CN117587046A - Recombinant hexokinase and preparation method and application thereof - Google Patents
Recombinant hexokinase and preparation method and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01001—Hexokinase (2.7.1.1)
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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Abstract
The application discloses a recombinant hexokinase and a preparation method and application thereof. Provided herein are polynucleotide sequences encoding hexokinase optimized for synonymous codon bias, which sequences are highly efficient in expression in E.coli; the vector adapting to the polynucleotide sequence is also provided, and the high-activity hexokinase with the specific activity as high as 111.8U/mg is prepared and obtained through the cooperative coordination of the vector and the polynucleotide sequence; in addition, the method for preparing the recombinant hexokinase not only improves the yield of the hexokinase, but also has the advantages of extremely high content of soluble protein of the obtained product, easy purification, simplified post-treatment steps and production cost saving.
Description
Technical Field
The invention relates to the field of biochemical detection, in particular to a recombinant hexokinase and a preparation method and application thereof.
Background
Hexokinase (HK), an evolutionarily conserved enzyme, phosphorylates hexoses (hexoses). Phosphorylated glucose (glucose-6-phosphate) is metabolized in the biosynthetic pathways of glycolysis, glycogenesis, pentose phosphate and hexosamine, plays a key role in ATP synthesis, NADH synthesis and protein glycosylation, and is the rate-limiting enzyme in the first step of glucose metabolism. In bacteria, hexokinase has a molecular weight of 32-37kDa. In vertebrates, hexokinases share four distinct subtypes, HK1, HK2, HK3, HK4, respectively, where HK1-3 has a molecular weight of 100kDa and HK4 is 50kDa. HK1 is ubiquitously present in most tissues, HK2 is less expressed than HK1, and is widely present in heart, skeletal muscle and adipose tissue, as well as in tumor cells. Hexokinase is closely related to glucose metabolism, and the conversion of glucose from a stable state to an active state can provide energy for tumor cells and a carbon source necessary for substance synthesis, so that hexokinase has been an important potential target for treating cancer cells. In clinical diagnosis, the kit is also an important tool enzyme for detecting blood sugar content, and has very important application in clinical diagnostic reagents.
Most of the current industrialized hexokinase is derived from fermentation of original strains or cultivation of animal and plant cells, and the production method has unstable process, causes uneven enzyme quality, is unfavorable for purification and has complicated process. The foreign competitive products of hexokinase are monopoly and are expensive, so that the production of high-quality and low-cost domestic enzymes is imperative. The genetic engineering technology cuts the recombinant DNA with tool enzyme, combines the recombinant DNA according to thought, transfers the recombinant DNA into organisms for asexual reproduction, and finally obtains the protein which is expected to be produced. The method has high efficiency and is suitable for mass production. Therefore, it is important to develop a stable hexokinase preparation method based on genetic engineering.
Disclosure of Invention
The invention aims to provide a preparation method of recombinant hexokinase with high yield and good product activity.
It is another object of the present invention to provide a polynucleotide sequence encoding a hexokinase.
It is another object of the present invention to provide a vector adapted to a polynucleotide sequence encoding a hexokinase.
It is another object of the present invention to provide a kit comprising a polynucleotide sequence encoding hexokinase.
To solve the above technical problem, according to a first aspect of the present invention, there is provided a polynucleotide encoding hexokinase, the polynucleotide being codon-optimized, and the 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 a homology of greater than 95% with a sequence as shown 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 polynucleotide provided in the first aspect of the invention.
In some preferred embodiments, the expression vector comprises a polynucleotide sequence that expresses a His X6 tag, more preferably, the expression vector has the 3' end of the polynucleotide linked to a polynucleotide sequence that expresses a His X6 tag.
In some preferred embodiments, the expression vector is an E.coli expression vector, more preferably pET-28a (+).
In a third aspect 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 of preparing hexokinase, 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 hexokinase;
wherein the target protein has an amino acid sequence shown as SEQ ID NO. 2.
