CN116426546A

CN116426546A - Recombinant cholesterol oxidase, and preparation method and application thereof

Info

Publication number: CN116426546A
Application number: CN202111654036.8A
Authority: CN
Inventors: 蒋析文; 朱伟伟; 张伟; 孟晓琳; 郑桑桑
Original assignee: Guangzhou Da'an Gene Co ltd
Current assignee: Guangzhou Da'an Gene Co ltd
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2023-07-14

Abstract

The application discloses a recombinant cholesterol oxidase and a preparation method and application thereof. In the present application, a polynucleotide encoding a cholesterol oxidase is codon optimized and the polynucleotide is selected from any one of the following: (i) a polynucleotide having a sequence as set forth in SEQ ID NO. 1; (ii) A polynucleotide having a homology of greater than 95% with the sequence shown in SEQ ID NO. 1; and (iii) a polynucleotide having a sequence complementary to the polynucleotide described in (i) or (ii). The invention develops a cholesterol oxidase fermentation system based on genetic engineering, is more suitable for an escherichia coli expression system through a large number of codon optimization, greatly improves the preparation efficiency, and has high occupation ratio of the obtained soluble cholesterol oxidase, and is easy to purify and mass production.

Description

Recombinant cholesterol oxidase, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to recombinant cholesterol oxidase and a preparation method and application thereof.

Background

Cholesterol oxidase (Cholesterol oxidase or Chox for short) is an oxidoreductase of a flavoprotein which catalyzes cholesterol to cholest-4-en-3-one and hydrogen peroxide, is a key enzyme in cholesterol metabolism (Kreit J, sampson N.Cholesterol oxidase: physiological functions [ J ]. Febs J,2010,276 (23): 6844-6856), mainly catalyzes dehydrogenation of the cholesterol carbon 3 site, and is also an isomerase, and thus cholesterol oxidase is a bifunctional enzyme (Lata Kumari, shamsher Kanwar. Purification and characterization of an extracellular cholesterol oxidase of Bacillus subtilis isolated from tiger excreta [ J ]. Applied Biochemistry and Biotechnology,2016,178 (2): 353-367). Its molecular weight is 30kD, encoding 504 amino acids, and in clinical applications Chox is a useful analytical tool, which has been used for biocatalytically producing a number of steroids, as insecticidal proteins against bollworm larvae, in particular as diagnostic enzymes for determining serum cholesterol levels (Pollegioni L, piubelli L, molla G.Cholesterol oxidase: biotechnological applications. FEBS J.2009 Dec;276 (23): 6857-70.Epub 2009 Oct 16.PMID:19843167). In bacteria, chOx is the first enzyme in the catalytic pathway to produce propionic acid and acetate as end products. Furthermore, chOx has no homologous genes in mammals. An important aspect of cholesterol oxidase (3 b-hydroxysteroid oxidase) catalysis is its ability to bind to lipid bilayer containing sterol substrates. Efficient catalytic turnover is affected by the binding of proteins to membranes and the solubility of substrates in the lipid bilayer.

The traditional cholesterol oxidase production method comprises an extraction separation method, a chemical synthesis method and an animal and plant cell culture method. These methods have long production cycle, low efficiency and low activity of the product. The microorganism has the advantages of diversity, rapid propagation, easy generation of genetic variation, easy separation and purification of extracellular enzyme, and the produced protein has the advantages of wider pH, reaction temperature range and substrate specificity, thus being very suitable for mass synthesis of the protein. Therefore, it is important to develop a stable, high-yield preparation method of cholesterol oxidase based on a microbial fermentation system, which is easy to purify.

Disclosure of Invention

The invention aims to provide a preparation method of cholesterol oxidase, which ensures that the prepared cholesterol oxidase has good stability, higher yield, easy purification, easy fermentation mass production and lower cost.

To solve the above technical problem, the first aspect of the present invention provides a polynucleotide encoding cholesterol oxidase, which is codon optimized, and which is selected from any one of the following:

(i) A polynucleotide having a sequence as shown in SEQ ID NO. 1;

(ii) A polynucleotide having a homology of greater than 95% with the sequence shown in SEQ ID NO. 1; and

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

In some preferred embodiments, the polynucleotide is an isolated polynucleotide.

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

In some preferred embodiments, the expression vector comprises a polynucleotide sequence that expresses a His X6 tag, more preferably, the expression vector has the 3' end of the polynucleotide linked to a polynucleotide sequence that expresses a His X6 tag.

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

In a third aspect the invention provides a host cell comprising an expression vector provided in accordance with 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.

In a fourth aspect, the present invention provides a method of preparing cholesterol oxidase, the 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 cholesterol oxidase;

wherein the target protein has an amino acid sequence shown as SEQ ID NO. 2.

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

A host cell according to the third aspect of the invention; or alternatively

Or cholesterol oxidase prepared according to the method of the fourth aspect of the invention.

