Process for the preparation and use of cystathionine-beta-synthase

Abstract

The application discloses a preparation method and application of cystathionine-beta-synthase. The expression method of the cystathionine-beta-synthase based on a prokaryotic expression system is developed in the application, the soluble expression amount of the prepared cystathionine-beta-synthase is large, the cystathionine-beta-synthase is suitable for industrial production, the product has good stability, and the activity is high.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the preparation method and application of cystathionine-β-synthase. Background Technology

[0002] Cystathionine β-synthase (CBS) is located in the cytoplasm and consists of a homotetramer composed of four identical subunits. It has a relative molecular weight of 63 kDa and is a pyridoxal phosphate (PLP)-dependent enzyme. The active site of CBS is composed of a polypeptide from glutamate (Glu) at position 37 to arginine (Arg) at position 413, a highly conserved sequence. In addition to substrate binding sites such as homocysteine ​​(Hcy) and serine, the active site of CBS also contains three ligand binding sites: PLP, S-adenosylmethionine, and heme.

[0003] Catalyzed by vitamin B6, cystathionine-β-synthetase synthesizes cystathionine from serine and homocysteine ​​(HCY). As a key enzyme affecting Hcy levels, cystathionine-β-synthetase plays a crucial role in the transsulfate pathway of Hcy to cystathionine. Elevated plasma homocysteine ​​concentrations are considered an independent risk factor for vascular diseases, including cerebrovascular disease, coronary heart disease, and thrombosis. Cyathionine-β-synthetase is widely used to detect homocysteine ​​in blood to predict the occurrence of vascular diseases. However, the natural content of cystathionine-β-synthetase is very low, isolating large quantities of the natural product is difficult, and the cost of synthesizing the peptide is high, limiting its application prospects. Some researchers have also attempted to develop genetically engineered methods for preparing cystathionine-β-synthetase, but most of these methods have low yields and use inclusion body expression, resulting in poor practicality and hindering industrial production. Therefore, there is still a need in this field to develop a genetically engineered method for preparing cystathionine-β-synthase to improve yield and soluble expression content. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing cystathionine-β-synthase.

[0005] Another object of the present invention is to provide a multinucleotide sequence encoding cystathionine-β-synthase.

[0006] Another object of the present invention is to provide a vector adapted to a polynucleotide sequence encoding cystathionine-β-synthase.

[0007] Another object of the present invention is to provide a kit containing a polynucleotide sequence encoding cystathionine-β-synthase.

[0008] To address the aforementioned technical problems, in a first aspect, the present invention provides a polynucleotide encoding cystathionine-β-synthase, wherein the polynucleotide is codon-optimized and selected from any of the following:

[0009] (i) Polynucleotides with sequences as shown in SEQ ID NO.1-8;

[0010] (ii) polynucleotides with greater than 95% homology to sequences shown in SEQ ID NO. 1-8; and

[0011] (iii) A polynucleotide complementary to the polynucleotide sequence described in (i) or (ii).

[0012] In some preferred embodiments, the cystathionine-β-synthase is derived from Saccharomyces cerevisiae or from human sources.

[0013] In a second aspect, the present invention provides an expression vector comprising the polynucleotide provided in the first aspect of the present invention.

[0014] In some preferred embodiments, the expression vector is an Escherichia coli expression vector, more preferably pET-28a(+).

[0015] In a third aspect, the present invention provides a host cell comprising the expression vector provided in the second aspect of the present invention; or

[0016] The host cell genome integrates polynucleotides as provided in the first aspect of the present invention.

[0017] In some preferred embodiments, the host cell is *Escherichia coli*.

[0018] In some preferred embodiments, the host cell is Escherichia coli BL21(DE3) strain.

[0019] A fourth aspect of this invention provides a method for preparing cystathionine-β-synthase, the method comprising the steps of: culturing the host cells described in the third aspect of this invention to express the target protein; and

[0020] The target protein is isolated to obtain the cystathionine-β-synthase;

[0021] In some preferred embodiments, the host cell is obtained by transforming Escherichia coli with a plasmid containing the polynucleotide described in the first aspect of the invention.

[0022] In some preferred embodiments, the host cells are cultured in SB, TB, LB, or SOC media, more preferably in TB and LB media, and most preferably in TB media.

[0023] In some preferred embodiments, the host cells are cultured in an oscillating environment.

[0024] In some preferred embodiments, the host cells are cultured at a temperature of 16 to 19°C or 35 to 39°C.

[0025] In some preferred embodiments, the culture medium used to culture the host cells contains a kanamycin resistance gene.

[0026] In some preferred embodiments, IPTG is used to induce the expression of the target protein when culturing the host cells.

[0027] In some preferred embodiments, the host cells are cultured until the OD600 is between 0.6 and 0.8, and then induced with IPTG to express the target protein.

[0028] In some preferred embodiments, the step of isolating the target protein includes:

[0029] The supernatant of the lysed target protein was eluted by passing it through a chromatography column while the flow was constant, and the eluent was collected.

[0030] In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column (Ni-NTA).

[0031] A fifth aspect of the present invention provides a kit comprising: a polynucleotide as provided in the first aspect of the present invention; or

[0032] Such as the expression vector provided in the second aspect of the present invention; or

[0033] The host cell as described in the third aspect of the present invention; or

[0034] Alternatively, cystathionine-β-synthetase prepared by the method according to the fourth aspect of the present invention.

[0035] Compared with the prior art, the present invention has at least the following advantages:

[0036] This invention provides a method for expressing cystathionine-β-synthase based on a prokaryotic expression system. The prepared cystathionine-β-synthase has a large soluble expression level, is suitable for industrial production, and has good product stability and high activity.

[0037] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0038] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0039] Figure 1 This is the SDS-PAGE identification result of a small-scale expression of cystathionine-β-synthase from Saccharomyces cerevisiae according to the embodiments of the present invention;

[0040] Figure 2 This is the SDS-PAGE identification result of another small expression of cystathionine-β-synthase from Saccharomyces cerevisiae according to the embodiments of the present invention;

[0041] Figure 3 SDS-PAGE identification results of honeybee-derived cystathionine-β-synthase in an embodiment of the present invention;

[0042] Figure 4 This is the SDS-PAGE identification result of low-level expression of human cystathionine-β-synthase according to the embodiments of the present invention;

[0043] Figure 5 This is an electrophoresis image of cystathionine-β-synthase derived from Saccharomyces cerevisiae according to an embodiment of the present invention;

[0044] Figure 6 This is a nickel column electrophoresis image of human-derived cystathionine-β-synthase according to an embodiment of the present invention;

[0045] Figure 7 This is a standard curve diagram of cystathionine-β-synthase according to an embodiment of the present invention. Detailed Implementation

[0046] Through extensive and in-depth research, the inventors screened and obtained optimized codon sequences that are soluble and can be expressed in large quantities in Escherichia coli. In addition, the inventors also developed an expression system for cystathionine-β-synthetase based on a prokaryotic system. The expressed cystathionine-β-synthetase has high activity and good stability.

[0047] This invention obtains the target protein sequence from a large number of bacteria containing cystathionine-β-synthase, and analyzes the target protein sequence to obtain the polynucleotide sequence / gene sequence encoding the target protein. Several obtained polynucleotide sequences / gene sequences are then optimized using different synonymous codon bias methods to obtain several optimized codons. A vector is constructed using these optimized codons and introduced into host cells to induce expression of the recombinant target protein.

[0048] Obtain the nucleic acid sequence related to the target gene / protein.