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 temperature of the culture is 36 to 38℃or 16 to 20℃and more preferably 17 to 19℃when the host cells are cultured.
In some preferred embodiments, the host cell is cultured in a shaking environment.
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 a HisTrapTMFF.
In a fifth aspect, the invention provides a kit comprising: a polynucleotide as provided in the first aspect of the invention; or alternatively
An expression vector as provided in the second aspect of the invention; or alternatively
A host cell according to the third aspect of the invention; or alternatively
Or hexokinase prepared according to the method of the fourth aspect of the invention.
Compared with the prior art, the invention has at least the following advantages:
(1) The invention provides a polynucleotide sequence for coding hexokinase 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 hexokinase is prepared and obtained through the cooperative coordination of the vector and the polynucleotide sequence, and the specific activity is as high as 111.8U/mg;
(3) The invention provides a method for preparing recombinant hexokinase, which not only improves the yield of hexokinase, but also ensures that the obtained product has extremely high content of soluble protein, is easy to purify and simplifies the post-treatment steps.
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 SDS-PAGE identification of hexokinase according to an embodiment of the present invention;
FIG. 2 is a hexokinase according to an embodiment of the invention via HisTrap TM Electrophoresis after FF affinity purification;
FIG. 3 is a graph of absorbance versus hexokinase concentration for an example according to the invention.
Detailed Description
The inventor provides an expression system for preparing hexokinase by efficiently expressing heterologous genes in microorganisms through extensive and intensive research, and the polynucleotide sequences matched with a target escherichia coli vector are obtained through codon preference optimization in the expression system, so that the yield of the hexokinase is greatly improved, the subsequent purification steps are simplified, the economic cost is saved, and the activity of the recombinant hexokinase obtained by a hexokinase preparation method based on the expression system is greatly improved, so that the expression system has good application prospect. Embodiments of the present invention construct a method for efficiently preparing a highly active hexokinase, comprising the steps of: 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 protein hexokinase of interest; 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 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.
In the invention, a large number of screening is carried out on the sources of target proteins, and three strain sources suitable for an escherichia coli expression system are selected, namely saccharomyces cerevisiae, pseudomonas aeruginosa and longan. The amino acid sequence of hexokinase is analyzed by NCBI database to obtain the target gene sequence for encoding the hexokinase.
Synonymous codon bias optimization
To overcome the potential problem of reduced yield when expressing heterologous proteins in E.coli, the present invention relates to synonymous codon-biased optimized polynucleotide sequences. The target gene sequence obtained based on Saccharomyces cerevisiae source 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 another embodiment of the present invention, the target gene sequence obtained from Pseudomonas aeruginosa source is optimized for synonymous codon preference, and the target gene sequence (SEQ ID NO: 3) optimized for synonymous codon preference can express soluble protein with the expression amount of at least about 10%, but the enzyme activity is lower after purification.
The invention also relates to polynucleotides having a homology of more than 95% with the sequence shown in SEQ ID NO. 3; and a polynucleotide complementary to the sequence shown in SEQ ID No. 1.
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 linked to the exogenous gene, and then ligating the codon optimized fragment of the gene of interest to the vector, optionally with a single restriction site cohesive end ligation, double restriction site directional cloning, cohesive end ligation of different restriction sites, blunt end ligation, artificial linker ligation, or end ligation with an oligonucleotide.
In one embodiment of the invention, the vector further comprises a His X6 tag expressing polynucleotide sequence, preferably the His X6 tag expressing polynucleotide sequence is ligated to the 3' end (C-terminus) of the gene sequence of interest.
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. Depending on the host cell used, the medium used in the culture may be selected from various conventional media commonly used in the art. 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 present invention, the host cells are cultured at a low temperature at room temperature, and the resulting soluble protein is in a high proportion, and the temperature of the culture is more preferably 17 to 19 ℃.
