CN117587049A - Recombinant D-3-hydroxybutyrate dehydrogenase and preparation method and application thereof - Google Patents

Abstract

The application discloses a recombinant D-3-hydroxybutyrate dehydrogenase and a preparation method and application thereof. The application provides a polynucleotide sequence for encoding D-3-hydroxybutyrate dehydrogenase with optimized synonymous codon preference, which has high expression efficiency in escherichia coli; the vector is also provided, and the high-activity D-3-hydroxybutyrate dehydrogenase is prepared and obtained through the cooperative coordination of the vector and the polynucleotide sequence, wherein the specific activity is as high as 362.87U/mg; in addition, the method for preparing the recombinant D-3-hydroxybutyrate dehydrogenase not only improves the yield of the D-3-hydroxybutyrate dehydrogenase, but also has the advantages of extremely high content of soluble protein of the obtained product, easy purification, simplified post-treatment steps and saved production cost.

Description

Recombinant D-3-hydroxybutyrate dehydrogenase and preparation method and application thereof

Technical Field

The invention relates to the field of biochemical detection, in particular to recombinant D-3-hydroxybutyrate dehydrogenase and a preparation method and application thereof.

Background

D-3-hydroxybutyrate dehydrogenase, also known as 3-hydroxybutyrate dehydrogenase, 3-hydroxybutyrate dehydrogenase or D-3-hydroxybutyrate dehydrogenase, is an oxidoreductase acting on the CH-OH group of a donor with NAD+ or NADP+ as an acceptor, and is an enzyme capable of catalyzing the stereospecific oxidation of 3-hydroxybutyrate/(R) -3-hydroxybutyrate/3-hydroxybutyrate (3-HB) to acetoacetate. The enzyme is involved in ketone body synthesis, degradation and butyrate metabolism and can be used to determine ketone bodies in clinical samples, such as blood samples.

Ketone bodies are particularly important for brains without other substantial non-glucose derived energy sources, and are largely divided into two types, 3-hydroxybutyric acid and acetoacetic acid. Biochemically, abnormalities in ketone body metabolism can be subdivided into three categories: ketosis (ketosis), ketosis hypo-glycemia (hypoketotic hypoglycemia), and abnormal 3-hydroxybutyrate/acetoacetate ratio. The pathological causes of ketosis include diabetes, ketogenic hypoglycemia in childhood, corticosteroid or growth hormone deficiency, alcoholism or salicylic acid poisoning, and several congenital metabolic errors. Ketone bodies can be used as targets in clinical diagnostics, particularly diabetes. Thus, there is a current need to build a robust and sensitive test system for ketone bodies, especially 3-hydroxybutyrate, whereas building a robust 3-hydroxybutyrate test system requires a large amount of D-3-hydroxybutyrate dehydrogenase, however the inventors have found in research that the gene encoding D-3-hydroxybutyrate dehydrogenase is cloned into a vector and after expression in e.coli host cells purified, the resulting D-3-hydroxybutyrate dehydrogenase is inactivated in the absence of phospholipid activation. Therefore, there is still a need in the art to develop a method for preparing highly efficient and high-enzymatic D-3-hydroxybutyrate dehydrogenase.

Disclosure of Invention

The invention aims to provide a preparation method of recombinant D-3-hydroxybutyrate dehydrogenase with high yield and good product activity.

Another objective of the invention is to provide a polynucleotide sequence encoding a D-3-hydroxybutyrate dehydrogenase.

It is another object of the present invention to provide a vector adapted to a polynucleotide sequence encoding a D-3-hydroxybutyrate dehydrogenase.

Another objective of the invention is to provide a kit comprising a polynucleotide sequence encoding a D-3-hydroxybutyrate dehydrogenase.

To solve the above technical problems, in a first aspect of the present invention, there is provided a polynucleotide encoding a D-3-hydroxybutyrate dehydrogenase, 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 or SEQ ID NO. 3;

(ii) Polynucleotides having greater than 95% homology to the sequences as set forth in SEQ ID NO.1 or SEQ ID NO. 3; and

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

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

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

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

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

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

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

In a fourth aspect, the present invention provides a method for producing D-3-hydroxybutyrate dehydrogenase, 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 D-3-hydroxybutyrate dehydrogenase.