[0049] In this invention, the full-length nucleotide sequence or fragments of the target protein or its elements can typically be obtained using PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on publicly available nucleotide sequences, especially open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order.

[0050] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0051] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0052] The method of amplifying DNA / RNA using PCR technology is preferred for obtaining the gene of the present invention. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0053] Proteins of the same species derived from different organisms have different amino acid sequences. Gene sequences obtained based on target proteins from different sources often have unpredictable functional activities in their recombinant expression products. In one embodiment of the present invention, the amino acid sequences of target proteins from different sources are analyzed using the NCBI database to obtain target gene sequence information from different sources. In some embodiments, cystathionine-β-synthetaases from *Saccharomyces cerevisiae*, bees, and humans are analyzed using the NCBI database to obtain cystathionine-β-synthetaase gene sequence information from *Saccharomyces cerevisiae*, bees, and humans.

[0054] Synonymous codon preference optimization

[0055] To overcome the potential problem of reduced yield when expressing heterologous proteins in *E. coli*, this invention relates to polynucleotide sequences optimized by synonymous codon preference. Synonymous codon preference optimization is performed on the obtained target gene sequence, allowing target gene sequences from different sources to express the same amino acid sequence as the target protein. This invention has obtained a large number of cystathionine-β-synthease gene sequences from different sources. In some embodiments of this invention, by performing *E. coli* synonymous codon preference optimization on the *Saccharomyces cerevisiae* cystathionine-β-synthease gene sequence, codons as shown in SEQ ID NO:1 were obtained. In another embodiment, by performing *E. coli* synonymous codon preference optimization on the *Saccharomyces cerevisiae* cystathionine-β-synthease gene sequence, several optimized codons were obtained, exemplarily as optimized codon I as shown in SEQ ID NO:1, optimized codon IV as shown in SEQ ID NO:4, optimized codon V as shown in SEQ ID NO:5, and optimized codon VI as shown in SEQ ID NO:6. In another embodiment, several optimized codons are obtained by performing E. coli synonymous codon preference optimization on the gene sequence of bee-derived cystathionine-β-synthetase, exemplarily as optimized codon II shown in SEQ ID NO:2 and optimized codon VII shown in SEQ ID NO:7. In another embodiment, several optimized codons are obtained by performing E. coli synonymous codon preference optimization on the gene sequence of human-derived cystathionine-β-synthetase, exemplarily as optimized codon III shown in SEQ ID NO:3.

[0056] Homologous target proteins are optimized using synonymous codon preference in different ways to obtain several codons. Typically, while these codons can express target proteins with comparable activity, the expression levels, especially soluble expression levels, differ significantly when introduced into *E. coli* using different codons. In some embodiments of this invention, optimized codons I and III exhibit soluble expression, while other optimized codons, such as optimized codon II, are expressed as inclusion bodies.

[0057] The present invention also relates to polynucleotides having greater than 80%, preferably greater than 85%, more preferably greater than 90%, more preferably greater than 91%, and more preferably greater than 95% homology with the sequences shown in SEQ ID NO:1-8; and polynucleotides complementary to the sequences shown in SEQ ID NO:1-8.

[0058] Vector of the target gene

[0059] This invention also relates to vectors containing the polynucleotides of the invention. In this invention, "vector" refers to a linear or circular DNA molecule containing a fragment encoding a target protein, said target protein being operatively 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 optional markers, enhancers, polyadenylation signals, vectors, etc. The vector fragment may be derived from a host organism, another organism, plasmid or viral DNA, or may be synthetic. The vector may be any expression vector, either synthetic or readily manipulated with recombinant DNA, and the choice of vector generally depends on the host cell to which the vector is to be introduced. Thus, the vector may be a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be a vector that integrates into the host cell genome upon introduction into a host cell and replicates along with the chromosome into 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 the vector to obtain more efficient expression.

[0060] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the protein of the present invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Exemplarily, the vector DNA molecule is cleaved into linear molecules that can be linked to a foreign gene using a DNA endonuclease, and then a codon-optimized target gene fragment is ligated into the vector. The insertion of the foreign DNA fragment can be achieved by using sticky-end ligation at a single restriction enzyme site, directed cloning of double-restricted fragments, sticky-end ligation at different restriction enzyme sites, blunt-end ligation, artificial adapter ligation, or ligation to oligonucleotide ends.

[0061] Transform host cells with a vector containing the target gene

[0062] This invention also relates to host cells genetically engineered using the vector or fusion protein coding sequence of this invention. A vector containing a codon-optimized target gene can be inserted, transfected, or otherwise transformed into a host cell by known methods to obtain a transformant containing the codon-optimized target gene of this invention and capable of expressing the target protein. In this invention, "host cell" refers to a cell incorporating exogenous polynucleotides and / or a vector. The host cell can be a eukaryotic or prokaryotic host cell, preferably a bacterium, and more preferably *Escherichia coli* Rosetta(DE3) strain.

[0063] Methods for preparing target proteins

[0064] This invention also relates to a method for preparing the target protein, which can be used to express or produce recombinant proteins using the polynucleotide sequence of this invention. Generally, the method includes the following steps:

[0065] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding the protein of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0066] (2) Host cells cultured in a suitable culture medium;

[0067] (3) Isolate and purify proteins from culture media or cells.

[0068] In step (1), the recombinant expression vector containing the polynucleotide is transformed or transduced into a suitable host cell by conventional techniques known to those skilled in the art. When the host is Escherichia coli, heat shock and electroconversion methods can be used.

[0069] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media, preferably SB, TB, LB, or SOC media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature change or chemical induction), and the cells are cultured for a further period. In a preferred embodiment of this invention, to promote the expression of the target protein and increase the expression level of soluble proteins, host cells are cultured in TB or LB media containing a kanamycin resistance gene.

[0070] To further promote the soluble expression of the target protein, in a preferred embodiment of the present invention, host cells are cultured to OD0.05. 600After reaching a concentration between 0.6 and 0.8, induction was performed using IPTG, followed by further culture at 17 to 19°C or 35 to 39°C for approximately 8 to 12 hours. The expression content of the supernatant containing the polynucleotides of this invention was highest when host cells containing these polynucleotides were cultured in TB medium at 17 to 19°C, with a higher expression percentage than when cultured in LB medium at 17 to 19°C, TB medium at 35 to 39°C, or LB medium at 35 to 39°C.

[0071] The proteins described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, their physical, chemical, and other properties can be utilized to separate and purify them using various separation methods. Therefore, this invention, after successfully culturing the target protein, also involves steps of separating and purifying it, such as in step (3), where the protein is separated and purified from the culture medium to obtain a high-purity target protein. Methods for purifying the target protein are conventional techniques well-known to those skilled in the art, including but not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, percolation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof. A preferred embodiment of the present invention provides a method for separating a target protein, comprising the steps of: eluting the supernatant of the lysed target protein through a chromatography column simultaneously with a mobile phase, and collecting the eluent; the mobile phase comprising Buffer A, Buffer B, and / or Buffer C; wherein Buffer A comprises tris(hydroxymethyl)aminomethane (Tris) and NaCl solution (1M concentration); Buffer B comprises tris(hydroxymethyl)aminomethane (Tris), NaCl solution (1M concentration), and imidazole; Buffer C comprises tris(hydroxymethyl)aminomethane (Tris) and NaCl solution (1M concentration).