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

(1) According to the method provided by the invention, the cholesterol oxidase is successfully expressed by using an escherichia coli expression system through a codon optimization sequence, and the method has the advantages of high yield, short growth cycle and good product stability;

(2) The method provided by the invention adopts the recombinant fusion expression of the cholesterol oxidase of the genetically engineered escherichia coli, realizes high yield of the soluble expression protein in an escherichia coli system, is easy to purify, has catalytic activity, and is easy to transfer fermentation batch 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 diagram showing the structure of SDS-PAGE identification of cholesterol oxidase according to an embodiment of the present invention;

FIG. 2 is an electrophoresis diagram of purification of cholesterol oxidase Ni cartridge according to an embodiment of the present invention;

FIG. 3 is an electrophoresis chart obtained after dialysis by cholesterol oxidase according to an embodiment of the present invention;

FIG. 4 is a graph of cholesterol oxidase standard according to an embodiment of the present invention.

Detailed Description

In the prior art, the cholesterol oxidase has long production period and low product activity. The invention develops a cholesterol oxidase fermentation system based on genetic engineering, is more suitable for an escherichia coli expression system through a large number of codon optimization, greatly improves the preparation efficiency, and has high occupation ratio of the obtained soluble cholesterol oxidase, and is easy to purify and mass production.

In some embodiments of the invention there is provided an isolated polynucleotide encoding a cholesterol oxidase, said polynucleotide being codon optimized and said polynucleotide being selected from any one of the following:

(i) A polynucleotide having a sequence as shown in SEQ ID NO. 1;

In other embodiments of the invention there is provided an expression vector comprising a polynucleotide provided in accordance with the first aspect of the invention.

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

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

In other embodiments of the present invention, there is provided a method for preparing cholesterol oxidase, the method comprising the steps of:

separating the target protein to obtain the cholesterol oxidase;

wherein the target protein has an amino acid sequence shown as SEQ ID NO. 2.

In some preferred embodiments, the host cell is cultured to express the protein of interest by IPTG induction.

In some preferred embodiments, the step of isolating the protein of interest comprises: purification and/or dialysis using a Ni column.

In other embodiments of the invention, there is provided a kit comprising: a polynucleotide as provided in the first aspect of the invention; or alternatively

A host cell according to the third aspect of the invention; or alternatively

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 is inconsistent or inconsistent with the definition of that term described herein, the definition of the term described herein applies and the definition of the term in the reference does not apply.

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 blood 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 nucleotides 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, then its reverse complement is GTTCAT.

As used herein, the term "host cell" is a cell into which an exogenous polynucleotide and/or vector has been introduced. The host cell is a eukaryotic host cell or a prokaryotic host cell. Preferably a prokaryotic host cell, such as an E.coli cell.

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.

As used herein, the term "expression vector" refers to a linear or circular DNA molecule comprising a fragment encoding a polypeptide of interest operably linked to other fragments that provide 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 expression vector fragment may be derived from the host organism, another organism, plasmid or viral DNA, or may be synthetic. The expression vector may be any expression vector, either 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.

As used herein, the term "expression system" includes a vector comprising a host and a gene of interest, and a system that enables expression of the gene of interest in the host, in particular, by selection of successfully transfected recombinant host cells by a vector comprising a foreign gene encoding a protein of interest. Expression systems are divided into eukaryotic expression systems and prokaryotic expression systems, with a prokaryotic expression system being selected in one preferred embodiment herein. The prokaryotic expression system has the characteristics of rapid proliferation of host bacteria, simple culture, convenient operation, low price, definite genetic background, safe genetic genes, high protein expression level and the like. However, prokaryotic expression systems do not control expression time and expression levels. In addition, in the expression system of prokaryotes, since there is a possibility that the expression product exists as an enclosure, the biological activity is low, and the post-translational processing and modification system is incomplete (for example, glycosylation modification cannot be performed).

[ preparation of target protein ]

The full-length nucleotide sequence of the target protein or its element or a fragment thereof of the present invention can be usually obtained by PCR amplification, recombinant methods or artificial synthesis. 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.

The invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered with the vectors or fusion protein coding sequences of the invention, and methods for producing the proteins of the invention by recombinant techniques.

The polynucleotide sequences of the present invention may be used to express or produce recombinant proteins by conventional recombinant DNA techniques. 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.

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.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is E.coli, a heat shock method, an electrotransformation method or the like 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. The medium used in the culture may be selected from various conventional media depending on the host cell used. 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.

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. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, 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.

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, optimization of synonymous codon bias of E.coli and construction of plasmid

The amino acid sequence (shown as SEQ ID NO: 2) of Streptomyces cholesterol oxidase provided by NCBI is taken as a reference, and after optimization of the synonymous codon preference of Escherichia coli (the optimized base sequence is shown as SEQ ID NO:1 (3)), the ligation vector is ps28a, and the ligation vector is synthesized by Nanjing Jinsri biotechnology Co.