The protein in the above method may be expressed in the cell, or on the cell membrane, or secreted outside the cell. If desired, the proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods. In one embodiment of the invention, affinity chromatography is used to molecular target proteins.
In the present disclosure, any exemplary or exemplary language (e.g., ") provided for certain embodiments herein is used merely to better present the disclosure and does not limit the scope of the disclosure as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
If the definition or use of a term in a reference is inconsistent or inconsistent with the definition of that term described herein, the definition of the term described herein applies and the definition of the term in the reference does not apply.
Various terms used herein are shown below. If a term used in a claim is not defined below, the broadest definition persons in the pertinent art have given that term are given as reflected in publications or issued patents that are printed at the time of application.
As used herein, the term "isolated" refers to a nucleic acid or polypeptide that is separated from at least one other component (e.g., a nucleic acid or polypeptide) that the nucleic acid or polypeptide is found in its natural source. In one embodiment, the nucleic acid or polypeptide is found to be present only (if any) in solvents, buffers, ions or other components that are normally present in its solution. The terms "isolated" and "purified" do not include nucleic acids or polypeptides that are present in their natural source.
As used herein, the terms "polynucleotide" and "polynucleotide sequence" may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
The invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the invention. Variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.
As used herein, the term "codon optimization" refers to a manner of improving the efficiency of gene synthesis by avoiding the use of low-availability or rare codons according to codon usage differences exhibited by organisms (including e.coli, yeast, mammalian blood cells, plant cells, insect cells, etc.) that actually do protein expression or production.
As used herein, the terms "homology" and "identity" are used interchangeably to refer to the percentage of identical (i.e., identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by the following methods. The nucleotide or amino acid sequence of a polynucleotide or polypeptide is aligned, the number of positions in the aligned polynucleotide or polypeptide that contain the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotide or polypeptide that contain a different nucleotide or amino acid residue. Polynucleotides may differ at one position, for example, according to the inclusion of different nucleotides (i.e., substitutions or variations) or deletions of nucleotides (i.e., insertions or deletions of one or two nucleotides in the polynucleotide). The polypeptides may differ at one position, for example, by containing an amino acid (i.e., substitution or variation) or a deletion of an amino acid (i.e., an amino acid or deletion of an amino acid inserted into one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.
As used herein, the terms "sequence complementary" and "reverse sequence complementary" are used interchangeably to refer to a sequence that is opposite in direction to and complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is actaac, 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 hexokinase plasmid
(1) The amino acid sequence (SEQ ID NO: 2) of Saccharomyces cerevisiae-derived hexokinase provided by NCBI is taken as a reference, the target gene sequence is obtained after amino acid sequence analysis, and after synonymous codon preference optimization (SEQ ID NO: 1) is carried out on the target gene sequence, the linking vector is pET-28a (+), and the C-terminal fusion expression (His) 6 tag.
The amino acid sequence (SEQ ID NO: 4) of hexokinase of pseudomonas aeruginosa provided by NCBI is taken as a reference, the target gene sequence is obtained after amino acid sequence analysis, and after synonymous codon preference optimization (SEQ ID NO: 3) is carried out on the target gene sequence, the connection carrier is pET-28a (+), and the C-terminal fusion expression (His) 6 tag.
The amino acid sequence (SEQ ID NO: 6) of longan hexokinase provided by NCBI is taken as a reference, the target gene sequence is obtained after the amino acid sequence analysis, and after synonymous codon preference optimization (SEQ ID NO: 5) is carried out on the target gene sequence, the connection carrier is pET-28a (+), and the C-terminal fusion expression (His) 6 tag.
(2) E.coli BL21 (DE 3) was transformed with the recombinant plasmid, 1. Mu.L of the plasmid prepared in the step (1) was added to 30. Mu.L of E.coli competent BL21 (DE 3) under ice bath conditions, the mixture was left in ice bath for 20min, heat-shocked in a water bath at 42℃for 45s, immediately left on ice for 2min, 400. Mu.L of the SOC medium containing no antibiotic was added, and shaking culture was performed at 220rpm for 50min at 37 ℃. mu.L of the bacterial liquid was uniformly spread on LB plates containing 100. Mu.g/mL of kana resistance, and incubated overnight at 37 ℃.