[0072] As a preferred scheme for the proportions of components in the mobile phase, for example, the volume ratio of tris(hydroxymethyl)aminomethane (Tris) to NaCl solution (1M concentration) in Buffer A is 1:1; the volume ratio of tris(hydroxymethyl)aminomethane (Tris), NaCl solution (1M concentration), and imidazole in Buffer B is 1:1:10; the volume ratio of tris(hydroxymethyl)aminomethane (Tris) to NaCl solution (1M concentration) in Buffer A is 1:1; in Buffer B...

[0073] Preferably, in the elution step, the elution procedure includes a first stage and a second stage; in the first stage, the mobile phase used is Buffer A; in the second stage, the mobile phase used is a mixture of Buffer A and Buffer B, wherein the volume percentage of Buffer A gradually decreases from 100% to 40%, and the volume percentage of Buffer B gradually increases from 0% to 60%.

[0074] More preferably, the elution process further includes a third stage in which the mobile phase used is Buffer B.

[0075] The eluted and purified target protein product is dialyzed, and the dialyzed sample is collected. The concentration of the dialyzed sample can be measured using the BCA method to calculate the yield.

[0076] In this invention, any exemplary or illustrative terminology (e.g., “”) used with respect to certain embodiments herein is merely for the purpose of better presenting the invention and does not limit the scope of the invention as otherwise claimed. No terminology herein should be construed as indicating an element not described in the claims that is indispensable to the implementation of this invention.

[0077] If the definition or use of a term in the cited literature is inconsistent with or inconsistent with the definition of a term described herein, the definition of the term described herein shall be used instead of the definition of the term in the cited literature.

[0078] The various terms used herein are as follows. If a term used in the claims is not defined below, the broadest definition of that term given by a person skilled in the art should be given, as reflected in the publication printed at the time of application or in the published patent.

[0079] As used herein, the term "isolated" refers to a nucleic acid or polypeptide isolated from at least one other component (e.g., a nucleic acid or polypeptide) present in its natural source. In one embodiment, the nucleic acid or polypeptide is found to be present only (if any) in a solvent, buffer, ion, or other component normally present in its solution. The terms "isolated" and "purified" do not include nucleic acids or polypeptides present in their natural source.

[0080] As used herein, the terms "polynucleotide" and "polynucleotide sequence" can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding.

[0081] This invention also relates to variants of the aforementioned polynucleotides that encode protein fragments, analogs, and derivatives having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be the substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the encoded polypeptide.

[0082] As used in this article, the term "codon optimization" refers to the method of improving gene synthesis efficiency by avoiding the use of low-utilization or rare codons based on the differences in codon utilization exhibited by the actual organisms performing protein expression or production (including E. coli, yeast, mammalian blood cells, plant cells, insect cells, etc.).

[0083] As used herein, the terms “homology” and “identity” are used interchangeably and refer to the percentage of identical (i.e., same) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by arranging the nucleotide or amino acid sequences of the polynucleotide or polypeptide, scoring the number of positions in the arranged polynucleotide or polypeptide containing the same nucleotide or amino acid residues, and comparing this to the number of positions in the arranged polynucleotide or polypeptide containing different nucleotide or amino acid residues. Polynucleotides can differ at one position, for example, by containing different nucleotides (i.e., substitutions or variations) or by the deletion of nucleotides (i.e., the insertion or deletion of one or two nucleotides in the polynucleotide). Polypeptides can differ at one position, for example, by containing amino acids (i.e., substitutions or variations) or by the deletion of amino acids (i.e., the insertion of one or two amino acids in the polypeptide or the deletion of amino acids). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percentage identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

[0084] As used herein, the terms “sequence complement” and “reverse sequence complement” are used interchangeably and refer to a sequence that is in the opposite direction to the original polynucleotide sequence and is complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, then its reverse complementary sequence is GTTCAT.

[0085] As used herein, the term "expression" includes any step involved in the production of a polypeptide in a host cell, including but not limited to transcription, translation, post-translational modification, and secretion. Post-expression can be harvested, i.e., the host cell or the expressed product can be recovered.

[0086] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0087] Unless otherwise specified, the 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 pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0088] Example 1

[0089] In this embodiment, a plasmid containing optimized codons encoding cystathionine-β-synthase from Saccharomyces cerevisiae, bee-derived and human sources was synthesized and introduced into Escherichia coli culture to obtain a single clone.

[0090] (1) Construction of cystathionine-β-synthase plasmid

[0091] The gene sequence of cystathionine-β-synthase from Saccharomyces cerevisiae was obtained and optimized for Escherichia coli synonymous codon bias to obtain several optimized codons, such as optimized codon I (SEQ ID NO.1), optimized codon IV (SEQ ID NO.4), optimized codon V (SEQ ID NO.5), and optimized codon VI (SEQ ID NO.6), which were ligated into the pET-28a(+) vector and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0092] The gene sequence of cystathionine-β-synthase from bees was obtained and optimized using E. coli synonymous codon preference to obtain several optimized codons, exemplarily such as optimized codon II (SEQ ID NO.2) and optimized codon VII (SEQ ID NO.7). These were ligated into the pET-28a(+) vector and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0093] Human-derived cystathionine-β-synthetase was obtained and subjected to E. coli synonymous codon preference optimization to obtain several optimized codons, exemplarily shown in optimized codon III (SEQ ID NO.3). It was ligated into the pET-28a(+) vector and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0094] Human-derived cystathionine-β-synthetase (amino acid sequence SEQ ID NO.9) was obtained, and its gene sequence was analyzed. E. coli synonymous codon preference optimization was performed to obtain several optimized codons, exemplarily such as optimized codon VIII (SEQ ID NO.8). This codon was ligated into the pET-28a(+) vector and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0095] (2) Introduction of recombinant plasmids into host Escherichia coli

[0096] Take 1 μL of the expression plasmid prepared in step (1) above, add it to 30 μL of E. coli competent cells BL21(DE3) under ice bath conditions, incubate on ice for 30 min, incubate in water at 42℃ for 45 s, immediately place on ice for 2 min, add 400 μL of antibiotic-free SOC medium, and incubate at 37℃ and 230 rpm for 45 min with shaking. Take 100 μL of bacterial culture and spread it evenly on LB agar plates containing 100 μg / mL kanamycin resistance, and incubate overnight at 37℃.

