CN117737097A - Endonuclease VIII, and preparation method and application thereof - Google Patents

Abstract

The application discloses a preparation method and application of endonuclease VIII. The application provides a codon-optimized nucleotide sequence for encoding endonuclease VIII, which can realize a large amount of soluble expression in an escherichia coli system compared with other codon sequences, maintains high biocatalysis activity and is suitable for industrial production.

Description

Endonuclease VIII, and preparation method and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to endonuclease VIII, a preparation method and application thereof.

Background

Endonuclease VIII (Endonuclease VIII, nei) is an eighth endonuclease of E.coli, a monomeric protein of 262 amino acid residues in length and having a molecular weight of 29.7kDa. Endonuclease VIII has the activities of N-terminal glycosylase and AP lyase, is one of important DNA repair enzymes in procaryotic cells, and is mainly used for repairing damaged pyrimidine bases. Endonuclease VIII also has 5' -terminal phosphodiesterase activity and can scavenge 5-deoxyribose produced when processing AP site.

DNA is affected by deamination, oxidation and hydrocarbylation reactions in cells, and a large number of damaged bases are produced, which can lead to gene coding errors and genomic instability, and can lead to chronic diseases associated with aging and cancer. There is an endogenous base excision repair mechanism (BER) in the organism cell, which is started by the N-terminal glycosylase, recognizes and removes the damaged base, forms an AP site, and in the second step, the 3 'or 5' end of the AP site can be cleaved by the AP lyase activity, the AP site is removed, a base gap of 3 'and 5' phosphate is created, and finally repair is completed by DNA polymerase, ligase. Endonuclease VIII catalyzes a wide range of substrates including thymine glycol (Tg), uracil glycol, dihydrothymine, dihydrouracil (Dhu), 5-hydroxycytosine, 5-hydroxyuracil, and b-urea isobutyric acid, among others. In addition, studies have shown that endonuclease VIII has a certain activity on 8-oxoguanine (8-oxoguanine), and may play a standby role in the enzyme system for repairing oxidative damaged purines in DNA. Because of the diversity of endonuclease VIII catalytic substrates, it is of great importance for stabilizing the intracellular base excision repair mechanism system, preserving the integrity of the organism's genome.

Based on the core effect of endonuclease VIII in eliminating DNA damage in vivo, the method can be applied to single cell gel electrophoresis experiments to evaluate oxidative DNA damage in cells and in vitro. In the second generation sequencing technology, the enzyme generating the AP site is skillfully combined with Endonuclease VIII, the template chain containing the AP site is specifically excised, the double-end sequencing of DNA is realized, and the sequencing flux is greatly increased. DNA sequencing technology is the most reliable method for detecting DNA sequences, has huge market demand, and is expensive and imported in dependence despite the commercial endonuclease VIII, thereby being unfavorable for wide basic research and application. The prokaryotic expression system of the escherichia coli has the advantages of simplicity in operation, high yield, short growth period, clear research background, good product stability, easiness in purification, easiness in transfer fermentation batch production and the like, and is beneficial to localization and marketization of the endonuclease VIII, so that the realization of high expression and high activity of the endonuclease VIII by using the prokaryotic expression system has very important significance.

Disclosure of Invention

The invention aims to provide endonuclease VIII.

Another object of the present invention is to provide a method for producing endonuclease VIII.

It is another object of the present invention to provide a polynucleotide sequence encoding endonuclease VIII.

It is another object of the invention to provide a vector that is adapted to the polynucleotide sequence encoding endonuclease VIII.

It is another object of the invention to provide a kit comprising a polynucleotide sequence encoding endonuclease VIII.

To solve the above technical problem, according to a first aspect of the present invention, there is provided a polynucleotide encoding endonuclease VIII, said polynucleotide being codon optimized and said polynucleotide being selected from any one of the following:

(i) Polynucleotides of the sequence shown in SEQ ID NO. 2-3;

(ii) Polynucleotides having greater than 95% homology to the sequences shown in SEQ ID No. 2-3; and

(iii) A polynucleotide complementary to the polynucleotide sequence set forth in (i) or (ii).

In a second aspect of the invention there is provided an expression vector comprising a polynucleotide provided in the first aspect of the invention.

In some preferred embodiments, the expression vector is an E.coli expression vector, more preferably pET-28a (+).

In a third aspect of the invention there is provided a host cell comprising an expression vector provided in the second aspect of the invention; or alternatively

The host cell has integrated into its genome a polynucleotide as provided in the first aspect of the invention.