SEQ ID NO:1：
GTTCATCTGGGCCCGAAAAAACCGCAAGCTCGCAAAGGTTCTATGGCAGACGTCCCGAAAGAACTGATGCAGCAAATCGAAAACTTCGAAAAGATCTTTACCGTGCCAACGGAAACCCTGCAGGCTGTGACTAAACATTTCATCTCTGAACTGGAGAAAGGTCTGAGCAAAAAAGGTGGTAACATCCCAATGATCCCGGGTTGGGTGATGGATTTCCCGACTGGCAAAGAATCTGGCGACTTTCTGGCAATCGACCTGGGCGGTACCAACCTGCGCGTCGTTCTGGTTAAACTGGGTGGTGACCGCACCTTCGACACTACCCAGTCTAAATATCGCCTGCCGGATGCCATGCGTACTACTCAGAACCCAGATGAACTGTGGGAATTCATCGCTGACTCTCTGAAAGCATTCATCGATGAACAGTTCCCGCAGGGTATTTCTGAACCGATCCCTCTGGGCTTCACCTTTTCTTTCCCAGCTTCCCAGAACAAAATCAATGAAGGTATCCTGCAGCGCTGGACGAAAGGTTTCGACATCCCAAACATCGAAAACCACGATGTAGTGCCGATGCTGCAAAAACAGATTACCAAACGTAACATCCCAATCGAAGTTGTCGCGCTGATCAACGACACCACCGGTACCCTGGTTGCTTCTTATTACACCGACCCGGAAACTAAGATGGGCGTGATTTTCGGCACCGGTGTCAACGGTGCATACTATGATGTTTGCAGCGACATCGAAAAACTGCAGGGTAAACTGTCCGACGACATCCCGCCGTCTGCACCAATGGCAATCAACTGCGAATATGGCTCTTTCGATAACGAACACGTTGTCCTGCCGCGTACCAAATACGATATCACTATCGATGAAGAATCTCCGCGTCCGGGCCAGCAAACCTTTGAAAAAATGTCTTCCGGCTACTACCTGGGTGAAATCCTGCGCCTGGCGCTGATGGACATGTATAAACAGGGCTTCATTTTCAAAAACCAGGACCTGAGCAAGTTCGATAAACCGTTCGTCATGGACACCTCCTACCCAGCGCGTATCGAAGAAGACCCGTTTGAAAACCTGGAGGACACTGATGATCTGTTCCAAAACGAATTCGGTATCAATACCACCGTTCAGGAGCGTAAGCTGATCCGTCGTCTGTCTGAACTGATTGGCGCACGTGCTGCGCGTCTGTCTGTTTGTGGCATCGCTGCCATCTGCCAGAAACGTGGTTATAAAACCGGCCACATTGCTGCGGACGGTTCTGTGTATAACCGCTACCCGGGTTTTAAAGAGAAAGCGGCAAACGCGCTGAAAGATATCTACGGCTGGACCCAGACTAGCCTGGATGACTATCCGATCAAAATTGTGCCGGCAGAAGATGGTTCCGGTGCTGGTGCTGCTGTTATCGCAGCACTGGCTCAAAAACGTATCGCAGAAGGTAAAAGCGTAGGCATTATCGGCGCT
SEQ ID NO:2：