SEQ ID NO:1：

ATGAGTTCAAATGATGCTGAAAAACCCCTAGCGGCCTCCCGTCGTGATTTTCTGACCGGTTCGCTTAAGCTGGCGTCCGCAGGCGCGCTGGCCGGCTGGCTGCCGGCGGAGCGCATTATGGCAGGCAGACTGACGTGCTCGCAACCGAACAACTTTCCGGCGGAAATTCCGCTGTATAAGCAGTCTTTTAAAAATTGGGCCGGCGACATCAAAGTCGATGACGTGTGGACGTGCGCACCGCGTAGCGCGGATGAGGTCGTCAAGGTGGCTAACTGGGCAAAAGATAATGGCTACAAAGTACGCGCCCGTGGTATGATGCACAACTGGAGCCCGCTCACTCTCGCCGCGGGTGTCAGCTGTCCGGCGGTTGTTTTGCTGGACACCACGCGTTATCTGACCGCTATGAGCATCGATGCTTCCGGCCCTGTTGCGAAGGTTACGGCCCAAGCAGGTATCACCATGGAAGCACTGCTGACCGGCCTGGAAAAAGCTGGTCTGGGTGTTACCGCGGCTCCCGCGCCTGGTGATCTGACGTTAGGTGGTGTTTTAGCGATTAACGGTCATGGCACCGCGATCCCGGCAAAAGGTGAACGTCGCTTGGCTGGTGCATCCTATGGCAGCATCAGCAATCTGGTGTTGAGCTTGACCGCAGTCGTTTATGACAAGGCGTCTGGCGCGTACGCGCTGCGCAAATTCGCCCGTAATGATCCGCAGATTGCGCCGCTGCTGGCCCACGTGGGTCGTTCTCTGATTGTTGAGGCCACCCTGCAGGCTGCTCCGAACCAGAGATTGCGCTGCCAGAGCTGGTTTAACATTCCGTACGGCGAGATGTTTGCAGCGGCGGGCTCCGGTGGTCGTACCTTCGCCTCGTACCTGGATAGCGCAGGTAGGGTGGAGGCAATCTGGTTCCCGTTCACCAGCAATCCGTGGCTGAAGGTGTGGACCGTTACCCCGAACAAACCGCTGTTCTCTCGTCAGACCGATAAACCGTTCAACTATCCGTTCAGCGATAACCTGCCGGACGAGGTGACGGACCTGGCCAACAAAATCCTCAGCTTGGGCGACGGCAAGCTGACGCCGGCGTTTGGTAAAGCGCAATTTGCGGCCGCGTCAGCAGGTTTGGTTGCGACTGCGAGCTGGGACCTGTGGGGTTGGTCCAAGAACCTGCTGTTGTACGTGAAGCCGACCACCTTGCGTGTCACGGCAAACGGTTATGCCGTGCTAACTCGTCGTGAGAACGTTCAACGTGTGCTGAATGAATTTGTCACTTTCTACCAAGCTCGCGTGCAGGCGTATCAACAACAGGGTAGATACCCGATGAATGGACCAGTTGAGATCCGCGTGACCGGTCTCGACGACCCGAGCGAAGCGGCTTTGAGCGGTGGCGTTGCTCCAGCGTTGTCTGCGATCCGTCCGCGTCCGGATCATCCGGAGTGGAACGTGGCGGTTTGGCTGGACATCCTGACCTTACCGGGCACCCCTTACGCGAACCAGTTCTATCGTGAAATCGAGCAGTGGATTGAAGCGAACTTCAACGGTTCCTATGCTGCGGTGCGCCCAGAATGGTCAAAGGGTTGGGGCTACACCGACCAAGCGGCGTGGGCTGATAGCGCAATGCTGCAAACCACAATTCCGAATGCATTTCGTGCTGGCCAGCCGGCAGCGGCTAATTGGGATGCAGCTAAGGCGGCGCTGGCGGCGTACGACCCGTACCGCTTGTTCAGCTCTCCGCTGCTGGACTCCTTGGGCTTGTAA