In some preferred embodiments, the host cell is E.coli (Escherichia coli).

In some preferred embodiments, the host cell is an E.coli BL21 (DE 3) strain.

In a fourth aspect, the present invention provides a method for preparing endonuclease VIII, said method comprising the steps of: culturing the host cell of the third aspect of the invention to express the protein of interest; and

separating the target protein to obtain the endonuclease VIII;

in some preferred embodiments, the host cell is obtained by transforming E.coli with a plasmid comprising a polynucleotide according to the first aspect of the invention.

In some preferred embodiments, the host cells are cultured using SB medium.

In some preferred embodiments, the host cell is cultured in a shaking environment.

In some preferred embodiments, the host cell is cultured at a temperature of 16 to 19 ℃.

In some preferred embodiments, the medium used in culturing the host cell comprises a kanamycin resistance gene.

In some preferred embodiments, the host cell is cultured using IPTG to induce expression of the protein of interest.

In some preferred embodiments, the host cell is cultured to OD when it is cultured ₆₀₀ At 0.6 to 0.8, induction was then performed using IPTG to express the protein of interest.

In some preferred embodiments, the step of isolating the protein of interest comprises:

eluting the crushed target protein supernatant through a chromatographic column when the flow is the same as that of the target protein supernatant, and collecting the eluent.

In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column (Ni-NTA).

In some preferred embodiments, the mobile phase comprises Buffer A, buffer B, and Buffer C, each Buffer comprising Tris, naCl, and glycerol.

In some preferred embodiments Buffer A is 50mM Tris,50mM NaCl,5% glycerol.

In some preferred embodiments, buffer B is 50mM Tris,50mM NaCl,500mM Imidazole,5% glycerol.

In some preferred embodiments, buffer C:20mM Tris,1M NaCl,5% glycerol.

In some preferred embodiments, the pH of Buffer A, buffer B, and Buffer C are each 7.5.

In some preferred embodiments, the step of isolating the protein of interest further comprises:

the collected eluent is treated by an ion exchange column to obtain a treated liquid.

In some preferred embodiments, the step of isolating the protein of interest further comprises: the treatment solution was dialyzed.

In some preferred embodiments, the composition of the dialysate is as follows: 10mM Tris-HCl,250mM NaCl,0.1mM EDTA,50% glycerol, pH 8.0.

In a fifth aspect, the invention provides a kit comprising: a polynucleotide as provided in the first aspect of the invention; or alternatively

An expression vector as provided in the second aspect of the invention; or alternatively

The host cell according to the third aspect of the invention.

Compared with the prior art, the invention has at least the following advantages:

the invention provides a codon-optimized nucleotide sequence for encoding endonuclease VIII, which can realize a large amount of soluble expression in an escherichia coli system compared with other codon sequences, maintains high biocatalysis activity and is suitable for industrial production.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings.

FIG. 1 is a SDS-PAGE identification of endonuclease VIII products according to an embodiment of the present invention;

FIG. 2 is a diagram of the affinity purification of an endonuclease VIII nickel column in an embodiment in accordance with the present invention;

FIG. 3 is a graph showing the results of endonuclease VIII cation column purification in accordance with an embodiment of the present invention;

FIG. 4 is a graph showing the results of electrophoresis of the endonuclease VIII enzyme activity test in accordance with the embodiment of the present invention.

Detailed Description

In the prior art, the escherichia coli expressed endonuclease VIII enzyme product has low soluble expression and poor activity, and aiming at the problems, the inventor screens out optimized codons I and II for encoding the endonuclease VIII enzyme shown in the invention through a large number of experiments, and the optimized codons I and II can realize supernatant expression under an escherichia coli system, thereby completing the invention.

Furthermore, the inventors have further optimized the culture conditions, which are suitable for low-temperature expression of SB medium at 16-19℃and found that the soluble expression is highest under these conditions.

Coding endonuclease VIII polynucleotide sequences

The present invention relates to a polynucleotide sequence (gene sequence of interest) encoding endonuclease VIII.

As used herein, the term "endonuclease VIII" refers to E.coli eighth endonuclease, a monomeric protein of 262 amino acid residues in length and having a molecular weight of 29.7kDa. Endonuclease VIII has the activities of N-terminal glycosylase and AP lyase, is one of important DNA repair enzymes in procaryotic cells, and is mainly used for repairing damaged pyrimidine bases. Endonuclease VIII also has 5' -terminal phosphodiesterase activity and can scavenge 5-deoxyribose produced when processing AP site. In an embodiment of the invention, the amino acid sequence of endonuclease VIII is shown in SEQ ID NO. 1 of the invention.