VHLGPKKPQARKGSMADVPKELMQQIENFEKIFTVPTETLQAVTKHFISELEKGLSKKGGNIPMIPGWVMDFPTGKESGDFLAIDLGGTNLRVVLVKLGGDRTFDTTQSKYRLPDAMRTTQNPDELWEFIADSLKAFIDEQFPQGISEPIPLGFTFSFPASQNKINEGILQRWTKGFDIPNIENHDVVPMLQKQITKRNIPIEVVALINDTTGTLVASYYTDPETKMGVIFGTGVNGAYYDVCSDIEKLQGKLSDDIPPSAPMAINCEYGSFDNEHVVLPRTKYDITIDEESPRPGQQTFEKMSSGYYLGEILRLALMDMYKQGFIFKNQDLSKFDKPFVMDTSYPARIEEDPFENLEDTDDLFQNEFGINTTVQERKLIRRLSELIGARAARLSVCGIAAICQKRGYKTGHIAADGSVYNRYPGFKEKAANALKDIYGWTQTSLDDYPIKIVPAEDGSGAGAAVIAALAQKRIAEGKSVGIIGA
SEQ ID NO:3：
ATGTCTACTCCGGCGACTCCGACTAGCTGTGACATTCTGGTGTTCGGTGGCACCGGTGATCTGGCTCTGCATAAACTGCTGCCAGCGCTGTACCGTCTGCACCGTGAAGATCGTCTGCCGGCGGACACCCGTATCTTCGCTCTGGCTCGTTCCGCTCTGGATAACGCGGCTTTCCTGGCGCTGGCTGAACGTAACGTGCGCGCTGTCGTAGCGCGTTCTGAATTCGCCGCCGAACAGTGGAAAGGTTTCGCTGCACGTCTGGACTACCTGGCTATGGATGCAAGCCAGTCTGCTGATTTCGGCCGTCTGGCACGTCATCTGAAAAGCTCTGAAGGCCGTGTTCTGGTACACTATCTGGCGACTAGCCCATCTCTGTTCGCGCCAATCGCTCAAAACCTGTCTATCGCTGGTCTGGCCGGTCCACAGGCACGTATCGTGCTGGAGAAACCGCTGGGCCATTCTCTGGACAGCGCTCGTGCAATCAACCAGTCTATTGGTCGTGTATTTGACGAATCCCGCGTATTCCGTATCGATCACTATCTGGGCAAAGAAACCGTCCAGAACCTGATGGCGCTGCGTTTCGCGAACGCTCTGTTTGAACCGGTCTGGCGTGCTGCCCACATTGATCACGTCCAAATTAGCGTGAACGAAACCCTGGGCGTTGAAAACCGTGGTGGTTACTATGACCATGCAGGCGCCATGCGCGATATGGTTCAAAACCACCTGCTGCAGCTGCTGTGTCTGGTTGCTATGGAAGCTCCGGTTCGTTTTGACGCGGAGGCTGTGCGTAACGAAAAGCTGAAAGTTCTGCAGGCTCTGAAACCGATCAGCGGCCTGGACGTGCAGGATAAAACGGTTCGTGGTCAGTATGCGGCGGGTCGTATCGGCGGTCAGGAAGTTCCGGCGTATTACTTCGAAAAAAACGTCGATAATGACTCTGACACCGAGACCTTTGTCGCACTGCAAGCGGAAGTGGAAAATTGGCGCTGGGCTGGTGTTCCATTCTATCTGCGTACTGGCAAACGTATGGCGCGTAAATGCTCTGAAATTGTTATCCAGTTCAAACCGGTGCCGCACTCTCTGATTGATGGTGGTGGTGGTCCGGCTAACCGTCTGTGGATCCGCCTGCAACCAGAAGAACGTATTTCCGTGCAGCTGATGGCGAAAACTCCGGGTAAAGGCATGCAGCTGGAACCTGTCGAACTGGACCTGAACCTGGCAGAAGCTCTGTCCCGTAACAAACGCCGTTGGGATGCTTACGAACGCCTGCTGCTGGATGTCATCGAGGGCGATTCTACGCTGTTTATGCGCCGTGATGAAGTAGAAGCTGCATGGGGTTGGGTGGATCCGATCCTGGCAGGTTGGCGCGAACACTACCAATCTCCGCGTCCGTATCCTGCAGGCTCTAACGGTCCGGAACAGGCTCAGCTGCTGCTGGAACTGCAGGGCCGTCGTTGGCTGGAA