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

In some preferred embodiments, the host cells are cultured in TB medium.

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

In some preferred embodiments, the host cell is cultured at a temperature of 36 to 38℃or 16 to 19℃and more preferably 16 to 18 ℃.

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

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

In some preferred embodiments, the host cell is cultured until an OD600 of 0.6 to 0.8 is reached, and is then induced with IPTG to express the protein of interest.

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

passing the crushed target protein supernatant through a chromatographic column, eluting, and collecting the eluent.

In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column, such as Ni-NTA.

In a fifth aspect, the invention provides a kit comprising: a polynucleic acid 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

D-3-hydroxybutyrate dehydrogenase produced according to the method of the fourth aspect of the present invention.

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

(1) The invention provides a polynucleotide sequence for encoding D-3-hydroxybutyrate dehydrogenase with optimized synonymous codon preference, which has high expression efficiency in escherichia coli;

(2) The invention also provides a vector adapting to the polynucleotide sequence, and the high-activity D-3-hydroxybutyrate dehydrogenase is prepared and obtained through the cooperative matching of the vector and the polynucleotide sequence, wherein the specific activity is as high as 379U/mg;

(3) The invention provides a method for preparing recombinant D-3-hydroxybutyrate dehydrogenase, which optimizes the culture conditions, not only improves the yield of the D-3-hydroxybutyrate dehydrogenase, but also has extremely high content of soluble protein of the obtained product, is easy to purify, and simplifies the post-treatment steps;

(4) In a preferred embodiment of the present invention, a polynucleotide sequence obtained by codon optimization of a gene sequence corresponding to the mutated D-3-hydroxybutyrate dehydrogenase is provided, and the product obtained in the expression system of the present invention has extremely high enzymatic activity.

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 result of SDS-PAGE identification of D-3-hydroxybutyrate dehydrogenase according to an embodiment of the present invention;

FIG. 2 is a schematic illustration of the D-3-hydroxybutyrate dehydrogenase process according to an embodiment of the present invention ^TM Electrophoresis after FF affinity purification;

FIG. 3 is a graph of absorbance versus D-3-hydroxybutyrate dehydrogenase concentration in accordance with an embodiment of the present invention.

Detailed Description

The inventor provides an expression system for preparing D-3-hydroxybutyrate dehydrogenase by efficiently expressing heterologous genes in microorganisms through extensive and intensive research, wherein the expression system uses a polynucleotide sequence SEQ ID NO 1 matched with a target escherichia coli carrier and obtained through codon preference optimization, so that the yield of the D-3-hydroxybutyrate dehydrogenase is greatly improved, the subsequent purification steps are simplified, the economic cost is saved, and the activity of the recombinant D-3-hydroxybutyrate dehydrogenase obtained by the preparation method of the D-3-hydroxybutyrate dehydrogenase based on the expression system is greatly improved, and the expression system has good application prospect. Furthermore, the inventor obtains a polynucleotide sequence based on mutant D-3-hydroxybutyrate dehydrogenase, obtains a polynucleotide sequence SEQ ID NO.3 through codon preference optimization, wherein the SEQ ID NO.3 is more suitable for an escherichia coli expression system of the invention, and the obtained recombinant D-3-hydroxybutyrate dehydrogenase has higher activity.

The embodiment of the invention also constructs a method for efficiently preparing high-activity D-3-hydroxybutyrate dehydrogenase, which comprises the following steps: obtaining a target gene sequence; carrying out synonymous codon preference optimization on a target gene sequence; obtaining a vector of a target gene, and connecting the target gene to the vector; transforming a host cell with a vector comprising a gene of interest; culturing the host cell to express the desired protein D-3-hydroxybutyrate dehydrogenase; and separating the target protein.