In the present invention, the problem of reduced yield when expressing heterologous proteins in E.coli is overcome by synonymous codon bias optimization, and the present invention relates to synonymous codon bias optimized polynucleotide sequences. And (3) carrying out synonymous codon preference optimization on the obtained target gene sequence, wherein the target gene sequence subjected to synonymous codon preference optimization can express the amino acid sequence identical to the target protein. In some embodiments of the invention, the sequence after synonymous codon optimization is shown as optimized codon I (SEQ ID NO. 2), optimized codon II (SEQ ID NO. 3), optimized codon III (SEQ ID NO. 4), and optimized codon IV (SEQ ID NO. 5). In a more preferred embodiment, the endonuclease VIII encoding polynucleotide sequence is selected from any one of the following: (i) a polynucleotide having a sequence shown in SEQ ID NO. 2-3; (ii) A polynucleotide having a homology of more than 80% (more preferably more than 85%, more preferably more than 90%, more preferably more than 95%) with the sequence shown in SEQ ID No. 2-3; and (iii) a polynucleotide complementary to the polynucleotide sequence set forth in (i) or (ii). The inventors have found that transformation of E.coli with a plasmid containing optimized codon II can result in more supernatant expression.

The full-length sequence of the polynucleotide encoding endonuclease VIII of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining the genes of the present invention. Primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

Expression vector containing target gene

The invention also relates to vectors comprising the polynucleotides of the invention. "vector" in the present invention means a linear or circular DNA molecule comprising a fragment encoding a protein of interest operably linked to other fragments providing for its transcription. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, a vector, and the like. The vector fragment may be derived from the host organism, another organism, plasmid or viral DNA, or may be synthetic. The vector may be any expression vector, synthetic or conveniently subjected to recombinant DNA procedures, the choice of vector generally being dependent on the host cell into which the vector is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one that, when introduced into the host cell, integrates into the host cell genome and replicates with the chromosome with which it is integrated. In one embodiment, the vector of the invention is an expression vector. In one embodiment of the invention pET-28a (+) is selected as a vector to obtain higher expression efficiency.

Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequences of the proteins of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Illustratively, the insertion of the exogenous DNA fragment is accomplished by cleaving the vector DNA molecule with a DNA endonuclease into a linear molecule that can be linked to the exogenous gene, and then ligating the codon optimized fragment of the gene of interest to the vector, optionally with a single restriction site cohesive end ligation, double restriction site directional cloning, cohesive end ligation of different restriction sites, blunt end ligation, artificial linker ligation, or end ligation with an oligonucleotide.

Host cells containing genes of interest

The invention also relates to host cells genetically engineered with the vectors or coding sequences of the invention. The vector containing the codon-optimized gene of interest may be inserted, transfected or otherwise transformed into a host cell by known methods to obtain a transformant containing the codon-optimized gene of interest of the present invention and capable of expressing the protein of interest. A "host cell" in the present invention is a cell into which an exogenous polynucleotide and/or vector has been introduced. The host cell may be a eukaryotic host cell or a prokaryotic host cell, the host cell is preferably a bacterium, and is preferably E.coli, more preferably E.coli ROSETTA (DE 3) strain (Escherichia coli Rosetta (DE 3) strain).

Method for producing target protein

The invention also relates to a method for preparing the target protein, and the polynucleotide sequence can be used for expressing or producing recombinant protein. Generally, there are the following steps:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a protein of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) A host cell cultured in a suitable medium;

(3) Separating and purifying the protein from the culture medium or the cells.

Wherein, the transformation or transduction of the recombinant expression vector containing the polynucleotide of the step (1) into a suitable host cell can be performed by conventional techniques well known to those skilled in the art, and when the host is E.coli, a heat shock method, an electrotransformation method, etc. can be selected.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used in the culture may be selected from a variety of conventional media, preferably SB, TB, LB or SOC media. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time. In order to promote the expression of the target protein and to increase the expression level of the soluble protein, a preferred embodiment of the present invention uses a host cell cultured in SB medium, and the medium used contains a kanamycin resistance gene. The inventor has verified through experiments that the culture conditions are the same, and the yield of the target protein expressed by using the supernatant of the SB culture medium is higher than that of the SOC culture medium.

