure 3 shown.

[0025] 6. PvuI activity detection The enzyme activity of restriction endonuclease PvuI is defined as: the amount of enzyme required to completely digest 1 μg of plasmid in a 50 μl reaction system at 37°C for 1 hour is defined as 1 activity unit (U).

[0026] In a 50 μL enzyme digestion system, the purified PvuI protein sample was diluted in a gradient manner and digested with pBR322 DNA plasmid (containing one PvuI restriction site) as a substrate. Under the same enzyme activity, Figure 4As shown, the restriction endonuclease PvuI expressed and purified by the present invention has a better digestion effect than another well-known enzyme supplier (using the CN109706217A method).

[0027] Comparative test: M.SssI is used as a methyltransferase, which can methylate the C5 position of all cytosine nucleotide bases in unmethylated or hemimethylated double-stranded DNA in the context of 5'-CpG-3'. The recognition sequence of the restriction endonuclease PvuI is CGATCG. In theory, M.SssI methyltransferase can methylate cytosine C on the PvuI recognition sequence CGATCG. However, during the actual research and development implementation process, we found that the methylation degree of the CG sequence of the Escherichia coli genome by M.SssI was too high, which affected transcription and caused the Escherichia coli to grow very slowly, making it unsuitable for large-scale production.

[0028] It can be found that the traditional method directly separates and purifies the PvuI restriction endonuclease from bacteria, the purification process is difficult, and the final product purity and yield are not high; while the co-transformation method of the present invention, on the one hand, the expression plasmid containing the restriction endonuclease target gene is a recombinant plasmid, and a 6XHis tag is added to the C-terminus of the PvuI restriction endonuclease protein sequence, which is easier to purify the protein and the obtained protein purity is higher; on the other hand, the methylation protection plasmid containing the methylation transfer gene expresses the methylation transferase that can specifically methylate the recognition sequence CGATCG of PvuI, thereby avoiding the expressed restriction endonuclease PvuI from cutting the host DNA and causing the host to be unable to grow normally, and is suitable for large-scale production.

[0029] Although the above description is centered on the embodiment, it is only an example and does not limit the present invention. It is clear to those skilled in the art that various modifications and applications not illustrated above can be made within the scope of the essential characteristics of the present embodiment. For example, each component specifically shown in the embodiment can be implemented after being modified. Moreover, various differences related to such modifications and applications should be interpreted as being included in the scope of the present invention as defined in the attached claims.

Claims

1. A method for producing a restriction endonuclease PvuI, characterized in that: The method comprises the step of using methyltransferase M.XorII to methylate and protect the PvuI recognition sequence.

2. A method for producing a restriction endonuclease PvuI as claimed in claim 1, characterized in that: The method is to use an expression plasmid containing a restriction endonuclease target gene and a methylation protection plasmid containing a methylation transfer gene to co-transform.

3. A method for producing a restriction endonuclease PvuI as claimed in claim 1, characterized in that: The specific production method is as follows: S1. Construction of pACYC184-M.XorII methylation protection enzyme plasmid; S2. Construction of pBAD-PvuI expression plasmid; S3. After the successfully constructed pBAD-PvuI expression plasmid and pACYC184-M.XorII methylation protection enzyme plasmid were co-transformed into competent cells, the positive clones screened out were the PvuI expression strains; S4. Induce the expression of restriction endonuclease PvuI; S5. Purification of restriction endonuclease PvuI.

4. A method for producing a restriction endonuclease PvuI as claimed in claim 3, characterized in that: The specific method for constructing the pACYC184-M.XorII methylation protection enzyme plasmid is to optimize the DNA sequence of M.XorII for Escherichia coli codons, amplify the PCR product with synthetic primers and seamlessly clone it into the pACYC184 vector cut with XbaI / KpnI enzymes, transform competent cells, and construct the pACYC184-M.XorII methylation protection plasmid.

5. A method for producing a restriction endonuclease PvuI as claimed in claim 3, characterized in that: The specific method for constructing the pBAD-PvuI expression plasmid is to add a tag to the C-terminus of the PvuI amino acid sequence, optimize the Escherichia coli codon, amplify the PCR product with synthetic primers, seamlessly clone it into the pBAD vector cut with BamHI / BsaI, transform competent cells, and construct the pBAD-PvuI expression plasmid.

6. A method for producing a restriction endonuclease PvuI as claimed in claim 3, characterized in that: The specific method of co-transforming competent cells with the successfully constructed pBAD-PvuI expression plasmid and the pACYC184-M.XorII methylation protection enzyme plasmid to screen out positive clones is the PvuI expression strain, which is as follows: co-transforming competent cells with the successfully constructed pBAD-PvuI expression plasmid and the pACYC184-M.XorII methylation protection enzyme plasmid, coating on kanamycin and chloramphenicol double resistance LB plates to screen positive clones, the positive clones contain the two plasmids pBAD-PvuI and pACYC184-M.XorII, and the two plasmids are sequenced correctly after sequencing verification, and the positive clones with correct sequencing are the PvuI expression strains.

7. A method for producing a restriction endonuclease PvuI as claimed in claim 3, characterized in that: The specific method of inducing the expression of restriction endonuclease PvuI is as follows: after diluting the constructed PvuI expression strain with sterile LB liquid culture medium, applying the diluted bacterial solution on a kanamycin and chloramphenicol double-resistance LB plate, and culturing at 37°C overnight; picking a single clone on the plate and inoculating it in an LB liquid culture medium containing kanamycin and chloramphenicol, placing it in a 37°C constant temperature shaker and culturing it until the bacterial solution concentration OD 600 The expanded cultured bacterial solution was transferred to LB liquid medium containing kanamycin and chloramphenicol, placed in a 37°C constant temperature shaker, and cultured until the bacterial solution concentration OD 600 Not less than 1.2; add inducer and continue induction culture at 37℃.

8. A method for producing a restriction endonuclease PvuI as claimed in claim 3, characterized in that: The specific method for purifying the restriction endonuclease PvuI is to subject the bacterial cells after induction culture of the PvuI expression strain to high-pressure lysis, centrifugation and column chromatography to obtain the purified protein.

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