Acquisition of a Gene of interest/acquisition of a nucleic acid sequence related to a protein of interest

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.

In a preferred embodiment of the present invention, the target protein (D-3-hydroxybutyrate dehydrogenase) is derived from Alcaligenes faecalis, and the polynucleotide obtained by codon optimization is more suitable for the expression system of the present invention than the gene sequences corresponding to the target protein extracted from other strains, and the soluble protein proportion in the obtained product is high.

In one embodiment of the present invention, the amino acid sequence of the target protein (SEQ ID NO: 2) is analyzed by NCBI database to obtain the sequence information of the target gene. The amino acid sequence of the D-3-hydroxybutyrate dehydrogenase of Arthrobacter sphaeroides is analyzed, for example, by NCBI database to obtain the target gene sequence encoding the same.

In another embodiment of the present invention, the amino acid sequence of the mutated protein of interest (SEQ ID NO: 4) is analyzed by NCBI database to obtain the sequence information of the gene of interest.

Synonymous codon bias optimization

To overcome the potential problem of reduced yield when expressing heterologous proteins in E.coli, the present invention relates to polynucleotide sequences optimized for synonymous codon bias. The obtained target gene sequence is subjected to synonymous codon preference optimization, the target gene sequence (SEQ ID NO: 1) subjected to synonymous codon preference optimization can express the same amino acid sequence as the target protein, but the stability and efficiency of the expression process are improved, and finally the obtained target protein maintains higher activity.

The invention also relates to polynucleotides having a homology of more than 95% with the sequence shown in SEQ ID NO. 1; and a polynucleotide complementary to the sequence shown in SEQ ID No. 1.

In order to further increase the activity of the product, the polynucleotide sequence corresponding to the mutant target protein obtained unexpectedly by the inventor has higher enzyme activity after synonymous codon preference optimization (SEQ ID NO: 3).

Vector of target gene

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

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

Transformation of host cells with vectors containing genes of interest

The invention also relates to host cells genetically engineered with the vector or fusion protein coding sequences of the invention. The vector containing the codon-optimized gene of interest may be inserted, transfected or otherwise transformed into a host cell by known methods to obtain a transformant containing the codon-optimized gene of interest of the present invention and capable of expressing the protein of interest. A "host cell" in the present invention is a cell into which an exogenous polynucleotide and/or vector has been introduced. The host cell may be a eukaryotic host cell or a prokaryotic host cell, the host cell is preferably a bacterium, and is preferably E.coli, more preferably E.coli BL21 (DE 3) strain (Escherichia coli BL (DE 3) strain).

Method for producing target protein

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

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

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

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

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

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. 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. In order to promote expression of the target protein, a preferred embodiment of the present invention uses a host cell cultured in a TB medium, and the medium used contains a kanamycin resistance gene.

In a preferred embodiment of the invention, the temperature of the cultured host cells is below room temperature, more preferably between 16 and 19 ℃, at which the soluble protein expressed by the cultured host cells is high.

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 disruption, 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. In one embodiment of the invention, affinity chromatography is used to molecular proteins of interest.

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 printed or issued documents 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 nucleotide or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

As used herein, the terms "sequence complement" and "reverse sequence complement" are used interchangeably to refer to a sequence that is in the opposite direction to the original polynucleotide sequence and that is 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 "expression" includes any step involving the production of a polypeptide in a host cell, including, but not limited to, transcription, translation, post-translational modification, and secretion. After expression, the host cells or expression products can be harvested, i.e.recovered.

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

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

Example 1 construction of D-3-hydroxybutyrate dehydrogenase plasmid

(1) The amino acid sequence (SEQ ID NO: 2) of the alcaligenes D-3-hydroxybutyrate dehydrogenase provided by NCBI is taken as a reference, the target gene sequence is obtained after the amino acid sequence analysis, and the target gene sequence is subjected to synonymous codon preference optimization (SEQ ID NO: 1) and the connecting carrier is pET-28a.