To further promote soluble expression of the protein of interest, in a preferred embodiment of the invention, the host cell is cultured to OD ₆₀₀ After 0.6-0.8 induction with IPTG and incubation continued at low temperature for about 8 to 12 hours. The inventors have experimentally verified that other conditions, which are the same, allow higher soluble expression to be obtained in low temperature cultures, e.g., 17 to 19℃compared to high temperature cultures or room temperature cultures, e.g., about 37℃or about 25 ℃.

The protein in the above method may be expressed in the cell, or on the cell membrane, or secreted outside the cell. If desired, the proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Thus, in the present invention, after the successful culture to obtain the target protein, it also involves a step of separating and purifying it, for example, separating and purifying the protein from the culture medium to obtain the target protein in high purity. Although methods for purifying the protein of interest may be conventional means well known to those skilled in the art, including but not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods. In a preferred embodiment of the present invention, the expression product is purified using Ni column affinity chromatography and ion exchange. In affinity chromatography, the composition of the solution used affects the chromatographic effect, and in a preferred embodiment of the invention, the Buffer A solution used consists of: buffer A50mM Tris,50mM NaCl,5% glycerol, pH 7.5; buffer B50mM Tris,50mM NaCl,500mM Imidazole,5% glycerol, pH 7.5 and Buffer C:20mM Tris,1M NaCl,5% glycerol, pH 7.5; lysis Buffer:50mM Tris,300mM NaCl,5% glycerol, 30mM Imidazole,pH 7.5. In a preferred embodiment of the invention, the method further comprises a dialysis step after the Ni column affinity chromatography and the ion exchange method, wherein the dialysis solution comprises the following components: 10mM Tris-HCl,250mM NaCl,0.1mM EDTA,50% glycerol, pH 8.0.

In the present disclosure, any exemplary or exemplary language (e.g., ") provided for certain embodiments herein is used merely to better present the disclosure and does not limit the scope of the disclosure as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

If the definition or use of a term in a reference does not conform to the definition of the term as described herein, then the definition of the term as described herein is used, rather than the definition of the term in the reference.

Various terms used herein are shown below. If a term used in a claim is not defined below, the broadest definition persons in the pertinent art have given that term are given as reflected in publications or issued patents that are printed at the time of application.

As used herein, the term "isolated" refers to a nucleic acid or polypeptide that is separated from at least one other component (e.g., a nucleic acid or polypeptide) that the nucleic acid or polypeptide is found in its natural source. In one embodiment, the nucleic acid or polypeptide is found to be present only (if any) in solvents, buffers, ions or other components that are normally present in its solution. The terms "isolated" and "purified" do not include nucleic acids or polypeptides that are present in their natural source.

As used herein, the terms "polynucleotide" and "polynucleotide sequence" may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

The invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the invention. Variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.

As used herein, the term "codon optimization" refers to a manner of improving the efficiency of gene synthesis by avoiding the use of low-availability or rare codons according to codon usage differences exhibited by organisms (including e.coli, yeast, mammalian cells, plant cells, insect cells, etc.) that actually do protein expression or production.

As used herein, the terms "homology" and "identity" are used interchangeably to refer to the percentage of identical (i.e., identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by the following methods. The nucleotide or amino acid sequence of a polynucleotide or polypeptide is aligned, the number of positions in the aligned polynucleotide or polypeptide that contain the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotide or polypeptide that contain a different nucleotide or amino acid residue. Polynucleotides may differ at one position, for example, according to the inclusion of different nucleotides (i.e., substitutions or variations) or deletions of nucleotides (i.e., insertions or deletions of one or two nucleotides in the polynucleotide). The polypeptides may differ at one position, for example, by containing an amino acid (i.e., substitution or variation) or a deletion of an amino acid (i.e., an amino acid or deletion of an amino acid inserted into one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

As used herein, the terms "sequence complementary" and "reverse sequence complementary" are used interchangeably to refer to a sequence that is opposite in direction to and complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is actaac, its reverse complement is GTTCAGT.

As used herein, the term "expression" includes any step involving the production of a polypeptide in a host cell, including, but not limited to, transcription, translation, post-translational modification, and secretion. After expression, the host cells or expression products can be harvested, i.e.recovered.

The present invention will be further described with reference to specific embodiments in order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, it is to be noted that the terms used herein are used merely to describe specific embodiments and are not intended to limit the exemplary embodiments of this application.