SEQ ID NO:4：
MSTPATPTSCDILVFGGTGDLALHKLLPALYRLHREDRLPADTRIFALARSALDNAAFLALAERNVRAVVARSEFAAEQWKGFAARLDYLAMDASQSADFGRLARHLKSSEGRVLVHYLATSPSLFAPIAQNLSIAGLAGPQARIVLEKPLGHSLDSARAINQSIGRVFDESRVFRIDHYLGKETVQNLMALRFANALFEPVWRAAHIDHVQISVNETLGVENRGGYYDHAGAMRDMVQNHLLQLLCLVAMEAPVRFDAEAVRNEKLKVLQALKPISGLDVQDKTVRGQYAAGRIGGQEVPAYYFEKNVDNDSDTETFVALQAEVENWRWAGVPFYLRTGKRMARKCSEIVIQFKPVPHSLIDGGGGPANRLWIRLQPEERISVQLMAKTPGKGMQLEPVELDLNLAEALSRNKRRWDAYERLLLDVIEGDSTLFMRRDEVEAAWGWVDPILAGWREHYQSPRPYPAGSNGPEQAQLLLELQGRRWLE
SEQ ID NO:5：
ATGGGTAAAGTTGCCGTAGGTGCTGCAGTTGTATGTGCGGCTGCAGTGTGTGCAGCAACTGCACTGGTCGTTCGTCACCGTATGCAGTCTAGCGGCAAATGGGCACGTGCCATGGCAATCCTGAAGGACCTGGAAGACAAATGTGGCACCCCGATCGGTAAACTGCGTCAGATTGCCGACGCGATGACCGTTGAAATGCACGCAGGCCTGGCATCTGAAGGCGGTTCCAAACTGAAGATGCTGATTTCCTTCGTTGATAACCTGCCGACCGGCGACGAGAAAGGTCTGTTCTATGCGCTGGACCTGGGCGGTTCCAACTTTCGTGTCCTGCGTGTGCTGCTGGGTGGTAAAGAAGGTCACGTTGTGAAACAGGAATTCAAAGAGGTGTCCATCCCGCAGCACCTGATGGTGGGTAGCAGCCATGAACTGTTCAACTTCATCGCGGCGGCACTGGCAAAGTTTGTAGCAACGGAAGGTGAAGGTCTGCACGTTCCACCGGGTCGTCAGCGTGAACTGGGTTTCACCTTCTCCTTCCCGGTACGTCAGACCTCTATTTCCTCCGGTAACCTGATTAAATGGACTAAAGGTTTTAGCATCGAAGACGCCGTGGGCGAAGACGTGGTTGGCGAACTGACCAAAGCCATGGAACGTATCGGCCTGGATATGCGTGTTTCCGCGCTGGTCAACGATACCATCGGCACCCTGGCCGGTGGTCGTTATCACAACCAGGATGTTGTTGCCGCGGTTATTCTGGGCACCGGCACCAATGCTGCTTACGTAGAACGTGCTCACGCCATCCCGAAATGGCAGGGTCTGCTGCCGAAATCTGGTGAAATGGTAATCAACATGGAATGGGGTAACTTCCGTTCTTCTCACCTGCCGCTGACCGAATATGACCACGCACTGGACGAGGAATCCAAAAACCCTGAAGAGCAGATTTTCGAAAAAATGATTTCTGGCATGTACCTGGGTGAGGTCGTACGCCGCGTACTGTATCGTATGGCCGAGGAAGCTTCTTTCTTCGGCGACGCAGTTCCGCCGAAACTGAAAATTCCTTACATCCTGCGTACCCCGGATATGTCCGCTATGCACCACGATATGTCCTCCGACCTGAAAGTTGTAGGTAACAAACTGAAAGACATCCTGGAAATTTCCAACACTTCCCTGAAAATGCGTAAGGTAATCGTTGAAATTTGTGATATCGTTGCAACCCGTGGCGCACGTCTGTCTGCAGCTGGTATCGTGGGTATCCTGAAGAAACTGGGCCGCGATACGGTTCGTGATGGCGAAAAACAGAAGACCGTGATCGCTCTGGACGGCGGTCTGTTCGAACACTACAAAAAATTCTCTTCCTGCATGGAGAGCACCCTGAAAGAACTGCTGGGCGAAGAGGTTGCAGAAAACATTGTTATTGAACTGTCCAACGATGGCAGCGGTATCGGTGCTGCACTGCTGGCAGCGAGCCACTCTCAATATCTGGAGGAGCCG
SEQ ID NO:6：
MGKVAVGAAVVCAAAVCAATALVVRHRMQSSGKWARAMAILKDLEDKCGTPIGKLRQIADAMTVEMHAGLASEGGSKLKMLISFVDNLPTGDEKGLFYALDLGGSNFRVLRVLLGGKEGHVVKQEFKEVSIPQHLMVGSSHELFNFIAAALAKFVATEGEGLHVPPGRQRELGFTFSFPVRQTSISSGNLIKWTKGFSIEDAVGEDVVGELTKAMERIGLDMRVSALVNDTIGTLAGGRYHNQDVVAAVILGTGTNAAYVERAHAIPKWQGLLPKSGEMVINMEWGNFRSSHLPLTEYDHALDEESKNPEEQIFEKMISGMYLGEVVRRVLYRMAEEASFFGDAVPPKLKIPYILRTPDMSAMHHDMSSDLKVVGNKLKDILEISNTSLKMRKVIVEICDIVATRGARLSAAGIVGILKKLGRDTVRDGEKQKTVIALDGGLFEHYKKFSSCMESTLKELLGEEVAENIVIELSNDGSGIGAALLAASHSQYLEEP
EXAMPLE 2 expression of hexokinase target protein
Step 1 was selected for the monoclonal antibody, inoculated in 100. Mu.g/mL of a Carna resistant TB medium, shake-cultured at 37℃at 220rpm until OD600 was between 0.6 and 0.8, induced with IPTG (final concentration of 0.1 mM), placed at 18℃overnight, shake-cultured without the addition of the IPTG group as a control, and cultured at 37℃for 3 hours, and each experiment was repeated. SDS-PAGE identification was performed by sonication and the results are recorded in Table 1 and FIG. 1 (M: maker, NT: control, S: supernatant, P: pellet in FIG. 1).
The results in FIG. 1 show that E.coli transformed with the vector comprising SEQ ID NO.1 can be expressed in large amounts in the supernatant in TB medium at 18℃with a protein molecular weight of about 55kDa, which is substantially consistent with the predicted protein size (53.8 kDa) on the Expasy website.