The mutation optimization is carried out on the basis of the amino acid sequence (SEQ ID NO: 2) of the D-3-hydroxybutyrate dehydrogenase to obtain an amino acid sequence (SEQ ID NO: 4), and the connection carrier is pET-28a after synonymous codon preference optimization is carried out on the amino acid sequence (SEQ ID NO: 3).

(2) Recombinant plasmid transformed E.coli BL21 (DE 3)

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 ℃.

SEQ ID NO:1：

ATGCTGAAAGGTAAAAAAGCGGTGGTGACCGGTTCCACCAGCGGTATTGGTCTGGCAATGGCCACCGAACTGGCAAAAGC CGGTGCTGACGTTGTAATCAATGGCTTCGGTCAACCGGAAGATATTGAACGTGAGCGTAGCACTCTGGAATCTAAATTTG GTGTAAAAGCCTACTATCTGAACGCTGACCTGTCTGATGCGCAGGCTACCCGTGACTTTATTGCAAAAGCTGCTGAAGCG CTGGGTGGTCTGGATATCCTGGTTAACAATGCCGGCATCCAGTACACGGCGCCGATCGAAGAATTTCCGGTAGACAAATG GAATGCGATCATCGCGCTGAATCTGTCTGCGGTTTTTCATGGTACCGCGGCTGCACTGCCGATCATGCAGAAACAAGGCT GGGGTCGTATCATCAATATCGCGTCTGCACACGGTCTGGTCGCTTCTGTGAACAAAAGCGCCTACGTGGCGGCTAAACAC GGTGTTGTTGGTCTGACCAAAGTGACCGCCCTGGAAAACGCTGGCAAAGGTATCACCTGCAATGCGATCTGCCCGGGTTG GGTTCGTACCCCTCTGGTGGAAAAACAGATCGAAGCTATCTCCCAGCAAAAAGGTATCGACCTGGAAGCCGCTGCTCGTG AGCTGCTGGCTGAAAAACAGCCTAGCCTGCAGTTCGTTACTCCGGAACAGCTGGGCGGCGCTGCTGTTTTCCTGAGCTCT GCAGCAGCAGATCAGATGACCGGTACCACCCTGTCTCTGGATGGTGGCTGGACTGCGCGC

SEQ ID NO:2：

MLKGKKAVVTGSTSGIGLAMATELAKAGADVVINGFGQPEDIERERSTLESKFGVKAYYLNADLSDAQAT RDFIAKAAEALGGLDILVNNAGIQYTAPIEEFPVDKWNAIIALNLSAVFHGTAAALPIMQKQGWGRIINI ASAHGLVASVNKSAYVAAKHGVVGLTKVTALENAGKGITCNAICPGWVRTPLVEKQIEAISQQKGIDLEA AARELLAEKQPSLQFVTPEQLGGAAVFLSSAAADQMTGTTLSLDGGWTAR

SEQ ID NO:3：

ATGCTGAAGGGCAAAAAAGCTGTTGTTACGGGCAGCACTTCTGGTATCGGCCTGGCGATGGCAACCGAACTGGCGAAAGC TGGTGCAGATGTTGTGATTAACGGCTTCGGTCAACCGGAGGATATCGAACGTGAACGCAGCACCCTGGAGAGCAAATTCG GCGTTAAAGCGTACTATCTGAACGCGGACCTGTCTGACGCACAAGCTACCCGTGATTTCATCGCCAAAGCAGCGGAAGCC CTGGGTGGTCTGGATATTCTGGTGAACAACGCCGGCATCCAGCACACTGCTCCGATCGAAGAATTTCCTGTGGACAAATG GAACGCAATCATTGCGCTGAACCTGAGCGCGGTCTTCCACGGTACTGCCGCAGCGCTGCCAATCATGCAGAAACAGGGCT GGGGTCGCATCATCAACATCGCGTCCGCACACGGTCTGGTCGCAAGCGTCAACAAATCTGCGTACGTAGCGGCTAAACAC GGCGTTGTTGGTCTGACCAAAGTTACTGCGCTGGAAAACGCTGGCAAAGGTATTACTTGTAACGCAATCTGTCCGGGCTG GGTGCGTACCCCGCTGGTTGAAAAACAGATCGAAGCGATCAGCCAGCAGAAGGGCATCGACATCGAAGCAGCGGCACGTG AACTGCTGGCAGAAAAACAGCCGTCTCTGCAGTTCGTAACGCCGGAACAGCTGGGTGGTGCTGCGGTTTTCCTGTCCAGC GCCGCAGCAGACCAAATGACTGGTACCACTCTGTCCCTGGACGGTGGCTGGACCGCACGC