Example 1

In this example, a plasmid containing a target protein was prepared. The method comprises the following specific steps:

the protein sequence (SEQ ID NO: 1) of the escherichia coli endonuclease VIII provided by NCBI is taken as a reference, the experimental design requirement of the invention is combined, the escherichia coli codon optimization is carried out for a plurality of times on the basis of the base sequence of the escherichia coli endonuclease VIII, and after the optimization of the synonymous codon preference of the escherichia coli, a plurality of optimized codon sequences such as an endonuclease VIII optimized codon I (SEQ ID NO: 2), an endonuclease VIII optimized codon II (SEQ ID NO: 3), an endonuclease VIII optimized codon III (SEQ ID NO: 4) and an endonuclease VIII optimized codon IV (SEQ ID NO: 5) are obtained through the optimization of the synonymous codon preference of the escherichia coli, and the connecting vector is pET-28a (+) and is synthesized by Nanjing gold Style biotechnology Co.

Protein sequence (SEQ ID NO: 1):

MPEGPEIRRAADNLEAAIKGKPLTDVWFAFPQLKPYQSQLIGQHVTHVETRGKALLTHFSNDLTLYSHNQLYGVWRVVDT

GEEPQTTRVLRVKLQTADKTILLYSASDIEMLTPEQLTTHPFLQRVGPDVLDPNLTPEVVKERLLSPRFRNRQFAGLLLD

QAFLAGLGNYLRVEILWQVGLTGNHKAKDLNAAQLDALAHALLEIPRFSYATRGQVDENKHHGALFRFKVFHRDGEPCER

CGSIIEKTTLSSRPFYWCPGCQH

optimized codon I SEQ ID NO. 2:

ATGCCGGAAGGTCCGGAAATCCGTCGTGCTGCTGACAACCTGGAAGCTGCTATCAAAGGTAAACCGCTGACCGACGTTTG

GTTCGCTTTCCCGCAGCTGAAACCGTACCAGTCTCAGCTGATCGGTCAGCACGTTACCCACGTTGAAACCCGTGGTAAAG

CTCTGCTGACCCACTTCTCTAACGACCTGACCCTGTACTCTCACAACCAGCTGTACGGTGTTTGGCGTGTTGTTGACACC

GGTGAAGAACCGCAGACCACCCGTGTTCTGCGTGTTAAACTGCAGACCGCTGACAAAACCATCCTGCTGTACTCTGCTTC

TGACATCGAAATGCTGACCCCGGAACAGCTGACCACCCACCCGTTCCTGCAGCGTGTTGGTCCGGACGTTCTGGACCCGA

ACCTGACCCCGGAAGTTGTTAAAGAACGTCTGCTGTCTCCGCGTTTCCGTAACCGTCAGTTCGCTGGTCTGCTGCTGGAC

CAGGCTTTCCTGGCTGGTCTGGGTAACTACCTGCGTGTTGAAATCCTGTGGCAGGTTGGTCTGACCGGTAACCACAAAGC

TAAAGACCTGAACGCTGCTCAGCTGGACGCTCTGGCTCACGCTCTGCTGGAAATCCCGCGTTTCTCTTACGCTACCCGTG

GTCAGGTTGACGAAAACAAACACCACGGTGCTCTGTTCCGTTTCAAAGTTTTCCACCGTGACGGTGAACCGTGCGAACGT

TGCGGTTCTATCATCGAAAAAACCACCCTGTCTTCTCGTCCGTTCTACTGGTGCCCGGGTTGCCAGCAC

optimized codon II SEQ ID NO:3:

ATGCCAGAAGGTCCTGAAATTCGCCGTGCAGCTGATAACCTGGAAGCGGCGATCAAAGGTAAGCCGCTGACTGATGTCTG

GTTCGCTTTCCCGCAGCTGAAACCGTACCAGAGCCAACTGATCGGTCAACATGTGACTCACGTCGAAACCCGTGGTAAGG

CACTGCTGACCCATTTCTCCAACGATCTGACTCTGTACTCCCACAATCAGCTGTACGGTGTTTGGCGCGTTGTGGATACC

GGTGAAGAGCCTCAGACTACTCGCGTTCTGCGCGTAAAACTGCAAACCGCGGATAAAACCATCCTGCTGTACTCCGCGTC

CGACATCGAAATGCTGACCCCTGAACAACTGACTACGCACCCGTTTCTGCAGCGTGTGGGCCCGGACGTTCTGGATCCGA

ATCTGACCCCGGAAGTAGTAAAGGAGCGCCTGCTGTCTCCGCGTTTCCGTAACCGCCAGTTTGCTGGTCTGCTGCTGGAT

CAGGCATTCCTGGCTGGTCTGGGTAACTACCTGCGTGTTGAAATCCTGTGGCAGGTCGGCCTGACTGGCAACCACAAAGC

GAAGGATCTGAACGCCGCCCAGCTGGATGCACTGGCTCATGCGCTGCTGGAAATCCCGCGTTTCAGCTACGCTACTCGCG

GTCAGGTGGACGAAAACAAACACCACGGTGCACTGTTCCGTTTCAAGGTTTTTCACCGCGACGGCGAACCATGTGAACGC

TGTGGTAGCATCATCGAAAAGACCACGCTGTCCTCTCGCCCGTTCTACTGGTGCCCGGGCTGCCAGCAC

optimized codon III SEQ ID NO 4:

ATGCCGGAAGGCCCGGAAATTCGTCGTGCAGCGGATAATCTGGAAGCCGCGATTAAAGGTAAACCGCTGACCGATGTGTG

GTTTGCCTTCCCGCAGCTGAAACCGTATCAGAGCCAGCTGATCGGCCAGCATGTGACCCATGTGGAAACCCGCGGCAAAG

CGCTGCTGACCCATTTTAGTAATGATCTGACCCTGTATAGCCATAATCAGCTGTATGGCGTGTGGCGCGTGGTGGATACC

GGCGAAGAACCGCAGACGACCCGTGTGCTGCGCGTGAAACTGCAGACCGCGGATAAAACCATTCTGCTGTATAGCGCCAG

CGATATTGAAATGCTGACCCCGGAACAGCTGACCACCCACCCGTTTCTGCAGCGCGTGGGCCCGGATGTGCTGGATCCGA

ATCTGACCCCGGAAGTGGTGAAAGAACGCCTGCTGAGCCCGCGCTTTCGCAATCGCCAGTTCGCGGGCCTGCTGCTGGAC

CAGGCGTTTCTGGCAGGCCTGGGTAATTACCTGCGTGTGGAAATTCTGTGGCAAGTTGGCCTGACCGGTAACCATAAAGC

CAAAGATCTGAATGCGGCACAGCTGGATGCGCTGGCGCATGCGCTGCTGGAGATTCCGCGCTTTAGCTATGCGACCCGCG

GCCAGGTGGATGAAAACAAACATCATGGCGCCCTGTTTCGTTTTAAAGTCTTCCATCGCGATGGCGAACCGTGCGAACGT

TGCGGTAGCATTATTGAAAAAACCACCCTGAGCAGCCGCCCGTTTTACTGGTGCCCGGGCTGCCAGCAT

optimization codon IV SEQ ID NO:5:

ATGCCAGAAGGTCCAGAAATTCGTCGTGCTGCTGATAACTTAGAAGCTGCTATTAAAGGTAAACCATTAACTGATGTTTG

GTTCGCTTTCCCACAATTAAAACCATATCAATCTCAATTAATTGGTCAACACGTTACTCACGTTGAAACTCGTGGTAAAG

CTTTATTAACTCACTTCTCTAACGATTTAACTTTATATTCTCACAACCAATTATATGGTGTTTGGCGTGTTGTTGATACT

GGTGAAGAACCACAAACTACTCGTGTTTTACGTGTTAAATTACAAACTGCTGATAAAACTATTTTATTATATTCTGCTTC

TGATATTGAAATGTTAACTCCAGAACAATTAACTACTCACCCATTCTTACAACGTGTTGGTCCAGATGTTTTAGATCCAA

ACTTAACTCCAGAAGTTGTTAAAGAACGTTTATTATCTCCACGTTTCCGTAACCGTCAATTCGCTGGTTTATTATTAGAT

CAAGCTTTCTTAGCTGGTTTAGGTAACTATTTACGTGTTGAAATTTTATGGCAAGTTGGTTTAACTGGTAACCACAAAGC

TAAAGATTTAAACGCTGCTCAATTAGATGCTTTAGCTCACGCTTTATTAGAAATTCCACGTTTCTCTTATGCTACTCGTG

GTCAAGTTGATGAAAACAAACACCACGGTGCTTTATTCCGTTTCAAAGTTTTCCACCGTGATGGTGAACCATGTGAACGT

TGTGGTTCTATTATTGAAAAAACTACTTTATCTTCTCGTCCATTCTATTGGTGTCCAGGTTGTCAACAC

example 2

In this example, a host cell containing a host cell encoding a target protein was obtained by transforming a host cell with the plasmid prepared in example 1. The method comprises the following specific steps:

taking 1 mu L of plasmid, adding the plasmid into 30 mu L of escherichia coli competent cells BL21 (DE 3) under ice bath condition, standing for 20min in ice bath, carrying out heat shock for 45s in water bath at 42 ℃, standing for 2min on ice immediately, adding 400 mu L of SOC culture medium without antibiotics, and carrying out shaking culture at 220rpm for 50min at 37 ℃. mu.L of the bacterial liquid was uniformly spread on LB plates containing 100. Mu.g/mL of kana resistance, and incubated overnight at 37 ℃.