SEQ ID NO:3：

CATATGAGTTCAAATGATGCTGAAAAACCCCTAGCGGCCTCCCGTCGTGATTTTCTGACCGGTTCGCTTAAGCTGGCGTCCGCAGGCGCGCTGGCCGGCTGGCTGCCGGCGGAGCGCATTATGGCAGGCAGACTGACGTGCTCGCAACCGAACAACTTTCCGGCGGAAATTCCGCTGTATAAGCAGTCTTTTAAAAATTGGGCCGGCGACATCAAAGTCGATGACGTGTGGACGTGCGCACCGCGTAGCGCGGATGAGGTCGTCAAGGTGGCTAACTGGGCAAAAGATAATGGCTACAAAGTACGCGCCCGTGGTATGATGCACAACTGGAGCCCGCTCACTCTCGCCGCGGGTGTCAGCTGTCCGGCGGTTGTTTTGCTGGACACCACGCGTTATCTGACCGCTATGAGCATCGATGCTTCCGGCCCTGTTGCGAAGGTTACGGCCCAAGCAGGTATCACCATGGAAGCACTGCTGACCGGCCTGGAAAAAGCTGGTCTGGGTGTTACCGCGGCTCCCGCGCCTGGTGATCTGACGTTAGGTGGTGTTTTAGCGATTAACGGTCATGGCACCGCGATCCCGGCAAAAGGTGAACGTCGCTTGGCTGGTGCATCCTATGGCAGCATCAGCAATCTGGTGTTGAGCTTGACCGCAGTCGTTTATGACAAGGCGTCTGGCGCGTACGCGCTGCGCAAATTCGCCCGTAATGATCCGCAGATTGCGCCGCTGCTGGCCCACGTGGGTCGTTCTCTGATTGTTGAGGCCACCCTGCAGGCTGCTCCGAACCAGAGATTGCGCTGCCAGAGCTGGTTTAACATTCCGTACGGCGAGATGTTTGCAGCGGCGGGCTCCGGTGGTCGTACCTTCGCCTCGTACCTGGATAGCGCAGGTAGGGTGGAGGCAATCTGGTTCCCGTTCACCAGCAATCCGTGGCTGAAGGTGTGGACCGTTACCCCGAACAAACCGCTGTTCTCTCGTCAGACCGATAAACCGTTCAACTATCCGTTCAGCGATAACCTGCCGGACGAGGTGACGGACCTGGCCAACAAAATCCTCAGCTTGGGCGACGGCAAGCTGACGCCGGCGTTTGGTAAAGCGCAATTTGCGGCCGCGTCAGCAGGTTTGGTTGCGACTGCGAGCTGGGACCTGTGGGGTTGGTCCAAGAACCTGCTGTTGTACGTGAAGCCGACCACCTTGCGTGTCACGGCAAACGGTTATGCCGTGCTAACTCGTCGTGAGAACGTTCAACGTGTGCTGAATGAATTTGTCACTTTCTACCAAGCTCGCGTGCAGGCGTATCAACAACAGGGTAGATACCCGATGAATGGACCAGTTGAGATCCGCGTGACCGGTCTCGACGACCCGAGCGAAGCGGCTTTGAGCGGTGGCGTTGCTCCAGCGTTGTCTGCGATCCGTCCGCGTCCGGATCATCCGGAGTGGAACGTGGCGGTTTGGCTGGACATCCTGACCTTACCGGGCACCCCTTACGCGAACCAGTTCTATCGTGAAATCGAGCAGTGGATTGAAGCGAACTTCAACGGTTCCTATGCTGCGGTGCGCCCAGAATGGTCAAAGGGTTGGGGCTACACCGACCAAGCGGCGTGGGCTGATAGCGCAATGCTGCAAACCACAATTCCGAATGCATTTCGTGCTGGCCAGCCGGCAGCGGCTAATTGGGATGCAGCTAAGGCGGCGCTGGCGGCGTACGACCCGTACCGCTTGTTCAGCTCTCCGCTGCTGGACTCCTTGGGCTTGTAACTCGAG

SEQ ID NO:2：

MSSNDAEKPLAASRRDFLTGSLKLASAGALAGWLPAERIMAGRLTCSQPNNFPAEIPLYKQSFKNWAGDIKVDDVWTCAPRSADEVVKVANWAKDNGYKVRARGMMHNWSPLTLAAGVSCPAVVLLDTTRYLTAMSIDASGPVAKVTAQAGITMEALLTGLEKAGLGVTAAPAPGDLTLGGVLAINGHGTAIPAKGERRLAGASYGSISNLVLSLTAVVYDKASGAYALRKFARNDPQIAPLLAHVGRSLIVEATLQAAPNQRLRCQSWFNIPYGEMFAAAGSGGRTFASYLDSAGRVEAIWFPFTSNPWLKVWTVTPNKPLFSRQTDKPFNYPFSDNLPDEVTDLANKILSLGDGKLTPAFGKAQFAAASAGLVATASWDLWGWSKNLLLYVKPTTLRVTANGYAVLTRRENVQRVLNEFVTFYQARVQAYQQQGRYPMNGPVEIRVTGLDDPSEAALSGGVAPALSAIRPRPDHPEWNVAVWLDILTLPGTPYANQFYREIEQWIEANFNGSYAAVRPEWSKGWGYTDQAAWADSAMLQTTIPNAFRAGQPAAANWDAAKAALAAYDPYRLFSSPLLDSLGL