[0097] SEQ ID NO.1

[0098] ATGACTAAATCCGAACAGCAGGCTGACTCTCGTCACAACGTCATCGATCTGGTTGGCAACACCCGCTGATCGCGCTGAA

[0099] AAAACTGCCGAAAGCGCTGGGCATCAAGCCGCAAATCTATGCCAAACTGGAACTGTATAACCCGGGTTGGCAGCATTAAAG

[0100] ACCGCATCGCAAAAAGCATGGTCGAGGAAGCGGAAGCGTCTGGCCGCATTCACCCGTCCCGTAGCACCCTGATCGAGCCG

[0101] ACCTCTGGTAACACTGGCATCGGTCTGGCGCTGATCGGTGCGATCAAAGGCTACCGTACTATCATCACGCTGCCGGAAAA

[0102] AATGCTAACGAGAAAGTGTCCGTACTGAAAGCGCTGGGTGCCGAAATCATCCGTAACCCCGACCGCGGCTGCCTGGGACT

[0103] CTCCGGAAAGCCATATCGGTGTGGCTAAAAAACTGGAGAAAGAAATCCCGGGCGCCGTGATCCTGGACCAGTACAACAAC

[0104] ATGATGAACCCGGAAGCGCATTACTTCGGTACCGGTCGTGAGATCCAACGTCAGCTGGAGGACCTGAACCTGTTCGACAA

[0105] CCTGCGTGCGGTAGTGGCAGGCGCGGGCACCGGTGGCACTATCTCTGGCATCTCCAAATACCTGAAAGAACAAAACGATA

[0106] AAATCCAGATTGTTGGTGCCGACCCGTTCGGCTCTATTCTGGCACAGCCGGAAAACCTGAACAAAACTGACATCACTGAC

[0107] TACAAAGTAGAGGGCATCGGTTACGATTTTGTGCCTCAGGTTCTGGACCGTAAACTGATTGACGTTTGGTACAAAACCGA

[0108] CGATAAACCAAGCTTCAAATACGCACGTCAACTGATTTCCAACGAGGGTGTACTGGTCGGTGGCTCTTCCGGCAGCGCCT

[0109] TCACCGCTGTTGTAAAATACTGTGAAGATCACCCGGAGCTGACCGAAGATGATGTTATCGTGGCAATCTTTCCGGACTCT

[0110] ATCCGTTCTTACCTGACTAAATTCGTCGATGATGAGTGGCTGAAAAAAAATAACCTGTGGGACGACGATGTCCTGGCTCG

[0111] TTTCGATTCCAGCAAACTGGAGGCTTCCACTACCAAATACGCGGACGTATTCGGCAATGCGACGGTCAAAGATCTGCATC

[0112] TGAAACCTGTAGTGTCTGGAAAGAAACCGCGAAGTAACGGACGTGATTAAAATCCTGAAAGACAACGGTTTTGACCAG

[0113] CTGCCGGTGCTGACCGAAGATGGCAAGCTGAGCGGTCTGGTTACGCTGTCCGAACTGCTGCGCAAACTGTCCATCAACAA

[0114] CTCCAACAATGACAACACTATTAAAGGTAAATACCTGGATTTCAAGAAACTGAACACTTTAACGATGTTTCTTCCTATA

[0115] ACGAAAAACAAATCTGGTAAAAAAAAGTTCATCAAATTCGATGAAAACAGCAAACTGTCCGATCTGAACCGTTTCTTCGAA

[0116] AAAAAACTCTTCTGCAGTTATCACCGACGGCCTGAAGCCGATCCACATCGTCACTAAAATGGATCTGCTGTCTTACCTGGC

[0117] G

[0118] SEQ ID NO.2

[0119] ATGGAATTCAAACAGCCGAACCGTCCGTCTTATTGCACTTGGGAACTGAACGCCACTAACTCCCCGCACACCCTGTCGTAC

[0120] TAAAAACGGTGACTACACCAAGATCATGCCTGACATCCTGACCGCGATCGGCCAGACTCCGCTGATTAAACTGAATAATA

[0121] TCCCAAAAAGCTACGGCATCAAATGCGAAATCTACGGAAAATGCGAGTTCCTGAACCCAGGTGGTTCTGTCAAGGACCGC

[0122] ATCGCGTATCGTATGATCCAGGATGCGGAGGACAAAGGCCTGCTGAAACCTGGCTGTACCATCATTGAACCTACCTCTGG

[0123] CAACACTGGCATCGGTCTGGCAATGGCTGCTGCTGTTCGTGGTTACAAATGCATTATCGTGATGCCGGAAAAAATGTCTG

[0124] ACGAAAAAATTTCCACCCTGTATGCGCTGGGTGCGAAGATCATCCGCACTCCGACCGAAGCTTCTTGGCACTCTCCTGAA

[0125] GCGCATATTAGCGTAGCTCAGAAACTGCAGAAAGAAATCCCGAACTCTATCATCCTGGATCAGTACACCAACCCGGGCAA

[0126] CCCACTGGCACATTACGACCAGACTGCTATCGAAATTTGGAAACAGTGCGAGGGTAAGATCGATTACCTGGTTGCAGGCG

[0127] CTGGTACCGGCGGTACCATTAGCGGCATTGGTCGTAAACTGAAAGAACTGTCCCCGAACATCAAAATCATCGCCGTGGAT

[0128] CCGAAAGGTTCTATCCTGGACCCGTCCTCTGATTCTCAGAACGAAGTGGGTTTCTACGAAGTAGAGGGTATCGGTTACGA

[0129] TTTCATTCCGACTGTTCTGGATCGCAACGTAATCGATAAATGGATCAAAACCGAAGATAACGAATCTCTGAACGCGGCTC

[0130] GCATGCTGATCCGCCAGGAAGGCCTGCTGTGTGGCGGTTCTTCCGGCGCCGCGCTGATTGCAGCTCTGAAGATTGCGAAA

[0131] GACATCCCGGAAGAAAAGCGTATGGTGATTATCCTGCCGGATGGTATCCGTAACTATCTGACCAAATTCGTGAGCGAATA

[0132] CTGGATGGAAACCCGTGGTTTCCTGCAGCCGGTGTGTCAGAACGAAATGAACAAATGGTGGTGGAACATGAAAATTAGCA

[0133] ATCTGTCCTTTGACAAACAGTCTCTGCTGAAAGAAAACACCGTCACCTGCCAGGAAGCCATGCACATGCTGAAGAACGCG

[0134] GACTCTCAGCTGCTGGTAATCAGCGACGACAACATTCACATCAAAGGTGTTATCTCCCTGAACAAGCTGACTTCTTACGT

[0135] TATCTCCGGCATCGTTAAATGCACCGATTTTGTTGATAAAGCCATGGTGAAACAGTATGTAAAAGTTAAACACTCCGCGA

[0136] CCCTGGGCTACATCTCTCGCGTGCTGGAAAAAGAACCGTACGTGATCATTCTGGATGACGAACACGATGACGCGTTCATT

[0137] GGTATTGTTAATCAATTTCACATTCTGCAATTCATTACCAAAAATGGTACTTCTAACAACTACCTGATCAACSEQ ID NO.3

[0138] ATGCCATCCGAAACTCCTCAGGCTGAAGTAGGTCCAACTGGTTGCCCGCACCGTTCTGGTCCACACTCTGCTAAGGGCAG