Example 3

In this example, endonuclease VIII enzyme was prepared to be expressed in a soluble manner.

The monoclonal of example 2 was picked, inoculated in SB medium containing 100. Mu.g/mL of kana resistance, and shake-cultured at 37℃at 220rpm to OD ₆₀₀ Between 0.6 and 0.8, IPTG was induced (final concentration 0.1 mM), and the culture was carried out at 18℃overnight with shaking, without addition of IPTG as a control, for 3 hours, and each experiment was repeated. Sampling ultrasonic disruption is used for SDS-PAGE identification, and the identification result is shown in figure 1.

In FIG. 1, lanes 2 and 3 are bands corresponding to the monoclonal culture product containing the optimized codon I, lanes 4 and 5 are bands corresponding to the monoclonal culture product containing the optimized codon II, lanes 8 and 9 are bands corresponding to the monoclonal culture product containing the optimized codon III, and lanes 10 and 11 are bands corresponding to the monoclonal culture product containing the optimized codon IV. The results showed that at 18℃the expression of Endonuclease VIII enzyme was soluble in the supernatant in SB medium with the optimized codon I and optimized codon II, while the expression levels in SB medium were very low with the optimized codon III and optimized codon IV. The expression level of the monoclonal culture supernatant containing the optimized codon II is highest and higher than that of other optimized codons. The protein molecular weight was about 37Kda and the predicted protein size (30 Kda) was substantially consistent on the Expasy website.

Example 4

In this example, endonuclease VIII enzyme prepared in example 3 was purified.

Taking a monoclonal containing an optimized codon II, culturing 1.5L of bacterial liquid in an SB culture medium in a shaking flask, wherein the expression condition is consistent with the expression of the target protein in the example 3. And (5) centrifuging and collecting thalli. About 20g of the cells were weighed, and 100ml of Lysis Buffer was added thereto to resuspend the cells on ice. Crushing with homogenizer, centrifuging at 12000rpm and 4deg.C for 50min, collecting supernatant, and filtering with 0.22 μm needle filter to obtain supernatant. Subjecting the supernatant to Ni-column affinity chromatography using HisTrap as purification column ^TM HP,0-80% buffer B, and subjecting to ion exchange eluting with HisTrap ^TM SP-HP was eluted with 0-60% buffer C to give 56ml of protein, which was dialyzed overnight in a dialysis solution, and then the concentration was measured by BCA method, and the electrophoresis chart was shown in FIG. 2. The expression content of the target protein is calculated to be 0.3mg/mL, and the protein yield is very high. The concentrations of the solutions used are as follows:

Buffer A:50mM Tris，50mM NaCl，5％Glycerol，pH 7.5；

Buffer B:50mM Tris，50mM NaCl，500mM Imidazole，5％Glycerol，pH 7.5；

Buffer C：50mM Tris，1M NaCl，5％Glycerol，pH 7.5；

Lysis Buffer：50mM Tris，300mM NaCl，5％Glycerol，30mM Imidazole，pH 7.5；

dialysis Buffer:10mM Tris-HCl,250mM NaCl,0.1mM EDTA,50%Glycerol,pH 8.0.

Example 5

In this example, the activity of Endonuclease VIII enzyme purified in example 4 was examined. The method comprises the following specific steps:

(1) Experimental materials

1) Sample: endo VIII enzyme (20221011 batch)

2) And (3) equipment: PCR amplification instrument, chemiluminescence fluorescence imaging system.

3) Measuring a movable bottom object:

Test1：FMA-GATTTCATTTTTATTUATAACTTTACTTATATTG-FAM(SEQ ID NO:6)

Test2：CAATATAAGTAAAGTTATAAATAAAAATGAAATC(SEQ ID NO:7)

the 2 substrates were synthesized and purified by HPLC.

4) Reagent: self-produced UDG enzyme (HY 202201 batch, 10U/. Mu.L), rCutSmart ^TM Buffer (NEB), endo VIII (NEB, 10U/. Mu.L), formamide Loading Buffer, urea PAGE gel.