Introduction of the recombinant plasmid into the host E.coli: taking 1 mu L of expression plasmid, adding the expression plasmid into 30 mu L of escherichia coli competent BL21 (DE 3) under ice bath condition, standing in ice bath for 30min, standing in water bath at 42 ℃ for 45s, standing on ice immediately for 2min, adding 400 mu L of SOC culture medium without antibiotics, and culturing at 37 ℃ and 230rpm for 45min in a shaking way. 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 2 expression of the protein of interest (cholesterol oxidase)

Coli transformed with the recombinant plasmid prepared in example 1 was picked, inoculated in 100. Mu.g/mL of kana-resistant TB medium in a sterile procedure, each medium was subjected to 2-tube replicates, designated TB (1), TB (2), shaking culture at 37℃at 220rpm until OD600 was between 0.6 and 0.8, induction with IPTG, shaking culture at 37℃and 18℃overnight, respectively. SDS-PAGE identification by sonication of samples, the results of FIG. 1 show that a large number of protein soluble expression occurs in TB medium at 18 ℃. The sample was sonicated for SDS-PAGE identification and the results are shown in FIG. 1.

According to FIG. 1, a large number of protein soluble expressions occurred in TB medium at 18 ℃. The target protein is expressed in a small amount in the background, expressed in a large amount in the supernatant at 37 ℃, expressed in a large amount in the sediment, expressed in a large amount in the supernatant at 18 ℃, and expressed in a large amount in the sediment.

EXAMPLE 3 Ni column purification of target protein

Buffers were prepared as in table 1 below.

TABLE 1

Reagent(s)	BufferA	BufferB	Lysis Buffer
				Tris	50mM	50mM	50mM
NaCl	50mM	50mM	300mM
				Glycerol
	5％	5％	5％
				Imidazole	-	500mM	-
pH	8.0	8.0	8.0

4g of cells were sonicated, then 0.22 μm was subjected to membrane filtration, and purified with 1mL of Ni-NTA. The flow rate was 0.5mL/min, and the loading was done using 20mL Lysis Buffer rinse UV and conductance to baseline. The elution procedure included: step 1:0% B,8CV,1mL/min; step 2, 0-60% B,20CV,1mL/min; step 3:100% B,8CV,1mL/min. The electrophoresis pattern after purification and sampling is shown in FIG. 2.

In FIG. 2, 1-12 are crude, permeate, and eluents of different BufferB concentrations, respectively. Wherein, 0% B (sample No. 3-6) is insufficient in 1mL Ni column load, and part of the sample is penetrated out; 26% B-100% B (sample No. 7-12) eluted a large amount of the target protein. Collecting the 5-6 tube and the 8-12 tube respectively, wherein the sample collection volume of the 5-6 tube is 2.7mL; the sample collection volume of the No. 8-12 tube is 9.3mL.

And (3) enzyme cutting: the volume of the sample collected by the No. 5-6 tube is 2.7mL, 27 mu L of UPLC enzyme is added for overnight digestion, the volume of the sample collected by the No. 8-12 tube is 9.3mL, and 93 mu L of UPLC enzyme is added for overnight digestion.

The result of electrophoresis after cleavage is shown in FIG. 3. The UPLC enzyme of Nos. 5-6 was slightly insufficient and the sample showed a band at 18 kD. Placing into dialysis bag for overnight dialysis after enzyme digestion, wherein the volume after dialysis is 3.6mL, the concentration measured by BCA is 16.84mg/mL, the yield is 11.54mg, R ² ＝0.996。

Example 4 recombinant cholesterol oxidase Activity assay

Step 1, preparing a solution

13mM cholesterol solution: 0.25g cholesterol (purchased from Sigma) was weighed, added to 2.5ml of LTriton X-100 and dissolved by heating. Adding 20mL of deionized water, boiling for 30-60 s, cooling, and gently stirring until the solution becomes clear. After adding 2g of sodium cholate for dissolution, deionized water was added to make up 50mL.

0.1% 4-aminoantipyrine (4.92 mM): 0.05g of 4-aminoantipyrine (purchased from Sigma) powder was weighed into 50mL of deionized water and stored in the dark.

60mM phenol: 0.056g of phenol powder was weighed and dissolved in 10mL of deionized water and stored in a dark place.

0.2M PBS pH 7.0: firstly, preparing 0.2M mother solution of sodium dihydrogen phosphate and disodium hydrogen phosphate, and mixing according to the proportion of 1:1.56.

5mL of the working solution was prepared as shown in Table 2 below.

TABLE 2

Reagent(s)	Volume added	Final concentration
			0.1% 4-aminoantipyrine	1.42mL	1.4mM
60mM phenol	1.66mL	20mM
			Peroxidase (2U/. Mu.L)	12.5μL	25U
0.2M PBS pH 7.0	1.25mL	50mM
			Cholesterol solution	0.35mL	0.89mM
ddH ₂ O	307.5μL	-

Step 2, preparing a solution

Preparation of positive enzyme (purchased from Shanghai Seiyaka Biotech Co., ltd.): 200U positive enzyme was dissolved in 0.2mL of PBS pH 7.4 buffer (containing 50% glycerol) to prepare 1U/. Mu.L of enzyme solution, which was gradually diluted again according to the gradient, and the dilution was PBS pH 7.4 buffer.