SEQ ID NO:4：

MLKGKKAVVTGSTSGIGLAMATELAKAGADVVINGFGQPEDIERERSTLESKFGVKAYYLNADLSDAQAT RDFIAKAAEALGGLDILVNNAGIQHTAPIEEFPVDKWNAIIALNLSAVFHGTAAALPIMQKQGWGRIINI ASAHGLVASVNKSAYVAAKHGVVGLTKVTALENAGKGITCNAICPGWVRTPLVEKQIEAISQQKGIDIEA AARELLAEKQPSLQFVTPEQLGGAAVFLSSAAADQMTGTTLSLDGGWTAR

EXAMPLE 2 expression of the protein of interest by D-3-hydroxybutyrate dehydrogenase

Step of picking the monoclonal in example 1, aseptically inoculated into 100. Mu.g/mL of kana-resistant TB medium, each medium was subjected to 2-tube replicates, each medium was designated TB (1), TB (2), and shaking culture was performed at 37℃at 220rpm until OD600 was between 0.6 and 0.8, induction was performed with IPTG, and shaking culture was performed at 37℃and 18℃respectively overnight. SDS-PAGE identification was performed by sampling ultrasonication and the results are recorded in Table 1 and FIG. 1.

The results in FIG. 1 show that a large amount of soluble expression in TB medium is possible in the supernatant at 18℃and the protein molecular weight is about 27kDa.

TABLE 1

Sequences introduced into vectors	Culture conditions	Soluble protein ratio	Molecular weight of protein
				SEQ ID NO:1	37℃	40％	27kDa
	18℃	50％	27kDa
				SEQ ID NO:3	37℃	40％	27kDa
	18℃	50％	27kDa

EXAMPLE 3 purification of D-3-hydroxybutyrate dehydrogenase

After 4g of cells were sonicated, the cells were once treated with a 0.22 μm membrane and purified with 1mL of Ni-NTA. The flow rate was 0.5mL/min, and the loading was done using 90mL Lysis Buffer rinse UV and conductance to baseline. The elution procedure included: step 1:0%B,3CV, 1mL/min,96 deep hole plate automatic collection, 1mL per well; step 2, 0-60% B,20CV,1mL/min,96 deep well plates for automatic collection, 1mL per well; step 3:100% B,10CV,1mL/min,96 deep well plates were collected automatically, 1mL per well. The result of electrophoresis after sample collection is shown in FIG. 2. The molecular weight of the target protein is 27.10kDa, the main peak area is 11459mL mAu, and the average electric conductance is 8.76ms/cm; when going through Buffer A, the sample is eluted, and the sample quantity may exceed the load of the column; a total of 21mL of E4-F12 was collected for dialysis, 2.7mL of the sample was collected after dialysis, the sample concentration was 22.68582mg/mL (R2=0.992), and the yield was 61.251714mg. The expression level of the target protein in the case of SEQ ID NO. 4 introduced into the vector was calculated by the same method and recorded in Table 3. The concentrations of the solutions used are as follows:

Buffer A:100mM Tris,50mM NaCl,5％Glycerol,pH8.0

Buffer B:50mM Tris,50mM NaCl,500mM Imidazole,5％Glycerol,pH8.0

eluent: buffer linear elution of 0-60% B was performed at 20CV with a flow rate of 1mL/min

Lysis Buffer:50mM Tris,300mM NaCl,5％Glycerol,pH.0。

TABLE 3 Table 3

Sequences introduced into vectors	Expression level of target protein
		SEQ ID NO:1	81.9mg
SEQ ID NO:3	61.25mg