5) Endo VIII enzyme dilution: 10mM Tris-HCl,250mM NaCl,0.1mM EDTA,50%Glycerol pH 8@25 ℃.

(2) Experimental procedure

NEB positive enzyme was diluted as follows:

the Endo VIII prepared in the present invention was diluted to 2, 4, 8, 16, 32, 64, 128 as shown in the following Table

Gradient of	Sample to be diluted	Endo VIII enzyme dilution (ul)	Dilution factor
				1	Stock solution 10ul	10	2
2	Gradient 1 10ul	10	4
				3	Gradient 210 ul	10	8
4	Gradient 3 10ul	10	16
				5	Gradient 4. Mu.l	10	32
6	Gradient 5 10ul	10	64
				7	Gradient 6 10ul	10	128

Substrate formulation

Deionized water is added into the substrate according to the required amount of the manufacturer, the mixture is fully and uniformly mixed to prepare 100pmol/ul of mother liquor, 20ul of sterilized water is added into 100pmol/ul of mother liquor to prepare 10pmol/ul, and 20ul of sterilized water is added into 10pmol/ul to prepare 5pmol/ul. Placing the substrate at 95 ℃ for 3min, and naturally cooling and annealing for standby.

Reaction system configuration (Single reaction system)

Reagent(s)	1 person/ul
		10x rCutSmart Buffer	1
Substrate (5 pmol/ul)	1.2
		ddH2O	5.8
UDG(1U/ul)	1

The system is configured according to the volume of (N+3), N is the number of diluted samples, 9 mu L/hole of the prepared reaction system is added into 8 connecting pipes, 1 ul/pipe of each sample to be tested is added into a flick mixing system, and the mixture is centrifuged until bubbles basically disappear. An NTC set was additionally provided and 1. Mu.L of enzyme dilution was added to 9. Mu.L of the reaction system. The PCR amplification apparatus was set to a reaction program of 37℃for 1h and 95℃for 15min, and 8 sets were discharged until the reaction was completed. Adding 10 mu L of formamide Loading Buffer into the reacted system, uniformly mixing, naturally cooling at 95 ℃ for 10min, and carrying out urea PAGE (voltage: 120V, time: 40-50 min). After electrophoresis, urea PAGE gels were scanned for blue light channels on a ChampChemi 910PLUS chemiluminescent fluorescence imaging system.

The results of the electrophoresis of Endo VIII enzyme assay are shown in FIG. 4 below. Analysis of results: the Endo VIII enzyme prepared in the invention has consistent trend after dilution by 32 times and dilution by 2 times with positive enzyme, and the activity is 16 times of that of the positive enzyme, and the activity of the enzyme prepared in the invention is 160U/. Mu.L.

It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples of carrying out the invention and that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Claims (10)

1. An isolated polynucleotide encoding endonuclease VIII, wherein said polynucleotide is codon optimized and said polynucleotide is selected from any one of the following:

(i) A polynucleotide as shown in SEQ ID NO. 2;

(ii) A polynucleotide having a homology of greater than 95% with the sequence shown in SEQ ID NO. 2; and

(iii) A polynucleotide complementary to the sequence set forth in (i) or (ii).

2. An expression vector comprising the polynucleotide of claim 1.

3. The expression vector according to claim 2, characterized in that it is an escherichia coli expression vector, preferably pET-28a (+).

4. A host cell comprising the expression vector of claim 2 or 3; or alternatively

The polynucleotide of claim 1 integrated into the genome of the host cell.

5. A method for preparing endonuclease VIII, said method comprising the steps of:

transforming a host cell with a vector comprising the polynucleotide of claim 1;

culturing the host cell to express the endonuclease VIII.

6. The method of claim 5, wherein the host cell is cultured using SB medium.

7. The method of claim 5, wherein the host cell is cultured at a temperature of 16 to 19 ℃.

8. The method according to claim 5, wherein the medium used in culturing the host cell comprises a kanamycin resistance gene;

and/or, when the host cell is cultured, the target protein is expressed by IPTG induction.

9. The method according to claim 6, characterized in that the method further comprises the step of: isolating the protein of interest, the step of isolating the protein of interest comprising: eluting the crushed target protein supernatant with a chromatographic column when the flow is the same, collecting the eluent,

the mobile phase comprises Buffer A, buffer B and Buffer C, and each Buffer contains Tris, naCl and glycerol.

10. A kit, comprising: the polynucleotide of claim 1; or alternatively

The expression vector of claim 2 or 3; or alternatively

The host cell of claim 4.