The microplate reader was preheated for 30min and the incubation temperature was set at 37 ℃.

100. Mu.L of working solution was added to the 96-well ELISA plate, OD 1 was measured at 500nm, 1. Mu.L of each concentration of enzyme was then added thereto, and the reaction was carried out for 3 minutes, and OD 2 was measured at 500 nm.

The standard curve is drawn by taking the enzyme concentration as the X axis and the corresponding absorbance value as the Y axis, and the result of the cholesterol oxidase standard curve is shown in figure 4. A2-A1 was calculated.

The absorbance of the cholesterol oxidase prepared in the example was measured according to the above method, and the results are shown in table 3 below:

TABLE 3 Table 3

The stock solution had a concentration of 16.84mg/mL and an activity of 0.038U/. Mu.L, so that the specific activity was (0.038X 1000)/16.84=2.23U/mg.

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.

SEQUENCE LISTING

<110> Guangzhou da An Gene Co., ltd

<120> recombinant cholesterol oxidase, and preparation method and application thereof

<130> P210839-1CNCNA9

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 1755

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

atgagttcaa atgatgctga aaaaccccta gcggcctccc gtcgtgattt tctgaccggt 60

tcgcttaagc tggcgtccgc aggcgcgctg gccggctggc tgccggcgga gcgcattatg 120

gcaggcagac tgacgtgctc gcaaccgaac aactttccgg cggaaattcc gctgtataag 180

cagtctttta aaaattgggc cggcgacatc aaagtcgatg acgtgtggac gtgcgcaccg 240

cgtagcgcgg atgaggtcgt caaggtggct aactgggcaa aagataatgg ctacaaagta 300

cgcgcccgtg gtatgatgca caactggagc ccgctcactc tcgccgcggg tgtcagctgt 360

ccggcggttg ttttgctgga caccacgcgt tatctgaccg ctatgagcat cgatgcttcc 420

ggccctgttg cgaaggttac ggcccaagca ggtatcacca tggaagcact gctgaccggc 480

ctggaaaaag ctggtctggg tgttaccgcg gctcccgcgc ctggtgatct gacgttaggt 540

ggtgttttag cgattaacgg tcatggcacc gcgatcccgg caaaaggtga acgtcgcttg 600

gctggtgcat cctatggcag catcagcaat ctggtgttga gcttgaccgc agtcgtttat 660

gacaaggcgt ctggcgcgta cgcgctgcgc aaattcgccc gtaatgatcc gcagattgcg 720

ccgctgctgg cccacgtggg tcgttctctg attgttgagg ccaccctgca ggctgctccg 780

aaccagagat tgcgctgcca gagctggttt aacattccgt acggcgagat gtttgcagcg 840

gcgggctccg gtggtcgtac cttcgcctcg tacctggata gcgcaggtag ggtggaggca 900

atctggttcc cgttcaccag caatccgtgg ctgaaggtgt ggaccgttac cccgaacaaa 960

ccgctgttct ctcgtcagac cgataaaccg ttcaactatc cgttcagcga taacctgccg 1020

gacgaggtga cggacctggc caacaaaatc ctcagcttgg gcgacggcaa gctgacgccg 1080

gcgtttggta aagcgcaatt tgcggccgcg tcagcaggtt tggttgcgac tgcgagctgg 1140

gacctgtggg gttggtccaa gaacctgctg ttgtacgtga agccgaccac cttgcgtgtc 1200

acggcaaacg gttatgccgt gctaactcgt cgtgagaacg ttcaacgtgt gctgaatgaa 1260

tttgtcactt tctaccaagc tcgcgtgcag gcgtatcaac aacagggtag atacccgatg 1320

aatggaccag ttgagatccg cgtgaccggt ctcgacgacc cgagcgaagc ggctttgagc 1380

ggtggcgttg ctccagcgtt gtctgcgatc cgtccgcgtc cggatcatcc ggagtggaac 1440

gtggcggttt ggctggacat cctgacctta ccgggcaccc cttacgcgaa ccagttctat 1500

cgtgaaatcg agcagtggat tgaagcgaac ttcaacggtt cctatgctgc ggtgcgccca 1560

gaatggtcaa agggttgggg ctacaccgac caagcggcgt gggctgatag cgcaatgctg 1620

caaaccacaa ttccgaatgc atttcgtgct ggccagccgg cagcggctaa ttgggatgca 1680

gctaaggcgg cgctggcggc gtacgacccg taccgcttgt tcagctctcc gctgctggac 1740

tccttgggct tgtaa 1755

<210> 2

<211> 584

<212> PRT

<213> person (Homo sapiens)