TABLE 2
| Sequences introduced into vectors | Culture conditions | Soluble protein ratio | Molecular weight of protein |
| SEQ ID NO:1 | 37℃ | 30％ | 55KD |
| 18℃ | 40％ | 55KD | |
| SEQ ID NO:3 | 37℃ | 10％ | 55KD |
| 18℃ | 15％ | 55KD | |
| SEQ ID NO:5 | 37℃ | 5％ | 55KD |
| 18℃ | 5％ | 55KD |
EXAMPLE 3 purification of hexokinase
TB medium shake flask culture of 1.5L bacterial liquid, expression conditions were consistent with the expression of the target protein in example 2. And centrifugally collecting thalli, wherein the wet weight is as follows: 30g. About 4g of the cells were weighed, and 35ml of Lysis Buffer was added thereto to resuspend on ice. Centrifuging at 20000rpm at 4deg.C for 30min after ultrasonic disruption, collecting supernatant, and filtering with 0.22 μm needle filter to obtain supernatant. Subjecting the supernatant to Ni-column affinity chromatography, performing linear elution with 0-60% buffer B, dialyzing with eluent with main peak, eluting to obtain 17ml protein, and electrophoresing the protein with an electrophoresis chart shown in figure 2, wherein M: maker, S, cell lysis supernatant, FT, affinity penetration solution. The target protein expression level was calculated 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
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 | 5.34mg/g bacteria |
| SEQ ID NO:3 | 2.01mg/g of bacteria |
Example 4 determination of recombinant hexokinase Activity
The activity of the hexokinase prepared in the example was measured on the sample using a hexokinase activity detection kit (soribao BC 0745). Hexokinase (2.5 KU) positive enzyme was purchased from sigma, dissolved in 1mL PBS pH 7.4 buffer, and then diluted to 0.01-0.32U/. Mu.L. The living testing working solution is prepared according to the instruction. The reagent II and the reagent III are fully mixed according to the proportion of 18:1 to prepare enzyme activity determination working solution (the reagent II and the reagent III are provided by a glutamate dehydrogenase activity detection kit).
100. Mu.L of the working solution for measuring activity was added to the ELISA plate, and 1. Mu.L of positive enzyme was added to each concentration, and the absorbance was read at 340nm by reaction at 25 ℃. A standard curve (FIG. 3) was drawn with the enzyme concentration on the X-axis and the corresponding absorbance on the Y-axis, so that R was 2 And more than or equal to 0.95. The absorbance was measured by the method described above, and the experiment was repeated 3 times to average 1. Mu.L of hexokinase prepared in the example. If the average absorbance is within the standard curve range, the enzyme activity is calculated by taking into a linear regression equation. If the concentration is not within the standard curve, the sample is concentrated or diluted and then measured.
The hexokinase prepared in the example was subjected to absorbance measurement according to the method described above, and the results are shown in table 4 below:
TABLE 4 Table 4
| Sequences introduced into vectors | Enzyme concentration | Average OD450 | Enzyme activity (U/mu L) |
| SEQ ID NO:2 | Stock solution | 0.492366667 | 0.183573848 |
| SEQ ID NO:4 | Stock solution | 0.119566667 | 0.024407252 |
When the sequence insert introduced into the vector was SEQ ID NO. 2, the activity of the resulting enzyme was 0.18U/. Mu.L, and the stock concentration was 1.642mg/mL, so that the specific activity was (0.18X 1000)/1.642=111.8U/mg.
When the sequence insert introduced into the vector was SEQ ID NO. 4, the activity of the resulting enzyme was 0.024U/. Mu.L, and the stock concentration was 1.483mg/mL, so that the specific activity was (0.024X 1000)/1.483=16.46U/mg.
It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples of carrying out the invention and that various changes in form and details may be made therein without departing from the spirit and scope of the invention.
Claims (10)
1. A polynucleotide encoding a hexokinase, wherein the polynucleotide is codon optimized and the 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 a homology of greater than 95% with a sequence as shown 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 of claim 2, wherein the expression vector comprises a polynucleotide sequence that expresses a His x 6 tag.
4. The expression vector according to claim 2, characterized in that it is an e.coli expression vector, preferably pS28a (+).
5. A host cell comprising the expression vector of any one of claims 2 to 4; or alternatively
The polynucleotide of claim 1 integrated into the genome of the host cell.
6. A method of preparing hexokinase, the 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 hexokinase.
7. The method according to claim 6, wherein the temperature of the culturing is 36 to 38 ℃ or 16 to 20 ℃, more preferably 17 to 19 ℃ when culturing the host cell.
8. The method according to claim 6, wherein the medium used in culturing the host cell comprises a kanamycin resistance gene.
9. The method of claim 6, 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 any one of claims 2 to 4; or alternatively
The host cell of claim 5; or alternatively
A recombinant creatine kinase isoenzyme prepared by the method of any one of claims 6 to 9.
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