Example 4 determination of recombinant D-3-hydroxybutyrate dehydrogenase Activity

1) Solution preparation

1M Tris-HCl pH 8.5: tris powder 121.14g was weighed out, poured into a 1L beaker and sterilized purified water was added to 800 mL. After stirring uniformly, the pH was adjusted to 8.5 with concentrated hydrochloric acid at 25℃and then the volume was set to 1L. Filtering with 0.22 μm, and storing at 4deg.C;

3-hydroxybutyric acid (1M): the mother solution concentration is 10.8M,92.5 mu L of mother solution and 907.5 mu L of deionized water are fully and uniformly mixed;

nad+ (100 mM): 0.066g of NAD+ powder was weighed and dissolved in 1mL of deionized water and stored protected from light.

The working fluid for measuring the enzyme activity was prepared according to the following table 4:

TABLE 4 Table 4

Reagent(s)	Volume added	Final concentration
			1M Tris-HCl	0.25mL	50mM
NAD(100mM)	0.125mL	2.5mM
			3-hydroxybutyric acid (1M)	0.125mL	25mM
ddH2O	4.5mL

2) Drawing of a Standard Curve

Configuration of positive enzymes: 110KU 3-hydroxybutyrate dehydrogenase was dissolved in 5mL of PBS pH 7.4 buffer (containing 50% glycerol) to prepare 22U/. Mu.L of enzyme solution, which was gradually diluted according to a gradient, and the diluted solution was PBS pH 7.4 buffer.

The enzyme label instrument is preheated for 30min. Program setting of the enzyme label instrument: 100. Mu.L of the reaction solution was added, the absorbance A1 was measured at 340nm, and then 1. Mu.L of the enzyme solution was added at each concentration, and the reaction was carried out for 5 minutes, and the absorbance A2 was measured at 340 nm. The OD difference A2-A1D-3-hydroxybutyrate dehydrogenase standard curve before and after the reaction was calculated and the result is shown in FIG. 3.

The absorbance of the D-3-hydroxybutyrate dehydrogenase prepared from example was measured as described above, and the results are shown in Table 5 below:

TABLE 5

Sequences introduced into vectors	Enzyme concentration	Average OD	Enzyme activity
				SEQ ID NO:1	Diluted 30 times	0.2101	285.7U/mg
SEQ ID NO:3	Diluted 30 times	0.28	379U/mg

When the sequence insert introduced into the vector was SEQ ID NO.1, the stock concentration was 27.3mg/mL and the average viability was 7.8. 7.8U/. Mu.L, the specific viability was (7.8.times.1000)/27.3= 285.7U/mg

When the sequence inserted sequence introduced into the vector is SEQ ID NO.3, the stock solution concentration is 22.68mg/mL, the average activity is 8.6U/μL, and the specific activity is (8.6×1000)/22.68 =379U/mg.

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

Claims (10)

1. A polynucleotide encoding a D-3-hydroxybutyrate dehydrogenase, 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 or SEQ ID NO. 3;

(ii) Polynucleotides having greater than 95% homology to the sequences as set forth in SEQ ID NO.1 or SEQ ID NO. 3; and

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

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

3. The expression vector according to claim 2, characterized in that it is an e.coli expression vector, preferably pS28a.

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

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

5. A method for preparing D-3-hydroxybutyrate dehydrogenase, said method comprising the steps of:

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

culturing the host cell to express the D-3-hydroxybutyrate dehydrogenase.

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

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

8. The method of claim 5, wherein the culturing of the host cell is at a temperature of 36 to 38 ℃ or 16 to 19 ℃.

9. A method according to any one of claims 5 to 8, wherein the host cell is cultured to express the protein of interest by IPTG induction.

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

The expression vector of claim 2 or 3; or alternatively

The host cell of claim 4; or alternatively

A recombinant D-3-hydroxybutyrate dehydrogenase produced by the method of any one of claims 5-9.