<400> 2

Met Ser Ser Asn Asp Ala Glu Lys Pro Leu Ala Ala Ser Arg Arg Asp

1 5 10 15

Phe Leu Thr Gly Ser Leu Lys Leu Ala Ser Ala Gly Ala Leu Ala Gly

20 25 30

Trp Leu Pro Ala Glu Arg Ile Met Ala Gly Arg Leu Thr Cys Ser Gln

35 40 45

Pro Asn Asn Phe Pro Ala Glu Ile Pro Leu Tyr Lys Gln Ser Phe Lys

50 55 60

Asn Trp Ala Gly Asp Ile Lys Val Asp Asp Val Trp Thr Cys Ala Pro

65 70 75 80

Arg Ser Ala Asp Glu Val Val Lys Val Ala Asn Trp Ala Lys Asp Asn

85 90 95

Gly Tyr Lys Val Arg Ala Arg Gly Met Met His Asn Trp Ser Pro Leu

100 105 110

Thr Leu Ala Ala Gly Val Ser Cys Pro Ala Val Val Leu Leu Asp Thr

115 120 125

Thr Arg Tyr Leu Thr Ala Met Ser Ile Asp Ala Ser Gly Pro Val Ala

130 135 140

Lys Val Thr Ala Gln Ala Gly Ile Thr Met Glu Ala Leu Leu Thr Gly

145 150 155 160

Leu Glu Lys Ala Gly Leu Gly Val Thr Ala Ala Pro Ala Pro Gly Asp

165 170 175

Leu Thr Leu Gly Gly Val Leu Ala Ile Asn Gly His Gly Thr Ala Ile

180 185 190

Pro Ala Lys Gly Glu Arg Arg Leu Ala Gly Ala Ser Tyr Gly Ser Ile

195 200 205

Ser Asn Leu Val Leu Ser Leu Thr Ala Val Val Tyr Asp Lys Ala Ser

210 215 220

Gly Ala Tyr Ala Leu Arg Lys Phe Ala Arg Asn Asp Pro Gln Ile Ala

225 230 235 240

Pro Leu Leu Ala His Val Gly Arg Ser Leu Ile Val Glu Ala Thr Leu

245 250 255

Gln Ala Ala Pro Asn Gln Arg Leu Arg Cys Gln Ser Trp Phe Asn Ile

260 265 270

Pro Tyr Gly Glu Met Phe Ala Ala Ala Gly Ser Gly Gly Arg Thr Phe

275 280 285

Ala Ser Tyr Leu Asp Ser Ala Gly Arg Val Glu Ala Ile Trp Phe Pro

290 295 300

Phe Thr Ser Asn Pro Trp Leu Lys Val Trp Thr Val Thr Pro Asn Lys

305 310 315 320

Pro Leu Phe Ser Arg Gln Thr Asp Lys Pro Phe Asn Tyr Pro Phe Ser

325 330 335

Asp Asn Leu Pro Asp Glu Val Thr Asp Leu Ala Asn Lys Ile Leu Ser

340 345 350

Leu Gly Asp Gly Lys Leu Thr Pro Ala Phe Gly Lys Ala Gln Phe Ala

355 360 365

Ala Ala Ser Ala Gly Leu Val Ala Thr Ala Ser Trp Asp Leu Trp Gly

370 375 380

Trp Ser Lys Asn Leu Leu Leu Tyr Val Lys Pro Thr Thr Leu Arg Val

385 390 395 400

Thr Ala Asn Gly Tyr Ala Val Leu Thr Arg Arg Glu Asn Val Gln Arg

405 410 415

Val Leu Asn Glu Phe Val Thr Phe Tyr Gln Ala Arg Val Gln Ala Tyr

420 425 430

Gln Gln Gln Gly Arg Tyr Pro Met Asn Gly Pro Val Glu Ile Arg Val

435 440 445

Thr Gly Leu Asp Asp Pro Ser Glu Ala Ala Leu Ser Gly Gly Val Ala

450 455 460

Pro Ala Leu Ser Ala Ile Arg Pro Arg Pro Asp His Pro Glu Trp Asn

465 470 475 480

Val Ala Val Trp Leu Asp Ile Leu Thr Leu Pro Gly Thr Pro Tyr Ala

485 490 495

Asn Gln Phe Tyr Arg Glu Ile Glu Gln Trp Ile Glu Ala Asn Phe Asn

500 505 510

Gly Ser Tyr Ala Ala Val Arg Pro Glu Trp Ser Lys Gly Trp Gly Tyr

515 520 525

Thr Asp Gln Ala Ala Trp Ala Asp Ser Ala Met Leu Gln Thr Thr Ile

530 535 540

Pro Asn Ala Phe Arg Ala Gly Gln Pro Ala Ala Ala Asn Trp Asp Ala

545 550 555 560

Ala Lys Ala Ala Leu Ala Ala Tyr Asp Pro Tyr Arg Leu Phe Ser Ser

565 570 575

Pro Leu Leu Asp Ser Leu Gly Leu

580

<210> 3

<211> 1764

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

catatgagtt caaatgatgc tgaaaaaccc ctagcggcct cccgtcgtga ttttctgacc 60

ggttcgctta agctggcgtc cgcaggcgcg ctggccggct ggctgccggc ggagcgcatt 120

atggcaggca gactgacgtg ctcgcaaccg aacaactttc cggcggaaat tccgctgtat 180

aagcagtctt ttaaaaattg ggccggcgac atcaaagtcg atgacgtgtg gacgtgcgca 240

ccgcgtagcg cggatgaggt cgtcaaggtg gctaactggg caaaagataa tggctacaaa 300

gtacgcgccc gtggtatgat gcacaactgg agcccgctca ctctcgccgc gggtgtcagc 360