[0139] CCTGGAAAAAGGCTCTCCGGAGGACAAAGAGGCGAAAGAACCGCTGTGGATTCGTCCGGATGCGCCTAGCCGTTGTACCT

[0140] GGCAGCTGGGTCGTCCGGCTTCTGAATCTCCACATCACCATACCGCTCCAGCTAAGTCCCCGAAAATTCTGCCGGATATC

[0141] CTGAAAAAAATCGGTGACACCCCGATGGTCCGTATCAACAAAATCGGCAAAAAGTTTGGTCTGAAATGCGAACTGCTGGC

[0142] GAAATGCGAATTCTTCAATGCAGGTGGCTCTGTTAAAGACCGTATTTCCCTGCGTATGATTGAAGACGCAGAACGTGACG

[0143] GTACTCTGAAACCGGGTGATACTATTATTGAGCCTACTTCTGGTAATACTGGTATTGGTCTGGCTCTGGCTGCTGCTGTT

[0144] CGTGGCTACCGCTGTATCATCGTTATGCCGGAAAAAATGTCCTCCGAAAAAGTTGACGTACTGCGTGCTCTGGGCGCTGA

[0145] GATTGTTCGCACCCCGACCAACGCACGCTTCGATTCTCCGGAATCCCACGTTGGTGTAGCTTGGCGCCTGAAAAACGAAA

[0146] TTCCGAACTCCCATATTCTGGATCAGTACCGCAACGCCTCCAACCCGCTGGCTCACTATGACACTACCGCGGATGAAATC

[0147] CTGCAGCAGTGCGATGGTAAACTGGACATGCTGGTTGCAAGCGTTGGTACCGGCGGTACTATCACCGGCATCGCACGTAA

[0148] ACTGAAGGAAAAATGTCCGGGTTGTCGTATCATTGGTGTCGATCCGGAAGGTTCTATCCTGGCCGAACCAGAAGAACTGA

[0149] ACCAGACGGAACAGACCACGTATGAGGTGGAGGGCATCGGCTACGACTTTATCCCGACTGTACTGGACCGTACCGTTGTG

[0150] GATAAATGGTTCAAAAGCAACGATGAAGAAGCGTTCACGTTTGCACGCATGCTGATCGCTCAAGAGGGTCTGCTGTGTGG

[0151] TGGTTCCGCAGGTTCTACCGTAGCAGTAGCAGTGAAAGCCGCTCAGGAGCTGCAAGAAGGCCAGCGTTGTGTGGTTATCC

[0152] TGCCGGATAGCGTGCGCAACTACATGACCAAATTCCTGTCCGACCGCTGGATGCTGCAGAAAGGCTTCCTGAAGGAAGAA

[0153] GACCTGACCGAGAAGAAACCGTGGTGGTGGCACCTGCGTGTTCAGGAACTGGGTCTGTCTGCACCGCTGACTGTTCTGCC

[0154] GACTATCACCTGTGGCCATACCATCGAGATCCTGCGTGAAAAAGGTTTCGACCAAGCGCCGGTGGTAGACGAAGCAGGTG

[0155] TTATCCTGGGCATGGTTACCCTGGGCAACATGCTGTCTTCCCTGCTGGCGGGCAAAGTTCAGCCGTCTGATCAGGTAGGC

[0156] AAAGTAATCTATAAACAGTTCAAACAGATCCGCCTGACCGATACTCTGGGCCGTCTGAGCCATATTCTGGAAATGGATCA

[0157] TTTCGCGCTGGTTGTTCACGAACAGATCCAGTACCATTCCACCGGCAAATCCTCTCAGCGTCAGATGGTTTTTGGTGTTG

[0158] TTACCGCCATCGACCTGCTGAACTTTGTGGCGGCTCAGGAACGTGATCAGAAA

[0159] SEQ ID NO.4:

[0160] ATGACAAAATCCGAACAGCAGGCCGATTCTCGGCATAACGTTATTGACCTCGTAGGGAACACGCCGCTTATTGCACTTAA

[0161] AAAATTGCCAAAGGCCTTGGGCATCAAACCGCAAATCTATGCGAAACTTGAACTTTATAACCCGGGAGGTAGTATTAAAG

[0162] ATCGAATTGCAAAATCGATGGTCGAAGAAGCGGAAGCCTCAGGCCGAATCCATCCGTCTAGAAGCACGCTCATTGAACCA

[0163] ACCTCCGGTAATACGGGAATTGGACTGGCGCTGATTGGAGCCATCAAGGGCTATAGAACAATTATTACATTGCCGGAAAA

[0164] AATGTCAAATGAAAAAGTTAGTGTTCTTAAAGCCTTAGGGGCGGAAATCATAAGAACACCGACTGCTGCGGCATGGGACT

[0165] CCCCTGAATCTCACATTGGTGTCGCCAAAAAGCTGGAAAAAGAAATTCCAGGAGCCGTCATTCTTGATCAGTATAACAAT

[0166] ATGATGAACCCGGAAGCACATTATTTTGGTACGGGCCGCGAAATCCAACGCCAACTCGAAGACCTTAACTTATTTGACAA

[0167] TCTCCGGGCGGTCGTCGCTGGAGCTGGAACCGGCGGGACGATCTCTGGTATTTCTAAATACCTGAAAGAACAGAATGATA

[0168] AAATTCAAATTGTTGGTGCAGACCCGTTTGGATCCATTCTCGCGCAGCCGGAAAATCTGAACAAGACTGATATTACAGAT

[0169] TATAAAGTCGAAGGCATCGGTTATGATTTCGTCCCGCAGGTTTTAGATCGTAAACTTATTGATGTATGGTATAAGACAGA

[0170] TGACAAGCCGTCATTTAAATACGCTCGTCAGCTTATTAGCAACGAAGGAGTCCTTGTGGGCGGCTCCTCTGGAAGCGCAT

[0171] TCACGGCGGTTGTCAAGTATTGCGAAGATCATCCTGAACTGACAGAGGATGATGTTATTGTTGCTATTTTTCCGGACAGC

[0172] ATTCGGTCTTACTTAACTAAGTTTGTAGATGATGAATGGCTGAAAAAAAATAATCTTTGGGACGATGACGTGCTCGCCAG

[0173] ATTCGATTCGTCAAAATTAGAAGCTTCCACAACCAAATATGCAGATGTTTTTGGCAACGCAACAGTGAAAGATCTTCATC

[0174] TCAAACCTGTCGTCTCGGTGAAAGAAACGGCTAAAGTTACAGACGTAATTAAAATACTTAAAGATAATGGCTTTGATCAA

[0175] TTACCAGTGTTAACCGAGGACGGGAAATTGTCGGGGTTAGTTACGCTGAGCGAACTGTTGCGTAAACTTTCGATAAACAA

[0176] CTCTAATAATGATAACACCATTAAGGGCAAATATTTGGACTTTAAAAAACTTAATAATTTTAACGACGTATCTTCATACA

[0177] ATGAGAATAAAAGCGGCAAAAAGAAATTCATCAAATTTGATGAGAATTCAAAATTGAGCGATCTGAATAGATTTTTTGAA

[0178] AAGAACAGCTCTGCTGTTATTACAGATGGACTTAAACCGATCCATATAGTCACTAAAATGGACCTGCTGTCTTATTTGGC

[0179] A

[0180] SEQ ID NO.5:

[0181] ATGACCAAATCAGAGCAACAGGCTGATTCCAGGCACAACGTGATAGATTTAGTTGGAAATACACCCTTGATAGCCTTAAA

[0182] GAAACTGCCAAAAGCTCTTGGTATCAAGCCTCAAATTTACGCCAAGTTGGAGTTATACAATCCCGGTGGATCTATCAAGG

[0183] ATAGGATCGCCAAGTCCATGGTAGAAGAAGCCGAAGCCTCCGGTCGAATTCATCCATCTAGATCCACTTTGATTGAGCCA

[0184] ACAAGTGGAAACACAGGTATTGGTCTGGCATTGATTGGTGCTATTAAGGGATATAGAACCATTATTACCTTGCCCGAAAA

[0185] GATGTCTAACGAAAAGGTTTCTGTTTTGAAAGCTTTGGGTGCTGAAATCATCAGAACCCCAACCGCTGCCGCCTGGGACT

[0186] CCCCAGAATCCCACATTGGTGTGGCTAAGAAGCTGGAGAAAGAGATCCCAGGAGCCGTAATCTTGGACCAATACAATAAC

[0187] ATGATGAACCCTGAGGCACATTACTTTGGAACTGGTAGAGAGATCCAAAGACAATTGGAAGATTTGAACTTGTTCGACAA

[0188] CTTGCGAGCCGTGGTTGCTGGAGCTGGCACCGGTGGTACAATTTCAGGTATTTCCAAATATTTGAAAGAGCAGAATGACA

[0189] AGATTCAGATCGTCGGAGCTGATCCATTCGGATCAATCCTGGCTCAACCAGAAAACTTGAACAAAACCGATATCACCGAT

[0190] TACAAGGTTGAAGGTATTGGTTACGATTTTGTTCCTCAAGTCTTGGACAGGAAGTTGATTGACGTTTGGTATAAAACAGA

[0191] CGATAAGCCAAGTTTTAAGTATGCACGTCAATTAATTTCTAATGAAGGCGTTTTGGTTGGTGGTTCTTCTGGATCAGCTT

[0192] TTACGGCCGTTGTTAAGTATTGTGAAGATCACCCTGAATTGACAGAAGATGACGTTATCGTTGCAATTTTCCCCGATTCA

[0193] ATCAGATCCTATCTTACCAAATTTGTCGATGATGAATGGTTGAAGAAGAACAACCTTTGGGACGATGATGTACTGGCTAG

[0194] ATTTGACTCCTCCAAGCTGGAGGCTAGTACTACAAAGTACGCTGACGTTTTTGGCAACGCTACGGTTAAAGATTTGCATC

[0195] TAAAACCCGTTGTCTCTGTCAAAGAGACAGCTAAGGTTACCGATGTAATAAAGATCTTGAAAGACAACGGTTTTGATCAA

[0196] CTGCCAGTTTTGACTGAGGACGGAAAATTGTCCGGCCTTGTTACTCTTTCTGAGTTGTTGCGAAAACTAAGTATCAACAA

[0197] TTCTAATAATGATAACACCATCAAGGGAAAATATCTAGATTTTAAGAAGCTAAACAATTTTAACGACGTCAGTTCCTACA

[0198] ACGAGAATAAATCTGGCAAGAAAAAATTTATAAAGTTCGATGAAAATAGTAAGTTATCAGATCTTAATCGATTTTTCGAG

[0199] AAAAATAGTTCCGCTGTCATTACAGATGGTTTGAAGCCAATTCATATTGTCACCAAAATGGACCTACTATCTTATCTTGC

[0200] T

[0201] SEQ ID NO.6:

[0202] ATGACCAAGTCAGAACAGCAAGCTGATTCTAGACACAATGTCATAGATCTGGTGGGAAATACCCCATTGATCGCTCTAAA

[0203] GAAATTGCCTAAGGCGTTGGGCATTAAACCACAAATATATGCTAAACTGGAACTATACAACCCAGGGGGGTTCCATCAAGG

[0204] ATAGAATTGCTAAGTCCATGGTCGAAGAGGCGGAGGCGTCTGGAAGAATCCATCCATCTAGGTCCACACTAATTGAACCT

[0205] ACATCCGGTAATACTGGGATTGGTTTAGCCCTAATCGGTGCCATCAAAGGTTACAGAACTATTATTACCTTGCCAGAGAA

[0206] AATGAGTAATGAGAAAGTTTCTGTACTTAAGGCTCTAGGTGCCGAAATTATAAGAACTCCAACTGCAGCTGCTTGGGATT

[0207] CTCCAGAAAGCCATATCGGGGTTGCAAAGAAGCTAGAAAAAGAAATTCCTGGCGCAGTTATTCTTGACCAGTACAATAAT

[0208] ATGATGAATCCAGAGGCCCACTACTTCGGTACTGGCCGTGAAATTCAAAGACAATTAGAAGATTTGAACTTGTTTGACAA

[0209] CCTGCGTGCGGTTGTTGCTGGTGCAGGAACCGGAGGTACTATAAGCGGTATCAGCAAATATCTTAAAGAGCAGAACGATA

[0210] AAATACAGATTGTTGGTGCCGATCCATTTGGTTCAATACTTGCACAACCCGAAAACCTGAATAAGACTGATATTACTGAT

[0211] TACAAAGTCGAAGGAATTGGATACGACTTCGTTCCTCAGGTGCTTGATAGAAAATTAATAGACGTGTGGTATAAGACAGA

[0212] TGACAAGCCATCTTTCAAATATGCTAGGCAGTTGATTTCCAACGAAGGAGTGTTAGTGGGTGGATCTTCAGGATCTGCGT

[0213] TCACGGCAGTCGTGAAATATTGCGAAGACCATCCAGAATTGACAGAAGATGATGTTATAGTAGCTATTTTTCCAGACTCT

[0214] ATCCGTAGTTATTTAACTAAGTTTGTTGACGATGAATGGTTAAAGAAAAACAACTTGTGGGACGACGATGTACTTGCTAG

[0215] ATTTGACTCCAGCAAATTGGAAGCTTCTACTACCAAATATGCCGATGTTTTTGGTAATGCCACTGTCAAGGATCTGCATC

[0216] TAAAACCTGTGGTCTCAGTGAAGGAAACAGCGAAGGTCACTGATGTGATCAAAATTTTAAAAGATAATGGTTTTGATCAA

[0217] TTACCAGTTTTGACGGAGGATGGTAAGTTGTCCGGGCTTGTCACCTTGTCAGAACTACTTAGAAAATTATCTATTAATAA

[0218] CAGTAACAATGATAATACCATAAAAGGTAAGTATTTAGATTTTAAGAAACTGAATAATTTTAATGACGTCTCCTCTTATA

[0219] ACGAGAATAAATCTGGCAAAAGAAGTTTATCAAATTTGATGAGAATTCTAAACTAAGCGATTTGAATAGATTTTTTGAA

[0220] AAGAACTCATCCGCTGTTATTACTGATGGTCTTAAACCTTATACATATTGTGACGAAAATGGATTTATTGTCTTATCTGGC

[0221] A

[0222] SEQ ID NO.7:

[0223] ATGGAATTTAAGCAACCGAATCGTCCGTCTTACTGTACGTGGGAACTGAATGCAACCAACAGTCCACACACGTGCAGAAC

[0224] GAAGAATGGAGACTATACCAAAATTATGCCAGATATCCTTACAGCGATCGGACAGACCCCGTTGATTAAACTTAATAATA

[0225] TCCCGAAATCATACGGAATTAAGTGTGAGATCTATGCGAAGTGCGAATTTCTCAATCCGGGTGGGTCGGTCAAAGATCGA

[0226] ATAGCATACCGAATGATACAGGATGCGGAAGATAAAGGGCTTCTCAAGCCGGGTTGTACGATCATCGAACCAACAAGTGG

[0227] CAATACAGGAATCGGGCTGGCGATGGCGGCGCCAGTCCGCGGATAAAATGTTATCATTGTTATCGCGGAAAAGATGTCAG

[0228] ACGAAAAAATTAGCACGCTTTATGCGCTGGGAGCTAAGATTATACGTACGCCTACAGAGGCATCGTGGCATTCACCGGAG

[0229] GCACATATCTCCGTTGCTCAAAAGCTTCAAAAAGAAATTCCGAATAGCATTATTCTCGATCAGTATACAAACCCGGGCAA

[0230] TCCTCTGGCTCATTATGATCAGACAGCTATCGAAATCTGGAAACAGTGTGAAGGTAAGATTGATTATCTTGTCGCTGGGG

[0231] CTGGTACAGGCGGGACAATCTCTGGTATTGGCCGTAAACTGAAAGAACTTTCCCCAAACATTAAGATTATCGCAGTGGAT

[0232] CCTAAAGGATCGATTCTTGATCCGAGCTCAGATAGCCAAAATGAAGTTGGCTTTTATGAAGTAGAAGGTATTGGATATGA

[0233] TTTTATTCCAACAGTGCTGGACAGAAACGTCATTGACAAGTGGATCAAAACTGAGGATAACGAAAGCTTAAATGCCGCTA

[0234] GAATGTTAATCCGCCAGGAGGGGCTGCTGTGCGGAGGATCGAGCGGGGCTGCGTTAATTGCGGCATTAAAAATTGCCAAA

[0235] GACATCCCGGAAGAAAAGCGTATGGTTATCATCTTACCTGACGGAATTCGTAATTATCTGACTAAATTCGTTTCTGAATA

[0236] TTGGATGGAGACACGTGGATTTTTGCAGCCAGTTTGCCAGAATGAGATGAACAAATGGTGGTGGAACATGAAGATCTCAA

[0237] ATTTGTCATTTGACAAACAAAGCCTCCTCAAAGAAAATACCGTCACGTGTCAGGAGGCGATGCACATGCTTAAAAATGCT

[0238] GATAGCCAGTTACTGGTAATCTCTGACGATAATATCCATATCAAAGGAGTTATTTCCTTAAACAAATTAACTAGCTATGT

[0239] CATCTCCGGCATTGTAAAGTGCACAGATTTTGTTGATAAAGCGATGGTAAAACAATATGTCAAAGTGAAACACTCTGCAA

[0240] CTCTTGGGTATATCAGCAGAGTCCTCGAAGGAACCTTATGTTATCATTTTAGATGATGAGCACGATGACGCGTTCATT

[0241] GGGATCTCAATCAGTTTCATATTCTGCAATTTATCACAAAAAATGGCACGAGCAATAACTATTTAATTAATSEQ ID NO.8:

[0242] ATGCCGTCTGAAACCCCACAAGCTGAAGTGGGTCCTACTGGTTGTCCTCATCGTTCTGGCCCACACTCCGCCAAAGGCTC

[0243] TCTGGAAGGTAGCCCGGAGGACAAAGAAGCTAAAGAACCGCTGTGGATCCGTCCGGATGCACCGTCTCGTTGCACTT

[0244] GGCAGCTGGGTCGTCCTGCGTCTGAATCTCCACACCACCACACCGCGCCAGCAAAGTCTCCGAAGATCCTGCCAGACATC

[0245] CTGAAGAAAATTGGTGATACCCCGATGGTTCGCATCAACAAAATCGGTAAAAAATTCGGTCTGAAATGCGAACTGCTGGC

[0246] AAAGGTGTGAATTCTTCAACGCTGGCGGTTCTGTTAAAGATCGCATTCTCTGCGTATGATCGAGGACGCTGAACGTGACG

[0247] GCACCCTGAAACCGGGCGACACTATTATTGAACCGACCTCCGGTAATACTGGTATCGGTCTGGCTCTGGCTGCCGCTGTA

[0248] CGTGGTTACCGTTGCATCATTGTAATGCCGGAAAAAATGTCTTCTGAAAAGGTCGACGTTCTGCGTGCACTGGGTGCAGA

[0249] GATCGTACGCACTCCGACCAACGCACGTTTCGATAGCCCGGAATCCCATGTTGGTGTGGCCTGGCGTCTGAAGAACGAAA

[0250] TTCCGAACAGCCACATCCTGGACCAGTACCGTAACGCGTCTAACCCACTGGCTCACTATGATACCACCGCGGACGAAATC

[0251] CTGCAGCAGTGCGATGGTAAACTGGACATGCTGGTAGCATCTGTGGGTACTGGTGGCACCATTACCGGTATCGCCCGTAA

[0252] ACTGAAAGAAAAATGCCCGGGTTGCCGTATTATCGGTGTTGATCCGGAAGGTTCTATCCTGGCGGAGCCGGAAGAACTGA

[0253] ACCAGACTGAGCAGACGACCTATGAAGTTGAAGGTATCGGTTATGACTTTATTCCGACCGTTCTGGACCGTACGGTTGTT

[0254] GACAAGTGGTTCAAATCCAACGATGAAGAAGCTTTCACCTTCGCGCGTATGCTGATCGCACAGGAAGGTCTGCTGTGCGG

[0255] TGGTTCTGCTGGTTCTACCGTGGCTGTTGCGGTTAAGGCGGCTCAAGAGCTGCAGGAAGGTCAGCGTTGTGTCGTTATTC

[0256] TGCCTGACAGCGTTCGCAACTATATGACGAAGTTCCTGTCCGACCGTTGGATGCTGCAGAAGGGTTTCCTGAAAGAAGAA

[0257] GACCTGACCGAAAAAAAACCGTGGTGGTGGCATCTGCGT

[0258] SEQ ID NO.9

[0259] MPSETPQAEVGPTGCPHRSGPHSAKGSLEKGSPEDKEAKEPLWIRPDAPSRCTWQLGRPASESPHHHTAPAKSPKILPDI

[0260] LKKIGDTPMVRINKIGKKFGLKCELLAKCEFFNAGGSVKDRISLRMIEDAERDGTLKPGDTIIEPTSGNTGIGLALAAAV

[0261] RGYRCIIVMPEKMSSEKVDVLRALGAEIVRTPTNARFDSPESHVGVAWRLKNEIPNSHILDQYRNASNPLAHYDTTADEI

[0262] LQQCDGKLDMLVASVGTGGTITGIARKLKEKCPGCRIIGVDPEGSILAEPEELNQTEQTTYEVEGIGYDFIPTVLDRTVV

[0263] DKWFKSNDEEAFTFARMLIAQEGLLCGGSAGSTVAVAVKAAQELQEGQRCVVILPDSVRNYMTKFLSDRWMLQKGFLKEE

[0264] DLTEKKPWWWHLR

[0265] Example 2

[0266] In this embodiment, three different monoclonal antibodies obtained in Example 1 were aseptically inoculated into LB medium containing 100 μg / mL kanamycin, with two replicates. The cultures were incubated at 37°C with shaking at 220 rpm until the OD600 reached between 0.6 and 0.8. IPTG induction was then performed, followed by overnight shaking incubation at 37°C and 18°C, respectively. Samples were then ultrasonically disrupted and analyzed by SDS-PAGE. The results are referenced below. Figure 1-4 The results of the low-level expression assays of cystathionine-β-synthase from three sources are shown in Table 1 below.

[0267] Figure 1 In the table, columns 3 and 5 show the expression results of monoclonal antibodies containing optimized codon I cultured at 37°C, columns 6 and 7 show the expression results of monoclonal antibodies containing optimized codon I cultured at 18°C, columns 10 and 11 show the expression results of monoclonal antibodies containing optimized codon IV cultured at 37°C, and columns 12 and 13 show the expression results of monoclonal antibodies containing optimized codon IV cultured at 18°C.

[0268] Figure 2 In the table, columns 4-5 show the expression results of monoclonal antibodies containing optimized codon V cultured at 37℃, columns 6-7 show the expression results of monoclonal antibodies containing optimized codon V cultured at 18℃, columns 10-11 show the expression results of monoclonal antibodies containing optimized codon VI cultured at 37℃, and columns 12-13 show the expression results of monoclonal antibodies containing optimized codon VI cultured at 18℃.

[0269] Figure 3 In the table, columns 3-4 show the expression results of single clones containing optimized codon II cultured at 37℃, columns 5-6 show the expression results of single clones containing optimized codon II cultured at 18℃, columns 9 and 11 show the expression results of single clones containing optimized codon VII cultured at 37℃, and columns 12-13 show the expression results of single clones containing optimized codon VII cultured at 18℃.

[0270] Figure 4 In the table, columns 3-4 show the expression results of single clones containing optimized codon III cultured at 18℃, and columns 6-7 show the expression results of single clones containing optimized codon VIII cultured at 18℃.

[0271] Table 1

[0272]

[0273] Table 1 shows that several optimized codons for cystathionine-β-synthase from *Saccharomyces cerevisiae* were expressed in soluble form at both 18℃ and 37℃, with the target protein having a molecular weight of approximately 56 kDa. Optimized codons for human cystathionine-β-synthase were also expressed in soluble form at 18℃. Optimized codons for bee-derived cystathionine-β-synthase were expressed as inclusion bodies at both 18℃ and 37℃.

[0274] Example 3

[0275] Single clones of *Saccharomyces cerevisiae* and human-derived cystathionine-β-synthase were selected for expansion culture and purification. Approximately 4 g of recombinant cells of *Saccharomyces cerevisiae* and human-derived cystathionine-β-synthase were weighed and added to 20 ml of Lysis Buffer, then dispersed on ice using a disperser. Cell disruption was performed by sonication: Ф10 probe, 10% power, 5.5 s on, 9.9 s off, for 30 min. The cells were centrifuged at 20000 rpm, 4℃ for 30 min, and the supernatant was collected and filtered through a 0.22 μm membrane. Purification was performed using 1 ml of Ni-NTA; the composition of the purification reagents is shown in Table 2. The mobile phase flow rate was 0.5 ml / min. After loading, the UV filter was rinsed with 20 ml of Lysis Buffer, and the conductivity was adjusted to baseline. The elution program included: Step 1: 0% B, 10 CV, 1.5 ml / min; Step 2: 0–60% B, 20 CV, 1.5 ml / min; Step 3: 100% B, 15 CV, 1.5 ml / min.

[0276] Table 2

[0277]

[0278] The purified products of recombinant cystathionine-β-synthetaase obtained from cell cultures containing Saccharomyces cerevisiae and optimized human codons were collected and subjected to electrophoresis. The Saccharomyces cerevisiae-derived recombinant cystathionine-β-synthetaase could be loaded onto a column and eluted with 100 mM imidazole. The eluent was collected based on SDS results, dialyzed, and the sample was collected. The concentration was determined using BCA assay, and the yield was calculated.

[0279] The yield statistics of recombinant bacterial culture purification products containing optimized codons I, IV, V, and VI are shown in Table 3 below.

[0280] Table 3

[0281] codon Concentration / mg / ml Yield / mg Yield / mg / g bacteria Optimized codon I 3.038 54.681 13.7 Optimized Codon IV 2.326 39.542 9.9 Optimize codon V 1.563 26.571 6.6 Optimize codon VI 2.458 41.786 10.4

[0282] Based on the SDS results, eluent 2D4-2E4 was selected for dialysis, yielding 18 ml of sample. The concentration was determined using BCA, and the result was: R 2 =0.997, its concentration is 3.038 mg / ml, the yield is 54.681 mg, and the yield is 13.7 mg / g bacteria. Representative electrophoresis patterns are shown below. Figure 5 ( Figure 5 (Electrophoresis image of recombinant cystathionine obtained from bacterial culture containing optimized codon I).

[0283] The recombinant human cystathionine-β-synthase was collected, purified, and placed in a 10 kDa dialysis bag. The bag was then immersed in 1000 ml of 1X PBS and dialyzed overnight. The next day, all the sample precipitated, indicating poor colloidal stability of the protein. A representative electrophoresis image is shown below. Figure 6 (Similarly, 6 is an electrophoresis image of recombinant cystathionine obtained from bacterial culture containing optimized codon III).

[0284] Example 4

[0285] In this embodiment, purified cystathionine-β-synthase from *Saccharomyces cerevisiae* was used for subsequent enzyme activity assays. The specific steps are as follows:

[0286] (1) Solution preparation

[0287] Ninhydrin solution: Accurately weigh 1g of ninhydrin and dilute to 100ml with H2O.

[0288] 0.1M serine solution: Accurately weigh 0.105g of serine and dilute to 10ml with H2O.

[0289] 12.5mM pyridoxal phosphate solution (PLP): Accurately weigh 0.03g of pyridoxal phosphate and dilute to 10ml with H2O.

[0290] 12.5mM S-adenosylmethionine: Accurately weigh 0.01865g of S-adenosylmethionine and bring the volume to 10ml with H2O.

[0291] Preparation of positive enzyme (cystathionine-β-synthase): Dissolve 1610U of positive enzyme in 1mL of PBS pH 7.4 buffer to prepare an enzyme solution of 1.6U / μL. Then, dilute stepwise according to the gradient, and the dilution buffer is PBS pH 7.4 buffer.

[0292] (2) Instrument testing

[0293] Preheat the microplate reader for 30 minutes. Prepare a 50 μL reaction system according to Table 3 below. The blank control is without enzyme. Incubate at 37℃ for 5 minutes, then add the reagents listed in Table 4 below to each tube. After incubating at 7℃ for 1 hour, incubate on ice for 5 minutes, add 3 mL of ninhydrin to each tube, incubate in boiling water for 5 minutes for color development, incubate on ice for 5 minutes to terminate the reaction, and let stand at room temperature for 20 minutes. Microplate reader program settings: Add 200 μL of detection working solution to the blank tube and the test tube respectively. Detect the absorbance at 453 nm as A1 and A2, respectively. Calculate the OD difference between the sample and the blank, A2-A1. The standard curve is shown below. Figure 7 .

[0294] Table 3

[0295] reagents Add volume 100mM Tris-HCl (pH 8.6) 2μL 0.1M serine 40μL 12.5mM PLP 4μL 12.5mM S-adenosylmethionine 4μL Cystathion-β-synthase 5μL

[0296] Table 4

[0297] reagents Add volume 0.2M homocysteine 20μL 1M DTT 0.5μL <![CDATA[dd H2O]]> 127μL

[0298] The absorbance of the cystathionine-β-synthase derived from Saccharomyces cerevisiae prepared in this invention was measured according to the above method, and the results are shown in Table 5 below.

[0299] Table 5

[0300]

[0301] The concentration of the purified brewer's yeast-derived cystathionine-β-synthase stock solution was 3.038 mg / mL, and the average activity was 0.79 U / μL. Therefore, the specific activity was (0.79*1000) / 3.038=260 U / mg.

[0302] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. An isolated polynucleotide encoding cystathionine-β-synthase, characterized in that, The polynucleotide is codon-optimized and is as shown in SEQ ID NO.

1.

2. An expression carrier, characterized in that, The expression vector comprises the polynucleotide as described in claim 1.

3. The expression vector according to claim 2, characterized in that, The expression vector includes a polynucleotide sequence expressing a His×6 tag.

4. The expression vector according to claim 3, characterized in that, The expression vector is an Escherichia coli expression vector.

5. The expression vector according to claim 3, characterized in that, The expression vector is pET-28a(+).

6. A host cell, characterized in that, The host cell comprises the expression vector as described in any one of claims 2 to 5; or The host cell genome contains the polynucleotides as described in claim 1.

7. A method for preparing cystathionine-β-synthase, characterized in that, The method includes the following steps: Transform host cells using a vector containing the polynucleotide as described in claim 1; The host cells are cultured to express the cystathionine-β-synthetase.

8. The method according to claim 7, characterized in that, The host cells were cultured in LB medium.

9. The method according to claim 7, characterized in that, When culturing the host cells, the target protein is expressed by IPTG induction.

10. A reagent kit, characterized in that, The kit comprises: the polynucleotide as described in claim 1; or The expression vector as described in any one of claims 2 to 5; or The host cell as described in claim 6.

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