tgtccggcgg ttgttttgct ggacaccacg cgttatctga ccgctatgag catcgatgct 420

tccggccctg ttgcgaaggt tacggcccaa gcaggtatca ccatggaagc actgctgacc 480

ggcctggaaa aagctggtct gggtgttacc gcggctcccg cgcctggtga tctgacgtta 540

ggtggtgttt tagcgattaa cggtcatggc accgcgatcc cggcaaaagg tgaacgtcgc 600

ttggctggtg catcctatgg cagcatcagc aatctggtgt tgagcttgac cgcagtcgtt 660

tatgacaagg cgtctggcgc gtacgcgctg cgcaaattcg cccgtaatga tccgcagatt 720

gcgccgctgc tggcccacgt gggtcgttct ctgattgttg aggccaccct gcaggctgct 780

ccgaaccaga gattgcgctg ccagagctgg tttaacattc cgtacggcga gatgtttgca 840

gcggcgggct ccggtggtcg taccttcgcc tcgtacctgg atagcgcagg tagggtggag 900

gcaatctggt tcccgttcac cagcaatccg tggctgaagg tgtggaccgt taccccgaac 960

aaaccgctgt tctctcgtca gaccgataaa ccgttcaact atccgttcag cgataacctg 1020

ccggacgagg tgacggacct ggccaacaaa atcctcagct tgggcgacgg caagctgacg 1080

ccggcgtttg gtaaagcgca atttgcggcc gcgtcagcag gtttggttgc gactgcgagc 1140

tgggacctgt ggggttggtc caagaacctg ctgttgtacg tgaagccgac caccttgcgt 1200

gtcacggcaa acggttatgc cgtgctaact cgtcgtgaga acgttcaacg tgtgctgaat 1260

gaatttgtca ctttctacca agctcgcgtg caggcgtatc aacaacaggg tagatacccg 1320

atgaatggac cagttgagat ccgcgtgacc ggtctcgacg acccgagcga agcggctttg 1380

agcggtggcg ttgctccagc gttgtctgcg atccgtccgc gtccggatca tccggagtgg 1440

aacgtggcgg tttggctgga catcctgacc ttaccgggca ccccttacgc gaaccagttc 1500

tatcgtgaaa tcgagcagtg gattgaagcg aacttcaacg gttcctatgc tgcggtgcgc 1560

ccagaatggt caaagggttg gggctacacc gaccaagcgg cgtgggctga tagcgcaatg 1620

ctgcaaacca caattccgaa tgcatttcgt gctggccagc cggcagcggc taattgggat 1680

gcagctaagg cggcgctggc ggcgtacgac ccgtaccgct tgttcagctc tccgctgctg 1740

gactccttgg gcttgtaact cgag 1764

Claims

1. A polynucleotide encoding a cholesterol oxidase, wherein said polynucleotide is codon optimized and said polynucleotide is selected from any one of the following:

(i) A polynucleotide having a sequence as shown in SEQ ID NO. 1;

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

3. The expression vector of claim 2, wherein the expression vector comprises a polynucleotide sequence that expresses a His x 6 tag. Preferably, in the expression vector, a polynucleotide sequence expressing a His×6 tag is ligated to the 3' end of the polynucleotide of claim 1.

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

5. A host cell comprising the expression vector of any one of claims 2 to 4; or alternatively

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

6. The host cell of claim 5, wherein the host cell is E.coli.

7. A method of preparing cholesterol oxidase, said method comprising the steps of:

culturing the host cell of claim 5 or 6 to express the protein of interest; and

separating the target protein to obtain the cholesterol oxidase;

wherein the target protein has an amino acid sequence shown as SEQ ID NO. 2.

8. The method of claim 7, wherein the host cell is cultured to express the protein of interest by IPTG induction.

9. The method of claim 7, wherein the step of isolating the protein of interest comprises isolating the protein of interest using a Ni column.

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

The expression vector of any one of claims 2 to 4; or alternatively

The host cell of claim 5 or 6; or alternatively

Or cholesterol oxidase prepared according to the method of any one of claims 7 to 9.